Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia.

PubMed

Entcheva, P; Liebl, W; Johann, A; Hartsch, T; Streit, W R

2001-01-01

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).

Analysis of expressed sequence tags generated from full-length enriched cDNA libraries of melon

PubMed Central

2011-01-01

Background Melon (Cucumis melo), an economically important vegetable crop, belongs to the Cucurbitaceae family which includes several other important crops such as watermelon, cucumber, and pumpkin. It has served as a model system for sex determination and vascular biology studies. However, genomic resources currently available for melon are limited. Result We constructed eleven full-length enriched and four standard cDNA libraries from fruits, flowers, leaves, roots, cotyledons, and calluses of four different melon genotypes, and generated 71,577 and 22,179 ESTs from full-length enriched and standard cDNA libraries, respectively. These ESTs, together with ~35,000 ESTs available in public domains, were assembled into 24,444 unigenes, which were extensively annotated by comparing their sequences to different protein and functional domain databases, assigning them Gene Ontology (GO) terms, and mapping them onto metabolic pathways. Comparative analysis of melon unigenes and other plant genomes revealed that 75% to 85% of melon unigenes had homologs in other dicot plants, while approximately 70% had homologs in monocot plants. The analysis also identified 6,972 gene families that were conserved across dicot and monocot plants, and 181, 1,192, and 220 gene families specific to fleshy fruit-bearing plants, the Cucurbitaceae family, and melon, respectively. Digital expression analysis identified a total of 175 tissue-specific genes, which provides a valuable gene sequence resource for future genomics and functional studies. Furthermore, we identified 4,068 simple sequence repeats (SSRs) and 3,073 single nucleotide polymorphisms (SNPs) in the melon EST collection. Finally, we obtained a total of 1,382 melon full-length transcripts through the analysis of full-length enriched cDNA clones that were sequenced from both ends. Analysis of these full-length transcripts indicated that sizes of melon 5' and 3' UTRs were similar to those of tomato, but longer than many other dicot plants. Codon usages of melon full-length transcripts were largely similar to those of Arabidopsis coding sequences. Conclusion The collection of melon ESTs generated from full-length enriched and standard cDNA libraries is expected to play significant roles in annotating the melon genome. The ESTs and associated analysis results will be useful resources for gene discovery, functional analysis, marker-assisted breeding of melon and closely related species, comparative genomic studies and for gaining insights into gene expression patterns. PMID:21599934

Systematic cloning of human minisatellites from ordered array charomid libraries.

PubMed

Armour, J A; Povey, S; Jeremiah, S; Jeffreys, A J

1990-11-01

We present a rapid and efficient method for the isolation of minisatellite loci from human DNA. The method combines cloning a size-selected fraction of human MboI DNA fragments in a charomid vector with hybridization screening of the library in ordered array. Size-selection of large MboI fragments enriches for the longer, more variable minisatellites and reduces the size of the library required. The library was screened with a series of multi-locus probes known to detect a large number of hypervariable loci in human DNA. The gridded library allowed both the rapid processing of positive clones and the comparative evaluation of the different multi-locus probes used, in terms of both the relative success in detecting hypervariable loci and the degree of overlap between the sets of loci detected. We report 23 new human minisatellite loci isolated by this method, which map to 14 autosomes and the sex chromosomes.

Microsatellite DNA capture from enriched libraries.

PubMed

Gonzalez, Elena G; Zardoya, Rafael

2013-01-01

Microsatellites are DNA sequences of tandem repeats of one to six nucleotides, which are highly polymorphic, and thus the molecular markers of choice in many kinship, population genetic, and conservation studies. There have been significant technical improvements since the early methods for microsatellite isolation were developed, and today the most common procedures take advantage of the hybrid capture methods of enriched-targeted microsatellite DNA. Furthermore, recent advents in sequencing technologies (i.e., next-generation sequencing, NGS) have fostered the mining of microsatellite markers in non-model organisms, affording a cost-effective way of obtaining a large amount of sequence data potentially useful for loci characterization. The rapid improvements of NGS platforms together with the increase in available microsatellite information open new avenues to the understanding of the evolutionary forces that shape genetic structuring in wild populations. Here, we provide detailed methodological procedures for microsatellite isolation based on the screening of GT microsatellite-enriched libraries, either by cloning and Sanger sequencing of positive clones or by direct NGS. Guides for designing new species-specific primers and basic genotyping are also given.

Application and comparison of large-scale solution-based DNA capture-enrichment methods on ancient DNA

PubMed Central

Ávila-Arcos, María C.; Cappellini, Enrico; Romero-Navarro, J. Alberto; Wales, Nathan; Moreno-Mayar, J. Víctor; Rasmussen, Morten; Fordyce, Sarah L.; Montiel, Rafael; Vielle-Calzada, Jean-Philippe; Willerslev, Eske; Gilbert, M. Thomas P.

2011-01-01

The development of second-generation sequencing technologies has greatly benefitted the field of ancient DNA (aDNA). Its application can be further exploited by the use of targeted capture-enrichment methods to overcome restrictions posed by low endogenous and contaminating DNA in ancient samples. We tested the performance of Agilent's SureSelect and Mycroarray's MySelect in-solution capture systems on Illumina sequencing libraries built from ancient maize to identify key factors influencing aDNA capture experiments. High levels of clonality as well as the presence of multiple-copy sequences in the capture targets led to biases in the data regardless of the capture method. Neither method consistently outperformed the other in terms of average target enrichment, and no obvious difference was observed either when two tiling designs were compared. In addition to demonstrating the plausibility of capturing aDNA from ancient plant material, our results also enable us to provide useful recommendations for those planning targeted-sequencing on aDNA. PMID:22355593

Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21.

PubMed Central

McCormick, M K; Campbell, E; Deaven, L; Moyzis, R

1993-01-01

Construction of chromosome-specific yeast artificial chromosome (YAC) libraries from sorted chromosomes was undertaken (i) to eliminate drawbacks associated with first-generation total genomic YAC libraries, such as the high frequency of chimeric YACs, and (ii) to provide an alternative method for generating chromosome-specific YAC libraries in addition to isolating such collections from a total genomic library. Chromosome-specific YAC libraries highly enriched for human chromosomes 16 and 21 were constructed. By maximizing the percentage of fragments with two ligatable ends and performing yeast transformations with less than saturating amounts of DNA in the presence of carrier DNA, YAC libraries with a low percentage of chimeric clones were obtained. The smaller number of YAC clones in these chromosome-specific libraries reduces the effort involved in PCR-based screening and allows hybridization methods to be a manageable screening approach. Images PMID:8430075

Phylogenetic analysis of TCE-dechlorinating consortia enriched on a variety of electron donors.

PubMed

Freeborn, Ryan A; West, Kimberlee A; Bhupathiraju, Vishvesh K; Chauhan, Sadhana; Rahm, Brian G; Richardson, Ruth E; Alvarez-Cohen, Lisa

2005-11-01

Two rapidly fermented electron donors, lactate and methanol, and two slowly fermented electron donors, propionate and butyrate, were selected for enrichment studies to evaluate the characteristics of anaerobic microbial consortia that reductively dechlorinate TCE to ethene. Each electron donor enrichment subculture demonstrated the ability to dechlorinate TCE to ethene through several serial transfers. Microbial community analyses based upon 16S rDNA, including terminal restriction fragment length polymorphism (T-RFLP) and clone library/sequencing, were performed to assess major changes in microbial community structure associated with electron donors capable of stimulating reductive dechlorination. Results demonstrated that five phylogenic subgroups or genera of bacteria were present in all consortia, including Dehalococcoides sp., low G+C Gram-positives (mostly Clostridium and Eubacterium sp.), Bacteroides sp., Citrobacter sp., and delta Proteobacteria (mostly Desulfovibrio sp.). Phylogenetic association indicates that only minor shifts in the microbial community structure occurred between the four alternate electron donor enrichments and the parent consortium. Inconsistent detection of Dehalococcoides spp. in clone libraries and T-RFLP of enrichment subcultures was resolved using quantitative polymerase chain reaction (Q-PCR). Q-PCR with primers specific to Dehalococcoides 16S rDNA resulted in positive detection of this species in all enrichments. Our results suggest that TCE-dechlorinating consortia can be stably maintained on a variety of electron donors and that quantities of Dehalococcoides cells detected with Dehalococcoides specific 16S rDNA primer/probe sets do not necessarily correlate well with solvent degradation rates.

A linear concatenation strategy to construct 5'-enriched amplified cDNA libraries using multiple displacement amplification.

PubMed

Gadkar, Vijay J; Filion, Martin

2013-06-01

In various experimental systems, limiting available amounts of RNA may prevent a researcher from performing large-scale analyses of gene transcripts. One way to circumvent this is to 'pre-amplify' the starting RNA/cDNA, so that sufficient amounts are available for any downstream analysis. In the present study, we report the development of a novel protocol for constructing amplified cDNA libraries using the Phi29 DNA polymerase based multiple displacement amplification (MDA) system. Using as little as 200 ng of total RNA, we developed a linear concatenation strategy to make the single-stranded cDNA template amenable for MDA. The concatenation, made possible by the template switching property of the reverse transcriptase enzyme, resulted in the amplified cDNA library with intact 5' ends. MDA generated micrograms of template, allowing large-scale polymerase chain reaction analyses or other large-scale downstream applications. As the amplified cDNA library contains intact 5' ends, it is also compatible with 5' RACE analyses of specific gene transcripts. Empirical validation of this protocol is demonstrated on a highly characterized (tomato) and an uncharacterized (corn gromwell) experimental system.

Fidelity by design: Yoctoreactor and binder trap enrichment for small-molecule DNA-encoded libraries and drug discovery.

PubMed

Blakskjaer, Peter; Heitner, Tara; Hansen, Nils Jakob Vest

2015-06-01

DNA-encoded small-molecule library (DEL) technology allows vast drug-like small molecule libraries to be efficiently synthesized in a combinatorial fashion and screened in a single tube method for binding, with an assay readout empowered by advances in next generation sequencing technology. This approach has increasingly been applied as a viable technology for the identification of small-molecule modulators to protein targets and as precursors to drugs in the past decade. Several strategies for producing and for screening DELs have been devised by both academic and industrial institutions. This review highlights some of the most significant and recent strategies along with important results. A special focus on the production of high fidelity DEL technologies with the ability to eliminate screening noise and false positives is included: using a DNA junction called the Yoctoreactor, building blocks (BBs) are spatially confined at the center of the junction facilitating both the chemical reaction between BBs and encoding of the synthetic route. A screening method, known as binder trap enrichment, permits DELs to be screened robustly in a homogeneous manner delivering clean data sets and potent hits for even the most challenging targets. Copyright © 2015 Elsevier Ltd. All rights reserved.

Construction of a Full-Length Enriched cDNA Library and Preliminary Analysis of Expressed Sequence Tags from Bengal Tiger Panthera tigris tigris

PubMed Central

Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

2013-01-01

In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers. PMID:23708105

Construction of a full-length enriched cDNA library and preliminary analysis of expressed sequence tags from Bengal Tiger Panthera tigris tigris.

PubMed

Liu, Changqing; Liu, Dan; Guo, Yu; Lu, Taofeng; Li, Xiangchen; Zhang, Minghai; Ma, Jianzhang; Ma, Yuehui; Guan, Weijun

2013-05-24

In this study, a full-length enriched cDNA library was successfully constructed from Bengal tiger, Panthera tigris tigris, the most well-known wild Animal. Total RNA was extracted from cultured Bengal tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.28 × 106 pfu/mL and 1.56 × 109 pfu/mL respectively. The percentage of recombinants from unamplified library was 90.2% and average length of exogenous inserts was 0.98 kb. A total of 212 individual ESTs with sizes ranging from 356 to 1108 bps were then analyzed. The BLASTX score revealed that 48.1% of the sequences were classified as a strong match, 45.3% as nominal and 6.6% as a weak match. Among the ESTs with known putative function, 26.4% ESTs were found to be related to all kinds of metabolisms, 19.3% ESTs to information storage and processing, 11.3% ESTs to posttranslational modification, protein turnover, chaperones, 11.3% ESTs to transport, 9.9% ESTs to signal transducer/cell communication, 9.0% ESTs to structure protein, 3.8% ESTs to cell cycle, and only 6.6% ESTs classified as novel genes. By EST sequencing, a full-length gene coding ferritin was identified and characterized. The recombinant plasmid pET32a-TAT-Ferritin was constructed, coded for the TAT-Ferritin fusion protein with two 6× His-tags in N and C-terminal. After BCA assay, the concentration of soluble Trx-TAT-Ferritin recombinant protein was 2.32 ± 0.12 mg/mL. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genome and transcriptome research of Bengal tigers.

Preparation of Low-Input and Ligation-Free ChIP-seq Libraries Using Template-Switching Technology.

PubMed

Bolduc, Nathalie; Lehman, Alisa P; Farmer, Andrew

2016-10-10

Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA-binding proteins and cross-linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross-linking reversal and DNA recovery. Finally, we describe a fast, ligation-free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Informatic and genomic analysis of melanocyte cDNA libraries as a resource for the study of melanocyte development and function.

PubMed

Baxter, Laura L; Hsu, Benjamin J; Umayam, Lowell; Wolfsberg, Tyra G; Larson, Denise M; Frith, Martin C; Kawai, Jun; Hayashizaki, Yoshihide; Carninci, Piero; Pavan, William J

2007-06-01

As part of the RIKEN mouse encyclopedia project, two cDNA libraries were prepared from melanocyte-derived cell lines, using techniques of full-length clone selection and subtraction/normalization to enrich for rare transcripts. End sequencing showed that these libraries display over 83% complete coding sequence at the 5' end and 96-97% complete coding sequence at the 3' end. Evaluation of the libraries, derived from B16F10Y tumor cells and melan-c cells, revealed that they contain clones for a majority of the genes previously demonstrated to function in melanocyte biology. Analysis of genomic locations for transcripts revealed that the distribution of melanocyte genes is non-random throughout the genome. Three genomic regions identified that showed significant clustering of melanocyte-expressed genes contain one or more genes previously shown to regulate melanocyte development or function. A catalog of genes expressed in these libraries is presented, providing a valuable resource of cDNA clones and sequence information that can be used for identification of new genes important for melanocyte development, function, and disease.

UPIC + GO: Zeroing in on informative markers

USDA-ARS?s Scientific Manuscript database

Microsatellites/SSRs (simple sequence repeats) have become a powerful tool in genomic biology because of their broad range of applications and availability. An efficient method recently developed to generate microsatellite-enriched libraries used in combination with high throughput DNA pyrosequencin...

Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

PubMed

Kollmann, Christopher S; Bai, Xiaopeng; Tsai, Ching-Hsuan; Yang, Hongfang; Lind, Kenneth E; Skinner, Steven R; Zhu, Zhengrong; Israel, David I; Cuozzo, John W; Morgan, Barry A; Yuki, Koichi; Xie, Can; Springer, Timothy A; Shimaoka, Motomu; Evindar, Ghotas

2014-04-01

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

Development of microsatellite markers from an enriched genomic library for genetic analysis of melon (Cucumis melo L.)

PubMed Central

Ritschel, Patricia Silva; Lins, Tulio Cesar de Lima; Tristan, Rodrigo Lourenço; Buso, Gláucia Salles Cortopassi; Buso, José Amauri; Ferreira, Márcio Elias

2004-01-01

Background Despite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species. Results Seven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups. Conclusions Genomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon. PMID:15149552

Microsatellites for Lindera species

Treesearch

Craig S. Echt; D. Deemer; T.L. Kubisiak; C.D. Nelson

2006-01-01

Microsatellite markers were developed for conservation genetic studies of Lindera melissifolia (pondberry), a federally endangered shrub of southern bottomland ecosystems. Microsatellite sequences were obtained from DNA libraries that were enriched for the (AC)n simple sequence repeat motif. From 35 clone sequences, 20 primer...

Characterization of Thermostable Cellulases Produced by Bacillus and Geobacillus Strains

USDA-ARS?s Scientific Manuscript database

Bacterial community composition of thermophilic (60 deg C) mixed cellulose-enrichment cultures was examined by constructing a 16S rDNA clone library which demonstrated major lineages affiliated to Actinobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Firmicutes, and Proteobacteria. A tot...

Surveying the repair of ancient DNA from bones via high-throughput sequencing.

PubMed

Mouttham, Nathalie; Klunk, Jennifer; Kuch, Melanie; Fourney, Ron; Poinar, Hendrik

2015-07-01

DNA damage in the form of abasic sites, chemically altered nucleotides, and strand fragmentation is the foremost limitation in obtaining genetic information from many ancient samples. Upon cell death, DNA continues to endure various chemical attacks such as hydrolysis and oxidation, but repair pathways found in vivo no longer operate. By incubating degraded DNA with specific enzyme combinations adopted from these pathways, it is possible to reverse some of the post-mortem nucleic acid damage prior to downstream analyses such as library preparation, targeted enrichment, and high-throughput sequencing. Here, we evaluate the performance of two available repair protocols on previously characterized DNA extracts from four mammoths. Both methods use endonucleases and glycosylases along with a DNA polymerase-ligase combination. PreCR Repair Mix increases the number of molecules converted to sequencing libraries, leading to an increase in endogenous content and a decrease in cytosine-to-thymine transitions due to cytosine deamination. However, the effects of Nelson Repair Mix on repair of DNA damage remain inconclusive.

Capture-SELEX: Selection of DNA Aptamers for Aminoglycoside Antibiotics

PubMed Central

2012-01-01

Small organic molecules are challenging targets for an aptamer selection using the SELEX technology (SELEX—Systematic Evolution of Ligans by EXponential enrichment). Often they are not suitable for immobilization on solid surfaces, which is a common procedure in known aptamer selection methods. The Capture-SELEX procedure allows the selection of DNA aptamers for solute targets. A special SELEX library was constructed with the aim to immobilize this library on magnetic beads or other surfaces. For this purpose a docking sequence was incorporated into the random region of the library enabling hybridization to a complementary oligo fixed on magnetic beads. Oligonucleotides of the library which exhibit high affinity to the target and a secondary structure fitting to the target are released from the beads for binding to the target during the aptamer selection process. The oligonucleotides of these binding complexes were amplified, purified, and immobilized via the docking sequence to the magnetic beads as the starting point of the following selection round. Based on this Capture-SELEX procedure, the successful DNA aptamer selection for the aminoglycoside antibiotic kanamycin A as a small molecule target is described. PMID:23326761

Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs.

PubMed Central

Travis, G H; Sutcliffe, J G

1988-01-01

To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, we developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA. Images PMID:2894033

Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

PubMed

Brown, Dean G; Brown, Giles A; Centrella, Paolo; Certel, Kaan; Cooke, Robert M; Cuozzo, John W; Dekker, Niek; Dumelin, Christoph E; Ferguson, Andrew; Fiez-Vandal, Cédric; Geschwindner, Stefan; Guié, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Schlenker, Oliver; Sigel, Eric A; Snijder, Arjan; Soutter, Holly T; Sundström, Linda; Troast, Dawn M; Wiggin, Giselle; Zhang, Jing; Zhang, Ying; Clark, Matthew A

2018-06-01

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons

PubMed Central

Krishnaswami, Suguna Rani; Grindberg, Rashel V; Novotny, Mark; Venepally, Pratap; Lacar, Benjamin; Bhutani, Kunal; Linker, Sara B; Pham, Son; Erwin, Jennifer A; Miller, Jeremy A; Hodge, Rebecca; McCarthy, James K; Kelder, Martin; McCorrison, Jamison; Aevermann, Brian D; Fuertes, Francisco Diez; Scheuermann, Richard H; Lee, Jun; Lein, Ed S; Schork, Nicholas; McConnell, Michael J; Gage, Fred H; Lasken, Roger S

2016-01-01

A protocol is described for sequencing the transcriptome of a cell nucleus. Nuclei are isolated from specimens and sorted by FACS, cDNA libraries are constructed and RNA-seq is performed, followed by data analysis. Some steps follow published methods (Smart-seq2 for cDNA synthesis and Nextera XT barcoded library preparation) and are not described in detail here. Previous single-cell approaches for RNA-seq from tissues include cell dissociation using protease treatment at 30 °C, which is known to alter the transcriptome. We isolate nuclei at 4 °C from tissue homogenates, which cause minimal damage. Nuclear transcriptomes can be obtained from postmortem human brain tissue stored at −80 °C, making brain archives accessible for RNA-seq from individual neurons. The method also allows investigation of biological features unique to nuclei, such as enrichment of certain transcripts and precursors of some noncoding RNAs. By following this procedure, it takes about 4 d to construct cDNA libraries that are ready for sequencing. PMID:26890679

A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing

DOE PAGES

Bent, Zachary W.; Poorey, Kunal; LaBauve, Annette E.; ...

2016-12-21

When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich formore » pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000–3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs.« less

A Rapid Spin Column-Based Method to Enrich Pathogen Transcripts from Eukaryotic Host Cells Prior to Sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bent, Zachary W.; Poorey, Kunal; LaBauve, Annette E.

When analyzing pathogen transcriptomes during the infection of host cells, the signal-to-background (pathogen-to-host) ratio of nucleic acids (NA) in infected samples is very small. Despite the advancements in next-generation sequencing, the minute amount of pathogen NA makes standard RNA-seq library preps inadequate for effective gene-level analysis of the pathogen in cases with low bacterial loads. In order to provide a more complete picture of the pathogen transcriptome during an infection, we developed a novel pathogen enrichment technique, which can enrich for transcripts from any cultivable bacteria or virus, using common, readily available laboratory equipment and reagents. To evenly enrich formore » pathogen transcripts, we generate biotinylated pathogen-targeted capture probes in an enzymatic process using the entire genome of the pathogen as a template. The capture probes are hybridized to a strand-specific cDNA library generated from an RNA sample. The biotinylated probes are captured on a monomeric avidin resin in a miniature spin column, and enriched pathogen-specific cDNA is eluted following a series of washes. To test this method, we performed an in vitro time-course infection using Klebsiella pneumoniae to infect murine macrophage cells. K. pneumoniae transcript enrichment efficiency was evaluated using RNA-seq. Bacterial transcripts were enriched up to ~400-fold, and allowed the recovery of transcripts from ~2000–3600 genes not observed in untreated control samples. These additional transcripts revealed interesting aspects of K. pneumoniae biology including the expression of putative virulence factors and the expression of several genes responsible for antibiotic resistance even in the absence of drugs.« less

Diff-seq: A high throughput sequencing-based mismatch detection assay for DNA variant enrichment and discovery

PubMed Central

Karas, Vlad O; Sinnott-Armstrong, Nicholas A; Varghese, Vici; Shafer, Robert W; Greenleaf, William J; Sherlock, Gavin

2018-01-01

Abstract Much of the within species genetic variation is in the form of single nucleotide polymorphisms (SNPs), typically detected by whole genome sequencing (WGS) or microarray-based technologies. However, WGS produces mostly uninformative reads that perfectly match the reference, while microarrays require genome-specific reagents. We have developed Diff-seq, a sequencing-based mismatch detection assay for SNP discovery without the requirement for specialized nucleic-acid reagents. Diff-seq leverages the Surveyor endonuclease to cleave mismatched DNA molecules that are generated after cross-annealing of a complex pool of DNA fragments. Sequencing libraries enriched for Surveyor-cleaved molecules result in increased coverage at the variant sites. Diff-seq detected all mismatches present in an initial test substrate, with specific enrichment dependent on the identity and context of the variation. Application to viral sequences resulted in increased observation of variant alleles in a biologically relevant context. Diff-Seq has the potential to increase the sensitivity and efficiency of high-throughput sequencing in the detection of variation. PMID:29361139

Construction of cDNA expression library of watermelon for isolation of ClWRKY1 transcription factors gene involved in resistance to Fusarium wilt.

PubMed

Yang, Bing-Yan; Huo, Xiu-Ai; Li, Peng-Fei; Wang, Cui-Xia; Duan, Hui-Jun

2014-08-01

Full-length cDNAs are very important for genome annotation and functional analysis of genes. The number of full-length cDNAs from watermelon remains limited. Here we report first the construction of a full-length enriched cDNA library from Fusarium wilt stressed watermelon (Citrullus lanatus Thunb.) cultivar PI296341 root tissues using the SMART method. The titer of primary cDNA library and amplified library was 2.21 x 10(6) and 2.0 x 10(10) pfu/ml, respectively and the rate of recombinant was above 85%. The size of insert fragment ranged from 0.3 to 2.0 kb. In this study, we first cloned a gene named ClWRKY1, which was 1981 bp long and encoded a protein consisting of 394 amino acids. It contained two characteristic WRKY domains and two zinc finger motifs. Quantitative real-time PCR showed that ClWRKY1 expression levels reached maximum level at 12 h after inoculation with Fusarium oxysporum f. sp. niveum. The full-length cDNA library of watermelon root tissues is not only essential for the cloning of genes which are known, but also an initial key for the screening and cloning of new genes that might be involved in resistance to Fusarium wilt.

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

PubMed

Gupta, Amita; Shrivastava, Nimisha; Grover, Payal; Singh, Ajay; Mathur, Kapil; Verma, Vaishali; Kaur, Charanpreet; Chaudhary, Vijay K

2013-01-01

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins.

Corexit 9500 Enhances Oil Biodegradation and Changes ...

EPA Pesticide Factsheets

While COREXIT 9500 is widely applied after oil spills for its reported dispersing activity, there is still a debate on the effectiveness on enhancing oil biodegradation and its potential toxic effect on microbial communities. To better understand the impact of COREXIT 9500 on the structure and activity levels of hydrocarbon degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at cryophilic and mesophilic conditions and using both DNA and RNA extracts as sequencing templates. Oil biodegradation patterns in both cryophilic and mesophilic enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). A slight increase in biodegradation was observed in the presence of COREXIT at both 25°C and 5°C experiments. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25°C consortia was dominated by unclassified members of the Vibrio, Pseudoidiomarina, Marinobacter, Alcanivorax, and Thallassospira species, while the 5°C consortia were dominated by several genera of Flavobacteria, Alcanivorax and Oleispira. With the exception of Vibrio-like species, members of these genera have been linked to hydrocarbon degradation and have been observed after oil spills. Colwellia and Cycloclasticus, known aromatic degraders, was also found in these enrichments. RNA-based sequencing of 25°C

Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

PubMed Central

Chatterjee, Aniruddha; Rodger, Euan J.; Stockwell, Peter A.; Weeks, Robert J.; Morison, Ian M.

2012-01-01

Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background. PMID:23193365

Selection of DNA aptamers against Human Cardiac Troponin I for colorimetric sensor based dot blot application.

PubMed

Dorraj, Ghamar Soltan; Rassaee, Mohammad Javad; Latifi, Ali Mohammad; Pishgoo, Bahram; Tavallaei, Mahmood

2015-08-20

Troponin T and I are ideal markers which are highly sensitive and specific for myocardial injury and have shown better efficacy than earlier markers. Since aptamers are ssDNA or RNA that bind to a wide variety of target molecules, the purpose of this research was to select an aptamer from a 79bp single-stranded DNA (ssDNA) random library that was used to bind the Human Cardiac Troponin I from a synthetic nucleic acids library by systematic evolution of ligands exponential enrichment (Selex) based on several selection and amplification steps. Human Cardiac Troponin I protein was coated onto the surface of streptavidin magnetic beads to extract specific aptamer from a large and diverse random ssDNA initial oligonucleotide library. As a result, several aptamers were selected and further examined for binding affinity and specificity. Finally TnIApt 23 showed beast affinity in nanomolar range (2.69nM) toward the target protein. A simple and rapid colorimetric detection assay for Human Cardiac Troponin I using the novel and specific aptamer-AuNPs conjugates based on dot blot assay was developed. The detection limit for this protein using aptamer-AuNPs-based assay was found to be 5ng/ml. Copyright © 2015 Elsevier B.V. All rights reserved.

Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

PubMed Central

Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

2013-01-01

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

Use of the melting curve assay as a means for high-throughput quantification of Illumina sequencing libraries.

PubMed

Shinozuka, Hiroshi; Forster, John W

2016-01-01

Background. Multiplexed sequencing is commonly performed on massively parallel short-read sequencing platforms such as Illumina, and the efficiency of library normalisation can affect the quality of the output dataset. Although several library normalisation approaches have been established, none are ideal for highly multiplexed sequencing due to issues of cost and/or processing time. Methods. An inexpensive and high-throughput library quantification method has been developed, based on an adaptation of the melting curve assay. Sequencing libraries were subjected to the assay using the Bio-Rad Laboratories CFX Connect(TM) Real-Time PCR Detection System. The library quantity was calculated through summation of reduction of relative fluorescence units between 86 and 95 °C. Results.PCR-enriched sequencing libraries are suitable for this quantification without pre-purification of DNA. Short DNA molecules, which ideally should be eliminated from the library for subsequent processing, were differentiated from the target DNA in a mixture on the basis of differences in melting temperature. Quantification results for long sequences targeted using the melting curve assay were correlated with those from existing methods (R (2) > 0.77), and that observed from MiSeq sequencing (R (2) = 0.82). Discussion.The results of multiplexed sequencing suggested that the normalisation performance of the described method is equivalent to that of another recently reported high-throughput bead-based method, BeNUS. However, costs for the melting curve assay are considerably lower and processing times shorter than those of other existing methods, suggesting greater suitability for highly multiplexed sequencing applications.

Display of disulfide-rich proteins by complementary DNA display and disulfide shuffling assisted by protein disulfide isomerase.

PubMed

Naimuddin, Mohammed; Kubo, Tai

2011-12-01

We report an efficient system to produce and display properly folded disulfide-rich proteins facilitated by coupled complementary DNA (cDNA) display and protein disulfide isomerase-assisted folding. The results show that a neurotoxin protein containing four disulfide linkages can be displayed in the folded state. Furthermore, it can be refolded on a solid support that binds efficiently to its natural acetylcholine receptor. Probing the efficiency of the display proteins prepared by these methods provided up to 8-fold higher enrichment by the selective enrichment method compared with cDNA display alone, more than 10-fold higher binding to its receptor by the binding assays, and more than 10-fold higher affinities by affinity measurements. Cotranslational folding was found to have better efficiency than posttranslational refolding between the two investigated methods. We discuss the utilities of efficient display of such proteins in the preparation of superior quality proteins and protein libraries for directed evolution leading to ligand discovery. Copyright © 2011 Elsevier Inc. All rights reserved.

Compositional Bias in Naïve and Chemically-modified Phage-Displayed Libraries uncovered by Paired-end Deep Sequencing.

PubMed

He, Bifang; Tjhung, Katrina F; Bennett, Nicholas J; Chou, Ying; Rau, Andrea; Huang, Jian; Derda, Ratmir

2018-01-19

Understanding the composition of a genetically-encoded (GE) library is instrumental to the success of ligand discovery. In this manuscript, we investigate the bias in GE-libraries of linear, macrocyclic and chemically post-translationally modified (cPTM) tetrapeptides displayed on the M13KE platform, which are produced via trinucleotide cassette synthesis (19 codons) and NNK-randomized codon. Differential enrichment of synthetic DNA {S}, ligated vector {L} (extension and ligation of synthetic DNA into the vector), naïve libraries {N} (transformation of the ligated vector into the bacteria followed by expression of the library for 4.5 hours to yield a "naïve" library), and libraries chemically modified by aldehyde ligation and cysteine macrocyclization {M} characterized by paired-end deep sequencing, detected a significant drop in diversity in {L} → {N}, but only a minor compositional difference in {S} → {L} and {N} → {M}. Libraries expressed at the N-terminus of phage protein pIII censored positively charged amino acids Arg and Lys; libraries expressed between pIII domains N1 and N2 overcame Arg/Lys-censorship but introduced new bias towards Gly and Ser. Interrogation of biases arising from cPTM by aldehyde ligation and cysteine macrocyclization unveiled censorship of sequences with Ser/Phe. Analogous analysis can be used to explore library diversity in new display platforms and optimize cPTM of these libraries.

Characterization of embryo-specific genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sung, Z.R.

1988-01-01

The objective of the proposed research is to characterize the structure and function of a set of genes whose expression is regulated in embryo development, and that are not expressed in mature tissues -- the embryogenic genes. In order to isolate these genes, we immunized a rabbit with total extracts of somatic embryos of carrot, and enriched the anti-embryo antiserum for antibodies reacting with extracts of carrot somatic embryos. Using this enriched antiserum, we screened a lambda gt11 cDNA library constructed from embryo poly A{sup +} RNA, and isolated 10 cDNA clones that detect embryogenic mRNAs. Monospecific antibodies have beenmore » purified for proteins corresponding to each cDNA sequence. Four cDNA clones were further characterized in terms of the expression of their corresponding mRNA and protein in somatic embryos of carrot. In some cases, comparable gene sequences or products have been detected in somatic and zygotic embryos of other plant species. The characteristics of these 4 cDNA clones -- clone Nos. 8, 59, and 66 -- are described in this report. 3 figs.« less

Peregrine

PubMed Central

Langevin, Stanley A.; Bent, Zachary W.; Solberg, Owen D.; Curtis, Deanna J.; Lane, Pamela D.; Williams, Kelly P.; Schoeniger, Joseph S.; Sinha, Anupama; Lane, Todd W.; Branda, Steven S.

2013-01-01

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows. PMID:23558773

Generation of Aptamers from A Primer-Free Randomized ssDNA Library Using Magnetic-Assisted Rapid Aptamer Selection

NASA Astrophysics Data System (ADS)

Tsao, Shih-Ming; Lai, Ji-Ching; Horng, Horng-Er; Liu, Tu-Chen; Hong, Chin-Yih

2017-04-01

Aptamers are oligonucleotides that can bind to specific target molecules. Most aptamers are generated using random libraries in the standard systematic evolution of ligands by exponential enrichment (SELEX). Each random library contains oligonucleotides with a randomized central region and two fixed primer regions at both ends. The fixed primer regions are necessary for amplifying target-bound sequences by PCR. However, these extra-sequences may cause non-specific bindings, which potentially interfere with good binding for random sequences. The Magnetic-Assisted Rapid Aptamer Selection (MARAS) is a newly developed protocol for generating single-strand DNA aptamers. No repeat selection cycle is required in the protocol. This study proposes and demonstrates a method to isolate aptamers for C-reactive proteins (CRP) from a randomized ssDNA library containing no fixed sequences at 5‧ and 3‧ termini using the MARAS platform. Furthermore, the isolated primer-free aptamer was sequenced and binding affinity for CRP was analyzed. The specificity of the obtained aptamer was validated using blind serum samples. The result was consistent with monoclonal antibody-based nephelometry analysis, which indicated that a primer-free aptamer has high specificity toward targets. MARAS is a feasible platform for efficiently generating primer-free aptamers for clinical diagnoses.

Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites.

PubMed Central

He, M; Taussig, M J

1997-01-01

We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries. PMID:9396828

Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites.

PubMed

He, M; Taussig, M J

1997-12-15

We describe a rapid, eukaryotic, in vitro method for selection and evolution of antibody combining sites using antibody-ribosome-mRNA (ARM) complexes as selection particles. ARMs carrying single-chain (VH/K) binding fragments specific for progesterone were selected using antigen-coupled magnetic beads; selection simultaneously captured the genetic information as mRNA, making it possible to generate and amplify cDNA by single-step RT-PCR on the ribosome-bound mRNA for further manipulation. Using mutant libraries, antigen-binding ARMs were enriched by a factor of 10(4)-10(5)-fold in a single cycle, with further enrichment in repeated cycles. While demonstrated here for antibodies, the method has the potential to be applied equally for selection of receptors or peptides from libraries.

By-product formation in repetitive PCR amplification of DNA libraries during SELEX.

PubMed

Tolle, Fabian; Wilke, Julian; Wengel, Jesper; Mayer, Günter

2014-01-01

The selection of nucleic acid aptamers is an increasingly important approach to generate specific ligands binding to virtually any molecule of choice. However, selection-inherent amplification procedures are prone to artificial by-product formation that prohibits the enrichment of target-recognizing aptamers. Little is known about the formation of such by-products when employing nucleic acid libraries as templates. We report on the formation of two different forms of by-products, named ladder- and non-ladder-type observed during repetitive amplification in the course of in vitro selection experiments. Based on sequence information and the amplification behaviour of defined enriched nucleic acid molecules we suppose a molecular mechanism through which these amplification by-products are built. Better understanding of these mechanisms might help to find solutions minimizing by-product formation and improving the success rate of aptamer selection.

Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices.

PubMed

Dellagnezze, Bruna Martins; Vasconcellos, Suzan Pantaroto de; Melo, Itamar Soares de; Santos Neto, Eugênio Vaz Dos; Oliveira, Valéria Maia de

2016-01-01

Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Transcription profiling using RNA-Seq demonstrates expression differences in the body walls of juvenile albino and normal sea cucumbers Apostichopus japonicus

NASA Astrophysics Data System (ADS)

Ma, Deyou; Yang, Hongsheng; Sun, Lina; Chen, Muyan

2014-01-01

Sea cucumbers Apostichopus japonicus are one of the most important aquaculture species in China. Their normal body color is black to fit their surroundings. Wild albinos are rare and hard to breed. To understand the differences between albino and normal (control) sea cucumbers at the transcriptional level, we sequenced the transcriptomes in their body-wall tissues using RNA-Seq high-throughput sequencing. Approximately 4.876 million (M) and 4.884 M 200-nucleotide-long cDNA reads were produced in the cDNA libraries derived from the body walls of albino and control samples, respectively. A total of 9 561 (46.89%) putative genes were identified from among the RNA-Seq reads in both libraries. After filtering, 837 significantly differentially regulated genes were identified in the albino library compared with in the control library, and 3.6% of the differentially expressed genes (DEGs) were found to have changed those more than five-fold. The expression levels of 10 DEGs were checked by real-time PCR and the results were in full accord with the RNA-Seq expression trends, although the amplitude of the differences in expression levels was lower in all cases. A series of pathways were significantly enriched for the DEGs. These pathways were closely related to phagocytosis, the complement and coagulation cascades, apoptosis-related diseases, cytokine-cytokine receptor interaction, and cell adhesion. The differences in gene expression and enriched pathways between the albino and control sea cucumbers offer control targets for cultivating excellent albino A. japonicus strains in the future.

Consequences of Normalizing Transcriptomic and Genomic Libraries of Plant Genomes Using a Duplex-Specific Nuclease and Tetramethylammonium Chloride

PubMed Central

Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

2013-01-01

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce. PMID:23409088

Consequences of normalizing transcriptomic and genomic libraries of plant genomes using a duplex-specific nuclease and tetramethylammonium chloride.

PubMed

Matvienko, Marta; Kozik, Alexander; Froenicke, Lutz; Lavelle, Dean; Martineau, Belinda; Perroud, Bertrand; Michelmore, Richard

2013-01-01

Several applications of high throughput genome and transcriptome sequencing would benefit from a reduction of the high-copy-number sequences in the libraries being sequenced and analyzed, particularly when applied to species with large genomes. We adapted and analyzed the consequences of a method that utilizes a thermostable duplex-specific nuclease for reducing the high-copy components in transcriptomic and genomic libraries prior to sequencing. This reduces the time, cost, and computational effort of obtaining informative transcriptomic and genomic sequence data for both fully sequenced and non-sequenced genomes. It also reduces contamination from organellar DNA in preparations of nuclear DNA. Hybridization in the presence of 3 M tetramethylammonium chloride (TMAC), which equalizes the rates of hybridization of GC and AT nucleotide pairs, reduced the bias against sequences with high GC content. Consequences of this method on the reduction of high-copy and enrichment of low-copy sequences are reported for Arabidopsis and lettuce.

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

PubMed

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material.

PubMed

Langevin, Stanley A; Bent, Zachary W; Solberg, Owen D; Curtis, Deanna J; Lane, Pamela D; Williams, Kelly P; Schoeniger, Joseph S; Sinha, Anupama; Lane, Todd W; Branda, Steven S

2013-04-01

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

Identification of tissue-specific, abiotic stress-responsive gene expression patterns in wine grape (Vitis vinifera L.) based on curation and mining of large-scale EST data sets

PubMed Central

2011-01-01

Background Abiotic stresses, such as water deficit and soil salinity, result in changes in physiology, nutrient use, and vegetative growth in vines, and ultimately, yield and flavor in berries of wine grape, Vitis vinifera L. Large-scale expressed sequence tags (ESTs) were generated, curated, and analyzed to identify major genetic determinants responsible for stress-adaptive responses. Although roots serve as the first site of perception and/or injury for many types of abiotic stress, EST sequencing in root tissues of wine grape exposed to abiotic stresses has been extremely limited to date. To overcome this limitation, large-scale EST sequencing was conducted from root tissues exposed to multiple abiotic stresses. Results A total of 62,236 expressed sequence tags (ESTs) were generated from leaf, berry, and root tissues from vines subjected to abiotic stresses and compared with 32,286 ESTs sequenced from 20 public cDNA libraries. Curation to correct annotation errors, clustering and assembly of the berry and leaf ESTs with currently available V. vinifera full-length transcripts and ESTs yielded a total of 13,278 unique sequences, with 2302 singletons and 10,976 mapped to V. vinifera gene models. Of these, 739 transcripts were found to have significant differential expression in stressed leaves and berries including 250 genes not described previously as being abiotic stress responsive. In a second analysis of 16,452 ESTs from a normalized root cDNA library derived from roots exposed to multiple, short-term, abiotic stresses, 135 genes with root-enriched expression patterns were identified on the basis of their relative EST abundance in roots relative to other tissues. Conclusions The large-scale analysis of relative EST frequency counts among a diverse collection of 23 different cDNA libraries from leaf, berry, and root tissues of wine grape exposed to a variety of abiotic stress conditions revealed distinct, tissue-specific expression patterns, previously unrecognized stress-induced genes, and many novel genes with root-enriched mRNA expression for improving our understanding of root biology and manipulation of rootstock traits in wine grape. mRNA abundance estimates based on EST library-enriched expression patterns showed only modest correlations between microarray and quantitative, real-time reverse transcription-polymerase chain reaction (qRT-PCR) methods highlighting the need for deep-sequencing expression profiling methods. PMID:21592389

Corexit 9500 Enhances Oil Biodegradation and Changes Active Bacterial Community Structure of Oil-Enriched Microcosms.

PubMed

Techtmann, Stephen M; Zhuang, Mobing; Campo, Pablo; Holder, Edith; Elk, Michael; Hazen, Terry C; Conmy, Robyn; Santo Domingo, Jorge W

2017-05-15

To better understand the impacts of Corexit 9500 on the structure and activity levels of hydrocarbon-degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at 5 and 25°C using both DNA and RNA extracts as the sequencing templates. Oil biodegradation patterns in both 5 and 25°C enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). Slight increases in biodegradation were observed in the presence of Corexit at both temperatures. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25°C consortia were dominated by Vibrio , Idiomarina , Marinobacter , Alcanivorax , and Thalassospira species, while the 5°C consortia were dominated by several species of the genera Flavobacterium , Alcanivorax , and Oleispira Most of these genera have been linked to hydrocarbon degradation and have been observed after oil spills. Colwellia and Cycloclasticus , known aromatic degraders, were also found in these enrichments. The addition of Corexit did not have an effect on the active bacterial community structure of the 5°C consortia, while at 25°C, a decrease in the relative abundance of Marinobacter was observed. At 25°C, Thalassospira , Marinobacter , and Idiomarina were present at higher relative abundances in the RNA than DNA libraries, suggesting that they were active in degradation. Similarly, Oleispira was greatly stimulated by the addition of oil at 5°C. IMPORTANCE While dispersants such as Corexit 9500 can be used to treat oil spills, there is still debate on the effectiveness on enhancing oil biodegradation and its potential toxic effect on oil-degrading microbial communities. The results of this study provide some insights on the microbial dynamics of hydrocarbon-degrading bacterial populations in the presence of Corexit 9500. Operational taxonomic unit (OTU) analyses indicated that several OTUs were inhibited by the addition of Corexit. Conversely, a number of OTUs were stimulated by the addition of the dispersant, many of which were identified as known hydrocarbon-degrading bacteria. The results highlight the value of using RNA-based methods to further understand the impact of dispersant on the overall activity of different hydrocarbon-degrading bacterial groups. Copyright © 2017 American Society for Microbiology.

Corexit 9500 Enhances Oil Biodegradation and Changes Active Bacterial Community Structure of Oil-Enriched Microcosms

PubMed Central

Zhuang, Mobing; Campo, Pablo; Holder, Edith; Elk, Michael; Conmy, Robyn

2017-01-01

ABSTRACT To better understand the impacts of Corexit 9500 on the structure and activity levels of hydrocarbon-degrading microbial communities, we analyzed next-generation 16S rRNA gene sequencing libraries of hydrocarbon enrichments grown at 5 and 25°C using both DNA and RNA extracts as the sequencing templates. Oil biodegradation patterns in both 5 and 25°C enrichments were consistent with those reported in the literature (i.e., aliphatics were degraded faster than aromatics). Slight increases in biodegradation were observed in the presence of Corexit at both temperatures. Differences in community structure were observed between treatment conditions in the DNA-based libraries. The 25°C consortia were dominated by Vibrio, Idiomarina, Marinobacter, Alcanivorax, and Thalassospira species, while the 5°C consortia were dominated by several species of the genera Flavobacterium, Alcanivorax, and Oleispira. Most of these genera have been linked to hydrocarbon degradation and have been observed after oil spills. Colwellia and Cycloclasticus, known aromatic degraders, were also found in these enrichments. The addition of Corexit did not have an effect on the active bacterial community structure of the 5°C consortia, while at 25°C, a decrease in the relative abundance of Marinobacter was observed. At 25°C, Thalassospira, Marinobacter, and Idiomarina were present at higher relative abundances in the RNA than DNA libraries, suggesting that they were active in degradation. Similarly, Oleispira was greatly stimulated by the addition of oil at 5°C. IMPORTANCE While dispersants such as Corexit 9500 can be used to treat oil spills, there is still debate on the effectiveness on enhancing oil biodegradation and its potential toxic effect on oil-degrading microbial communities. The results of this study provide some insights on the microbial dynamics of hydrocarbon-degrading bacterial populations in the presence of Corexit 9500. Operational taxonomic unit (OTU) analyses indicated that several OTUs were inhibited by the addition of Corexit. Conversely, a number of OTUs were stimulated by the addition of the dispersant, many of which were identified as known hydrocarbon-degrading bacteria. The results highlight the value of using RNA-based methods to further understand the impact of dispersant on the overall activity of different hydrocarbon-degrading bacterial groups. PMID:28283527

In Vitro Selection for Small-Molecule-Triggered Strand Displacement and Riboswitch Activity.

PubMed

Martini, Laura; Meyer, Adam J; Ellefson, Jared W; Milligan, John N; Forlin, Michele; Ellington, Andrew D; Mansy, Sheref S

2015-10-16

An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.

By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

PubMed Central

Tolle, Fabian; Wilke, Julian; Wengel, Jesper; Mayer, Günter

2014-01-01

The selection of nucleic acid aptamers is an increasingly important approach to generate specific ligands binding to virtually any molecule of choice. However, selection-inherent amplification procedures are prone to artificial by-product formation that prohibits the enrichment of target-recognizing aptamers. Little is known about the formation of such by-products when employing nucleic acid libraries as templates. We report on the formation of two different forms of by-products, named ladder- and non-ladder-type observed during repetitive amplification in the course of in vitro selection experiments. Based on sequence information and the amplification behaviour of defined enriched nucleic acid molecules we suppose a molecular mechanism through which these amplification by-products are built. Better understanding of these mechanisms might help to find solutions minimizing by-product formation and improving the success rate of aptamer selection. PMID:25490402

SSHscreen and SSHdb, generic software for microarray based gene discovery: application to the stress response in cowpea

PubMed Central

2010-01-01

Background Suppression subtractive hybridization is a popular technique for gene discovery from non-model organisms without an annotated genome sequence, such as cowpea (Vigna unguiculata (L.) Walp). We aimed to use this method to enrich for genes expressed during drought stress in a drought tolerant cowpea line. However, current methods were inefficient in screening libraries and management of the sequence data, and thus there was a need to develop software tools to facilitate the process. Results Forward and reverse cDNA libraries enriched for cowpea drought response genes were screened on microarrays, and the R software package SSHscreen 2.0.1 was developed (i) to normalize the data effectively using spike-in control spot normalization, and (ii) to select clones for sequencing based on the calculation of enrichment ratios with associated statistics. Enrichment ratio 3 values for each clone showed that 62% of the forward library and 34% of the reverse library clones were significantly differentially expressed by drought stress (adjusted p value < 0.05). Enrichment ratio 2 calculations showed that > 88% of the clones in both libraries were derived from rare transcripts in the original tester samples, thus supporting the notion that suppression subtractive hybridization enriches for rare transcripts. A set of 118 clones were chosen for sequencing, and drought-induced cowpea genes were identified, the most interesting encoding a late embryogenesis abundant Lea5 protein, a glutathione S-transferase, a thaumatin, a universal stress protein, and a wound induced protein. A lipid transfer protein and several components of photosynthesis were down-regulated by the drought stress. Reverse transcriptase quantitative PCR confirmed the enrichment ratio values for the selected cowpea genes. SSHdb, a web-accessible database, was developed to manage the clone sequences and combine the SSHscreen data with sequence annotations derived from BLAST and Blast2GO. The self-BLAST function within SSHdb grouped redundant clones together and illustrated that the SSHscreen plots are a useful tool for choosing anonymous clones for sequencing, since redundant clones cluster together on the enrichment ratio plots. Conclusions We developed the SSHscreen-SSHdb software pipeline, which greatly facilitates gene discovery using suppression subtractive hybridization by improving the selection of clones for sequencing after screening the library on a small number of microarrays. Annotation of the sequence information and collaboration was further enhanced through a web-based SSHdb database, and we illustrated this through identification of drought responsive genes from cowpea, which can now be investigated in gene function studies. SSH is a popular and powerful gene discovery tool, and therefore this pipeline will have application for gene discovery in any biological system, particularly non-model organisms. SSHscreen 2.0.1 and a link to SSHdb are available from http://microarray.up.ac.za/SSHscreen. PMID:20359330

Analysis of expressed sequence tags from the four main developmental stages of Trypanosoma congolense

PubMed Central

Helm, Jared R.; Hertz-Fowler, Christiane; Aslett, Martin; Berriman, Matthew; Sanders, Mandy; Quail, Michael A.; Soares, Marcelo B.; Bonaldo, Maria F.; Sakurai, Tatsuya; Inoue, Noboru; Donelson, John E.

2009-01-01

Trypanosoma congolense is one of the most economically important pathogens of livestock in Africa. Culture-derived parasites of each of the three main insect stages of the T. congolense life cycle, i.e., the procyclic, epimastigote and metacyclic stages, and bloodstream stage parasites isolated from infected mice, were used to construct stage-specific cDNA libraries and expressed sequence tags (ESTs or cDNA clones) in each library were sequenced. Thirteen EST clusters encoding different variant surface glycoproteins (VSGs) were detected in the metacyclic library and twenty-six VSG EST clusters were found in the bloodstream library, six of which are shared by the metacyclic library. Rare VSG ESTs are present in the epimastigote library, and none were detected in the procyclic library. ESTs encoding enzymes that catalyze oxidative phosphorylation and amino acid metabolism are about twice as abundant in the procyclic and epimastigote stages as in the metacyclic and bloodstream stages. In contrast, ESTs encoding enzymes involved in glycolysis, the citric acid cycle and nucleotide metabolism are about the same in all four developmental stages. Cysteine proteases, kinases and phosphatases are the most abundant enzyme groups represented by the ESTs. All four libraries contain T. congolense-specific expressed sequences not present in the T. brucei and T. cruzi genomes. Normalized cDNA libraries were constructed from the metacyclic and bloodstream stages, and found to be further enriched for T. congolense-specific ESTs. Given that cultured T. congolense offers an experimental advantage over other African trypanosome species, these ESTs provide a basis for further investigation of the molecular properties of these four developmental stages, especially the epimastigote and metacyclic stages for which it is difficult to obtain large quantities of organisms. The T. congolense EST databases are available at: http://www.sanger.ac.uk/Projects/T_congolense/EST_index.shtml. PMID:19559733

Validation and application of quantitative PCR assays using host-specific Bacteroidales genetic markers for swine fecal pollution tracking.

PubMed

Fan, Lihua; Shuai, Jiangbing; Zeng, Ruoxue; Mo, Hongfei; Wang, Suhua; Zhang, Xiaofeng; He, Yongqiang

2017-12-01

Genome fragment enrichment (GFE) method was applied to identify host-specific bacterial genetic markers that differ among different fecal metagenomes. To enrich for swine-specific DNA fragments, swine fecal DNA composite (n = 34) was challenged against a DNA composite consisting of cow, human, goat, sheep, chicken, duck and goose fecal DNA extracts (n = 83). Bioinformatic analyses of 384 non-redundant swine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode metabolism-associated, cellular processes and information storage and processing. After challenged against fecal DNA extracted from different animal sources, four sequences from the clone libraries targeting two Bacteroidales- (genes 1-38 and 3-53), a Clostridia- (gene 2-109) as well as a Bacilli-like sequence (gene 2-95), respectively, showed high specificity to swine feces based on PCR analysis. Host-specificity and host-sensitivity analysis confirmed that oligonucleotide primers and probes capable of annealing to select Bacteroidales-like sequences (1-38 and 3-53) exhibited high specificity (>90%) in quantitative PCR assays with 71 fecal DNAs from non-target animal sources. The two assays also demonstrated broad distributions of corresponding genetic markers (>94% positive) among 72 swine feces. After evaluation with environmental water samples from different areas, swine-targeted assays based on two Bacteroidales-like GFE sequences appear to be suitable quantitative tracing tools for swine fecal pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation of human simple repeat loci by hybridization selection.

PubMed

Armour, J A; Neumann, R; Gobert, S; Jeffreys, A J

1994-04-01

We have isolated short tandem repeat arrays from the human genome, using a rapid method involving filter hybridization to enrich for tri- or tetranucleotide tandem repeats. About 30% of clones from the enriched library cross-hybridize with probes containing trimeric or tetrameric tandem arrays, facilitating the rapid isolation of large numbers of clones. In an initial analysis of 54 clones, 46 different tandem arrays were identified. Analysis of these tandem repeat loci by PCR showed that 24 were polymorphic in length; substantially higher levels of polymorphism were displayed by the tetrameric repeat loci isolated than by the trimeric repeats. Primary mapping of these loci by linkage analysis showed that they derive from 17 chromosomes, including the X chromosome. We anticipate the use of this strategy for the efficient isolation of tandem repeats from other sources of genomic DNA, including DNA from flow-sorted chromosomes, and from other species.

Reexamining Content-Enriched Access: Its Effect on Usage and Discovery

ERIC Educational Resources Information Center

Tosaka, Yuji; Weng, Cathy

2011-01-01

Content-enriched metadata in bibliographic records is considered helpful to library users in identifying and selecting library materials for their needs. The paper presents a study, using circulation data from a medium-sized academic library, of the effect of content-enriched records on library materials usage. The study also examines OPAC search…

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Evidence of accelerated evolution and ectodermal-specific expression of presumptive BDS toxin cDNAs from Anemonia viridis.

PubMed

Nicosia, Aldo; Maggio, Teresa; Mazzola, Salvatore; Cuttitta, Angela

2013-10-30

Anemonia viridis is a widespread and extensively studied Mediterranean species of sea anemone from which a large number of polypeptide toxins, such as blood depressing substances (BDS) peptides, have been isolated. The first members of this class, BDS-1 and BDS-2, are polypeptides belonging to the β-defensin fold family and were initially described for their antihypertensive and antiviral activities. BDS-1 and BDS-2 are 43 amino acid peptides characterised by three disulfide bonds that act as neurotoxins affecting Kv3.1, Kv3.2 and Kv3.4 channel gating kinetics. In addition, BDS-1 inactivates the Nav1.7 and Nav1.3 channels. The development of a large dataset of A. viridis expressed sequence tags (ESTs) and the identification of 13 putative BDS-like cDNA sequences has attracted interest, especially as scientific and diagnostic tools. A comparison of BDS cDNA sequences showed that the untranslated regions are more conserved than the protein-coding regions. Moreover, the KA/KS ratios calculated for all pairwise comparisons showed values greater than 1, suggesting mechanisms of accelerated evolution. The structures of the BDS homologs were predicted by molecular modelling. All toxins possess similar 3D structures that consist of a triple-stranded antiparallel β-sheet and an additional small antiparallel β-sheet located downstream of the cleavage/maturation site; however, the orientation of the triple-stranded β-sheet appears to differ among the toxins. To characterise the spatial expression profile of the putative BDS cDNA sequences, tissue-specific cDNA libraries, enriched for BDS transcripts, were constructed. In addition, the proper amplification of ectodermal or endodermal markers ensured the tissue specificity of each library. Sequencing randomly selected clones from each library revealed ectodermal-specific expression of ten BDS transcripts, while transcripts of BDS-8, BDS-13, BDS-14 and BDS-15 failed to be retrieved, likely due to under-representation in our cDNA libraries. The calculation of the relative abundance of BDS transcripts in the cDNA libraries revealed that BDS-1, BDS-3, BDS-4, BDS-5 and BDS-6 are the most represented transcripts.

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens.

PubMed

Loudig, Olivier; Liu, Christina; Rohan, Thomas; Ben-Dov, Iddo Z

2018-05-05

-Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18-24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3' barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3' barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller.

PubMed

Xu, Chang; Nezami Ranjbar, Mohammad R; Wu, Zhong; DiCarlo, John; Wang, Yexun

2017-01-03

Detection of DNA mutations at very low allele fractions with high accuracy will significantly improve the effectiveness of precision medicine for cancer patients. To achieve this goal through next generation sequencing, researchers need a detection method that 1) captures rare mutation-containing DNA fragments efficiently in the mix of abundant wild-type DNA; 2) sequences the DNA library extensively to deep coverage; and 3) distinguishes low level true variants from amplification and sequencing errors with high accuracy. Targeted enrichment using PCR primers provides researchers with a convenient way to achieve deep sequencing for a small, yet most relevant region using benchtop sequencers. Molecular barcoding (or indexing) provides a unique solution for reducing sequencing artifacts analytically. Although different molecular barcoding schemes have been reported in recent literature, most variant calling has been done on limited targets, using simple custom scripts. The analytical performance of barcode-aware variant calling can be significantly improved by incorporating advanced statistical models. We present here a highly efficient, simple and scalable enrichment protocol that integrates molecular barcodes in multiplex PCR amplification. In addition, we developed smCounter, an open source, generic, barcode-aware variant caller based on a Bayesian probabilistic model. smCounter was optimized and benchmarked on two independent read sets with SNVs and indels at 5 and 1% allele fractions. Variants were called with very good sensitivity and specificity within coding regions. We demonstrated that we can accurately detect somatic mutations with allele fractions as low as 1% in coding regions using our enrichment protocol and variant caller.

Flow cytometric purification of Colletotrichum higginsianum biotrophic hyphae from Arabidopsis leaves for stage-specific transcriptome analysis.

PubMed

Takahara, Hiroyuki; Dolf, Andreas; Endl, Elmar; O'Connell, Richard

2009-08-01

Generation of stage-specific cDNA libraries is a powerful approach to identify pathogen genes that are differentially expressed during plant infection. Biotrophic pathogens develop specialized infection structures inside living plant cells, but sampling the transcriptome of these structures is problematic due to the low ratio of fungal to plant RNA, and the lack of efficient methods to isolate them from infected plants. Here we established a method, based on fluorescence-activated cell sorting (FACS), to purify the intracellular biotrophic hyphae of Colletotrichum higginsianum from homogenates of infected Arabidopsis leaves. Specific selection of viable hyphae using a fluorescent vital marker provided intact RNA for cDNA library construction. Pilot-scale sequencing showed that the library was enriched with plant-induced and pathogenicity-related fungal genes, including some encoding small, soluble secreted proteins that represent candidate fungal effectors. The high purity of the hyphae (94%) prevented contamination of the library by sequences derived from host cells or other fungal cell types. RT-PCR confirmed that genes identified in the FACS-purified hyphae were also expressed in planta. The method has wide applicability for isolating the infection structures of other plant pathogens, and will facilitate cell-specific transcriptome analysis via deep sequencing and microarray hybridization, as well as proteomic analyses.

Complementary DNA libraries: an overview.

PubMed

Ying, Shao-Yao

2004-07-01

The generation of complete and full-length cDNA libraries for potential functional assays of specific gene sequences is essential for most molecules in biotechnology and biomedical research. The field of cDNA library generation has changed rapidly in the past 10 yr. This review presents an overview of the method available for the basic information of generating cDNA libraries, including the definition of the cDNA library, different kinds of cDNA libraries, difference between methods for cDNA library generation using conventional approaches and a novel strategy, and the quality of cDNA libraries. It is anticipated that the high-quality cDNA libraries so generated would facilitate studies involving genechips and the microarray, differential display, subtractive hybridization, gene cloning, and peptide library generation.

Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response

PubMed Central

Sakurai, Tetsuya; Plata, Germán; Rodríguez-Zapata, Fausto; Seki, Motoaki; Salcedo, Andrés; Toyoda, Atsushi; Ishiwata, Atsushi; Tohme, Joe; Sakaki, Yoshiyuki; Shinozaki, Kazuo; Ishitani, Manabu

2007-01-01

Background Cassava, an allotetraploid known for its remarkable tolerance to abiotic stresses is an important source of energy for humans and animals and a raw material for many industrial processes. A full-length cDNA library of cassava plants under normal, heat, drought, aluminum and post harvest physiological deterioration conditions was built; 19968 clones were sequence-characterized using expressed sequence tags (ESTs). Results The ESTs were assembled into 6355 contigs and 9026 singletons that were further grouped into 10577 scaffolds; we found 4621 new cassava sequences and 1521 sequences with no significant similarity to plant protein databases. Transcripts of 7796 distinct genes were captured and we were able to assign a functional classification to 78% of them while finding more than half of the enzymes annotated in metabolic pathways in Arabidopsis. The annotation of sequences that were not paired to transcripts of other species included many stress-related functional categories showing that our library is enriched with stress-induced genes. Finally, we detected 230 putative gene duplications that include key enzymes in reactive oxygen species signaling pathways and could play a role in cassava stress response features. Conclusion The cassava full-length cDNA library here presented contains transcripts of genes involved in stress response as well as genes important for different areas of cassava research. This library will be an important resource for gene discovery, characterization and cloning; in the near future it will aid the annotation of the cassava genome. PMID:18096061

Sequence analysis of 497 mouse brain ESTs expressed in the substantia nigra

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stewart, G.J.; Savioz, A.; Davies, R.W.

1997-01-15

The use of subtracted, region-specific cDNA libraries combined with single-pass cDNA sequencing allows the discovery of novel genes and facilitates molecular description of the tissue or region involved. We report the sequence of 497 mouse expressed sequence tags (ESTs) from two subtracted libraries enriched for cDNAs expressed in the substantia nigra, a brain region with important roles in movement control and Parkinson disease. Of these, 238 ESTs give no database matches and therefore derive from novel genes. A further 115 ESTs show sequence similarity to ESTs from other organisms, which themselves do not yield any significant database matches to genesmore » of known function. Fifty-six ESTs show sequence similarity to previously identified genes whose mouse homologues have not been reported. The total number of ESTs reported that are new for the mouse is 407, which, together with the 90 ESTs corresponding to known mouse genes or cDNAs, contributes to the molecular description of the substantia nigra. 21 refs., 4 tabs.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in {approx}300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressedmore » for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.« less

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics1

PubMed Central

Weitemier, Kevin; Straub, Shannon C. K.; Cronn, Richard C.; Fishbein, Mark; Schmickl, Roswitha; McDonnell, Angela; Liston, Aaron

2014-01-01

• Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • Methods and Results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. • Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics. PMID:25225629

Isolation of anonymous DNA sequences from within a submicroscopic X chromosomal deletion in a patient with choroideremia, deafness, and mental retardation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nussbaum, R.L.; Lesko, J.G.; Lewis, R.A.

1987-09-01

Choroideremia, an X-chromosome linked retinal dystrophy of unknown pathogenesis, causes progressive nightblindness and eventual central blindness in affected males by the third to fourth decade of life. Choroideremia has been mapped to Xq13-21 by tight linkage to restriction fragment length polymorphism loci. The authors have recently identified two families in which choroideremia is inherited with mental retardation and deafness. In family XL-62, an interstitial deletion Xq21 is visible by cytogenetic analysis and two linked anonymous DNA markers, DXYS1 and DXS72, are deleted. In the second family, XL-45, an interstitial deletion was suspected on phenotypic grounds but could not be confirmedmore » by high-resolution cytogenetic analysis. They used phenol-enhanced reassociation of 48,XXXX DNA in competition with excess XL-45 DNA to generate a library of cloned DNA enriched for sequences that might be deleted in XL-45. Two of the first 83 sequences characterized from the library were found to be deleted in probands from family XL-45 as well as from family XL-62. Isolation of these sequences proves that XL-45 does contain a submicroscopic deletion and provides a starting point for identifying overlapping genomic sequences that span the XL-45 deletion. Each overlapping sequence will be studied to identify exons from the choroideremia locus.« less

Signatures from Tissue-specific MPSS Libraries Identify Transcripts Preferentially Expressed in the Mouse Inner Ear

PubMed Central

Peters, Linda M.; Belyantseva, Inna A.; Lagziel, Ayala; Battey, James F.; Friedman, Thomas B.; Morell, Robert J.

2007-01-01

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well-suited for identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome (MRT) online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome. PMID:17049805

Novel encoding methods for DNA-templated chemical libraries.

PubMed

Li, Gang; Zheng, Wenlu; Liu, Ying; Li, Xiaoyu

2015-06-01

Among various types of DNA-encoded chemical libraries, DNA-templated library takes advantage of the sequence-specificity of DNA hybridization, enabling not only highly effective DNA-templated chemical reactions, but also high fidelity in library encoding. This brief review summarizes recent advances that have been made on the encoding strategies for DNA-templated libraries, and it also highlights their respective advantages and limitations for the preparation of DNA-encoded libraries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Methods comparison for microsatellite marker development: Different isolation methods, different yield efficiency

NASA Astrophysics Data System (ADS)

Zhan, Aibin; Bao, Zhenmin; Hu, Xiaoli; Lu, Wei; Hu, Jingjie

2009-06-01

Microsatellite markers have become one kind of the most important molecular tools used in various researches. A large number of microsatellite markers are required for the whole genome survey in the fields of molecular ecology, quantitative genetics and genomics. Therefore, it is extremely necessary to select several versatile, low-cost, efficient and time- and labor-saving methods to develop a large panel of microsatellite markers. In this study, we used Zhikong scallop ( Chlamys farreri) as the target species to compare the efficiency of the five methods derived from three strategies for microsatellite marker development. The results showed that the strategy of constructing small insert genomic DNA library resulted in poor efficiency, while the microsatellite-enriched strategy highly improved the isolation efficiency. Although the mining public database strategy is time- and cost-saving, it is difficult to obtain a large number of microsatellite markers, mainly due to the limited sequence data of non-model species deposited in public databases. Based on the results in this study, we recommend two methods, microsatellite-enriched library construction method and FIASCO-colony hybridization method, for large-scale microsatellite marker development. Both methods were derived from the microsatellite-enriched strategy. The experimental results obtained from Zhikong scallop also provide the reference for microsatellite marker development in other species with large genomes.

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

PubMed

Janku, Filip; Zhang, Shile; Waters, Jill; Liu, Li; Huang, Helen J; Subbiah, Vivek; Hong, David S; Karp, Daniel D; Fu, Siqing; Cai, Xuyu; Ramzanali, Nishma M; Madwani, Kiran; Cabrilo, Goran; Andrews, Debra L; Zhao, Yue; Javle, Milind; Kopetz, E Scott; Luthra, Rajyalakshmi; Kim, Hyunsung J; Gnerre, Sante; Satya, Ravi Vijaya; Chuang, Han-Yu; Kruglyak, Kristina M; Toung, Jonathan; Zhao, Chen; Shen, Richard; Heymach, John V; Meric-Bernstam, Funda; Mills, Gordon B; Fan, Jian-Bing; Salathia, Neeraj S

2017-09-15

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did ( P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF ( P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03). Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR . ©2017 American Association for Cancer Research.

The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery.

PubMed

O'Reilly, Rachel K; Turberfield, Andrew J; Wilks, Thomas R

2017-10-17

Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify "successful" structures. The largest libraries made by conventional synthesis are currently of the order of 10 8 distinct molecules. To put this in context, there are 10 13 ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required. DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a system that can translate instructions coded as a sequence of DNA bases into a chemical structure-a process analogous to the action of the ribosome in living organisms but with the potential to create a much more chemically diverse set of products. It is also possible to ensure that each product molecule is tagged with its identifying DNA sequence. Compound libraries synthesized in this way can be exposed to selection against suitable targets, enriching successful molecules. The encoding DNA can then be amplified using the polymerase chain reaction and decoded by DNA sequencing. More importantly, the DNA instruction sequences can be mutated and reused during multiple rounds of amplification, translation, and selection. In other words, DTS could be used as the foundation for a system of synthetic molecular evolution, which could allow us to efficiently search a vast chemical space. This has huge potential to revolutionize materials discovery-imagine being able to evolve molecules for light harvesting, or catalysts for CO 2 fixation. The field of DTS has developed to the point where a wide variety of reactions can be performed on a DNA template. Complex architectures and autonomous "DNA robots" have been implemented for the controlled assembly of BBs, and these mechanisms have in turn enabled the one-pot synthesis of large combinatorial libraries. Indeed, DTS libraries are being exploited by pharmaceutical companies and have already found their way into drug lead discovery programs. This Account explores the processes involved in DTS and highlights the challenges that remain in creating a general system for molecular discovery by evolution.

The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery

PubMed Central

2017-01-01

Conspectus Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify “successful” structures. The largest libraries made by conventional synthesis are currently of the order of 108 distinct molecules. To put this in context, there are 1013 ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required. DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a system that can translate instructions coded as a sequence of DNA bases into a chemical structure—a process analogous to the action of the ribosome in living organisms but with the potential to create a much more chemically diverse set of products. It is also possible to ensure that each product molecule is tagged with its identifying DNA sequence. Compound libraries synthesized in this way can be exposed to selection against suitable targets, enriching successful molecules. The encoding DNA can then be amplified using the polymerase chain reaction and decoded by DNA sequencing. More importantly, the DNA instruction sequences can be mutated and reused during multiple rounds of amplification, translation, and selection. In other words, DTS could be used as the foundation for a system of synthetic molecular evolution, which could allow us to efficiently search a vast chemical space. This has huge potential to revolutionize materials discovery—imagine being able to evolve molecules for light harvesting, or catalysts for CO2 fixation. The field of DTS has developed to the point where a wide variety of reactions can be performed on a DNA template. Complex architectures and autonomous “DNA robots” have been implemented for the controlled assembly of BBs, and these mechanisms have in turn enabled the one-pot synthesis of large combinatorial libraries. Indeed, DTS libraries are being exploited by pharmaceutical companies and have already found their way into drug lead discovery programs. This Account explores the processes involved in DTS and highlights the challenges that remain in creating a general system for molecular discovery by evolution. PMID:28915003

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

PubMed Central

Kilimann, M W; DeGennaro, L J

1985-01-01

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:3933975

Characterisation of culture-independent and -dependent microbial communities in a high-temperature offshore chalk petroleum reservoir.

PubMed

Kaster, Krista M; Bonaunet, Kristin; Berland, Harald; Kjeilen-Eilertsen, Grethe; Brakstad, Odd Gunnar

2009-11-01

Recent studies have indicated that oil reservoirs harbour diverse microbial communities. Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in produced water samples of the Ekofisk oil field, a high temperature, and fractured chalk reservoir in the North Sea. DGGE analyses of 16S rRNA gene fragments were used to assess the microbial diversity of both archaeal and bacterial communities in produced water samples and enrichment cultures from 4 different wells (B-08, X-08, X-18 and X-25). Low diversity communities were found when 16S rDNA libraries of bacterial and archaeal assemblages were generated from total community DNA obtained from produced water samples and enrichment cultures. Sequence analysis of the clones indicated close matches to microbes associated with high-temperature oil reservoirs or other similar environments. Sequences were found to be similar to members of the genera Thermotoga, Caminicella, Thermoanaerobacter, Archaeoglobus, Thermococcus, and Methanobulbus. Enrichment cultures obtained from the produced water samples were dominated by sheathed rods. Sequence analyses of the cultures indicated predominance of the genera Petrotoga, Arcobacter, Archaeoglobus and Thermococcus. The communities of both produced water and enrichment cultures appeared to be dominated by thermophilic fermenters capable of reducing sulphur compounds. These results suggest that the biochemical processes in the Ekofisk chalk reservoir are similar to those observed in high-temperature sandstone reservoirs.

Diazotrophic bacterioplankton in a coral reef lagoon: phylogeny, diel nitrogenase expression and response to phosphate enrichment.

PubMed

Hewson, Ian; Moisander, Pia H; Morrison, Amanda E; Zehr, Jonathan P

2007-05-01

We investigated diazotrophic bacterioplankton assemblage composition in the Heron Reef lagoon (Great Barrier Reef, Australia) using culture-independent techniques targeting the nifH fragment of the nitrogenase gene. Seawater was collected at 3 h intervals over a period of 72 h (i.e. over diel as well as tidal cycles). An incubation experiment was also conducted to assess the impact of phosphate (PO(4)3*) availability on nifH expression patterns. DNA-based nifH libraries contained primarily sequences that were most similar to nifH from sediment, microbial mat and surface-associated microorganisms, with a few sequences that clustered with typical open ocean phylotypes. In contrast to genomic DNA sequences, libraries prepared from gene transcripts (mRNA amplified by reverse transcription-polymerase chain reaction) were entirely cyanobacterial and contained phylotypes similar to those observed in open ocean plankton. The abundance of Trichodesmium and two uncultured cyanobacterial phylotypes from previous studies (group A and group B) were studied by quantitative-polymerase chain reaction in the lagoon samples. These were detected as transcripts, but were not detected in genomic DNA. The gene transcript abundance of these phylotypes demonstrated variability over several diel cycles. The PO(4)3* enrichment experiment had a clearer pattern of gene expression over diel cycles than the lagoon sampling, however PO(4)3* additions did not result in enhanced transcript abundance relative to control incubations. The results suggest that a number of diazotrophs in bacterioplankton of the reef lagoon may originate from sediment, coral or beachrock surfaces, sloughing into plankton with the flooding tide. The presence of typical open ocean phylotype transcripts in lagoon bacterioplankton may indicate that they are an important component of the N cycle of the coral reef.

Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

DOEpatents

Soares, Marcelo Bento; Bonaldo, Maria de Fatima

1998-01-01

This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods.

Efficient and simpler method to construct normalized cDNA libraries with improved representations of full-length cDNAs

DOEpatents

Soares, M.B.; Fatima Bonaldo, M. de

1998-12-08

This invention provides a method to normalize a cDNA library comprising: (a) constructing a directionally cloned library containing cDNA inserts wherein the insert is capable of being amplified by polymerase chain reaction; (b) converting a double-stranded cDNA library into single-stranded DNA circles; (c) generating single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) by polymerase chain reaction with appropriate primers; (d) hybridizing the single-stranded DNA circles converted in step (b) with the complementary single-stranded nucleic acid molecules generated in step (c) to produce partial duplexes to an appropriate Cot; and (e) separating the unhybridized single-stranded DNA circles from the hybridized DNA circles, thereby generating a normalized cDNA library. This invention also provides a method to normalize a cDNA library wherein the generating of single-stranded nucleic acid molecules complementary to the single-stranded DNA circles converted in step (b) is by excising cDNA inserts from the double-stranded cDNA library; purifying the cDNA inserts from cloning vectors; and digesting the cDNA inserts with an exonuclease. This invention further provides a method to construct a subtractive cDNA library following the steps described above. This invention further provides normalized and/or subtractive cDNA libraries generated by the above methods. 25 figs.

Library Construction from Subnanogram DNA for Pelagic Sea Water and Deep-Sea Sediments

PubMed Central

Hirai, Miho; Nishi, Shinro; Tsuda, Miwako; Sunamura, Michinari; Takaki, Yoshihiro; Nunoura, Takuro

2017-01-01

Shotgun metagenomics is a low biased technology for assessing environmental microbial diversity and function. However, the requirement for a sufficient amount of DNA and the contamination of inhibitors in environmental DNA leads to difficulties in constructing a shotgun metagenomic library. We herein examined metagenomic library construction from subnanogram amounts of input environmental DNA from subarctic surface water and deep-sea sediments using two library construction kits: the KAPA Hyper Prep Kit and Nextera XT DNA Library Preparation Kit, with several modifications. The influence of chemical contaminants associated with these environmental DNA samples on library construction was also investigated. Overall, shotgun metagenomic libraries were constructed from 1 pg to 1 ng of input DNA using both kits without harsh library microbial contamination. However, the libraries constructed from 1 pg of input DNA exhibited larger biases in GC contents, k-mers, or small subunit (SSU) rRNA gene compositions than those constructed from 10 pg to 1 ng DNA. The lower limit of input DNA for low biased library construction in this study was 10 pg. Moreover, we revealed that technology-dependent biases (physical fragmentation and linker ligation vs. tagmentation) were larger than those due to the amount of input DNA. PMID:29187708

cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsubara, Y.; Kraus, J.P.; Rosenberg, L.E.

MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the ..beta..-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonalmore » monospecific antibody. Single-stranded (/sup 32/P)labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting.« less

Label-free detection of gliadin food allergen mediated by real-time apta-PCR.

PubMed

Pinto, Alessandro; Polo, Pedro Nadal; Henry, Olivier; Redondo, M Carmen Bermudo; Svobodova, Marketa; O'Sullivan, Ciara K

2014-01-01

Celiac disease is an immune-mediated enteropathy triggered by the ingestion of gluten. The only effective treatment consists in a lifelong gluten-free diet, requiring the food industry to tightly control the gluten contents of their products. To date, several gluten quantification approaches using antibodies are available and recommended by the legal authorities, such as Codex Alimentarius. However, whilst these antibody-based tests exhibit high sensitivity and specificity, the production of antibodies inherently requires the killing of host animals and is time-consuming and relatively expensive. Aptamers are structured single-stranded nucleic acid ligands that bind with high affinity and specificity to their cognate target, and aiming for a cost-effective viable alternative to the use of antibodies. Herein, we report the systematic evolution of ligands by exponential enrichment (SELEX)-based selection of a DNA aptamer against gliadin from a combinatorial DNA library and its application in a novel detection assay. Taking into account the hydrophobic nature of the gliadin target, a microtitre plate format was exploited for SELEX, where the target was immobilised via hydrophobic interactions, thus exposing aptatopes accessible for interaction with the DNA library. Evolution was followed using surface plasmon resonance, and following eight rounds of SELEX, the enriched DNA pool was cloned, sequenced and a clear consensus motif was identified. An apta-PCR assay was developed where competition for the aptamer takes place between the surface-immobilised gliadin and gliadin in the target sample, akin to an ELISA competitive format where the more target present in the sample, the less aptamer will bind to the immobilised gliadin. Following competition, any aptamer bound to the immobilised gliadin was heat-eluted and quantitatively amplified using real-time PCR, achieving a detection limit of approx. 2 nM (100 ng mL(-1)). The specificity of the selected aptamer was demonstrated and no cross-reactivity was observed with streptavidin, bovine serum albumin or anti-gliadin IgG.

[Diversity of uncultured actinomycetes in saline-alkali soil from Jiuquan area of Hexi Corridor].

PubMed

Li, Hai-yun; Niu, Shi-quan; Kong, Wei-bao; Yan, Wei-ru; Geng, Hui; Han, Cai-hong; Da, Wen-yan; Zhang, Ai-mei; Zhu, Xue-tai

2015-09-01

In order to more accurately understand community structure and diversity of actinomycetes in saline-alkali soil from Jiuquan area of Hexi Corridor, the community structure and diversity from three kinds of soil samples (primary, secondary saline alkali soil and farmland soil) were analyzed using uncultured methods. The results showed that the 16S rDNA clone library of actinomycetales from the primary saline-alkali soil belonged to 19 OTUs, Micrococcineae, Propionibacterineae, Corynebacterineae, Frankineae, Pseudonocardineae and unknown groups of Actinomycetales; the 16S r DNA clone library of actinomycetales from the secondary saline-alkali soil belonged to 14 OTUs, Micrococcineae, Propionibacterineae, Corynebacterineae, Frankineae, Pseudonocardineae and unknown groups of Actinomycetales; the 16S rDNA clone library of farmland soil belonged to 7 OTUs, Micrococcineae, Propionibacterineae, Corynebacterineae, Frankineae, Pseudonocardineae and unknown groups of Actinomycetales; Micrococcineae was the common population in the three soils, and also was the dominant population in primary saline alkali soil and farmland soil. The diversity index and rarefaction curves analysis showed that actinomycetes species richness was in order of primary saline-alkali soil > secondary saline-alkali soil > farmland soil. The dilution curves of primary saline-alkali soil and secondary saline-alkali soil were not leveled off, which indicated the actinomycetes diversity in saline-alkali soil was more enriched than the actual. The rich and diverse actinomycetes resources in saline-alkali soil from Jiuquan area of Hexi Corridor provide important data on the actinomycetes ecology distribution research, exploitation and utilization in saline-alkali soil.

High-throughput Identification of DNA-Encoded IgG Ligands that Distinguish Active and Latent Mycobacterium Tuberculosis Infections

PubMed Central

Ndungu, John Maina; Suponitsky-Kroyter, Irena; Cavett, Valerie J.; McEnaney, Patrick J.; MacConnell, Andrew B.; Doran, Todd. M.; Ronacher, Katharina; Stanley, Kim; Utset, Ofelia; Walzl, Gerhard; Paegel, Brian M.; Kodadek, Thomas

2017-01-01

The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover “epitope surrogates” (ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 106 beads, 448k unique compounds) using fluorescent anti-human IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens. PMID:27957856

Large-scale DNA Barcode Library Generation for Biomolecule Identification in High-throughput Screens.

PubMed

Lyons, Eli; Sheridan, Paul; Tremmel, Georg; Miyano, Satoru; Sugano, Sumio

2017-10-24

High-throughput screens allow for the identification of specific biomolecules with characteristics of interest. In barcoded screens, DNA barcodes are linked to target biomolecules in a manner allowing for the target molecules making up a library to be identified by sequencing the DNA barcodes using Next Generation Sequencing. To be useful in experimental settings, the DNA barcodes in a library must satisfy certain constraints related to GC content, homopolymer length, Hamming distance, and blacklisted subsequences. Here we report a novel framework to quickly generate large-scale libraries of DNA barcodes for use in high-throughput screens. We show that our framework dramatically reduces the computation time required to generate large-scale DNA barcode libraries, compared with a naїve approach to DNA barcode library generation. As a proof of concept, we demonstrate that our framework is able to generate a library consisting of one million DNA barcodes for use in a fragment antibody phage display screening experiment. We also report generating a general purpose one billion DNA barcode library, the largest such library yet reported in literature. Our results demonstrate the value of our novel large-scale DNA barcode library generation framework for use in high-throughput screening applications.

Phylogenetic and physiological diversity of microorganisms isolated from a deep greenland glacier ice core

NASA Technical Reports Server (NTRS)

Miteva, V. I.; Sheridan, P. P.; Brenchley, J. E.

2004-01-01

We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group. The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.

COBRA-Seq: Sensitive and Quantitative Methylome Profiling

PubMed Central

Varinli, Hilal; Statham, Aaron L.; Clark, Susan J.; Molloy, Peter L.; Ross, Jason P.

2015-01-01

Combined Bisulfite Restriction Analysis (COBRA) quantifies DNA methylation at a specific locus. It does so via digestion of PCR amplicons produced from bisulfite-treated DNA, using a restriction enzyme that contains a cytosine within its recognition sequence, such as TaqI. Here, we introduce COBRA-seq, a genome wide reduced methylome method that requires minimal DNA input (0.1–1.0 μg) and can either use PCR or linear amplification to amplify the sequencing library. Variants of COBRA-seq can be used to explore CpG-depleted as well as CpG-rich regions in vertebrate DNA. The choice of enzyme influences enrichment for specific genomic features, such as CpG-rich promoters and CpG islands, or enrichment for less CpG dense regions such as enhancers. COBRA-seq coupled with linear amplification has the additional advantage of reduced PCR bias by producing full length fragments at high abundance. Unlike other reduced representative methylome methods, COBRA-seq has great flexibility in the choice of enzyme and can be multiplexed and tuned, to reduce sequencing costs and to interrogate different numbers of sites. Moreover, COBRA-seq is applicable to non-model organisms without the reference genome and compatible with the investigation of non-CpG methylation by using restriction enzymes containing CpA, CpT, and CpC in their recognition site. PMID:26512698

SeSaM-Tv-II generates a protein sequence space that is unobtainable by epPCR.

PubMed

Mundhada, Hemanshu; Marienhagen, Jan; Scacioc, Andreea; Schenk, Alexander; Roccatano, Danilo; Schwaneberg, Ulrich

2011-07-04

Generating high-quality mutant libraries in which each amino acid is equally targeted and substituted in a chemically diverse manner is crucial to obtain improved variants in small mutant libraries. The sequence saturation mutagenesis method (SeSaM-Tv(+) ) offers the opportunity to generate such high-quality mutant libraries by introducing consecutive mutations and by enriching transversions. In this study, automated gel electrophoresis, real-time quantitative PCR, and a phosphorimager quantification system were developed and employed to optimize each step of previously reported SeSaM-Tv(+) method. Advancements of the SeSaM-Tv(+) protocol and the use of a novel DNA polymerase quadrupled the number of transversions, by doubling the fraction of consecutive mutations (from 16.7 to 37.1 %). About 33 % of all amino acid substitutions observed in a model library are rarely introduced by epPCR methods, and around 10 % of all clones carried amino acid substitutions that are unobtainable by epPCR. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[cDNA library construction from panicle meristem of finger millet].

PubMed

Radchuk, V; Pirko, Ia V; Isaenkov, S V; Emets, A I; Blium, Ia B

2014-01-01

The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

Microcystin-LR nanobody screening from an alpaca phage display nanobody library and its expression and application.

PubMed

Xu, Chongxin; Yang, Ying; Liu, Liwen; Li, Jianhong; Liu, Xiaoqin; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Liu, Xianjin

2018-04-30

Microcystin-LR (MC-LR) is a type of biotoxin that pollutes the ecological environment and food. The study aimed to obtain new nanobodies from phage nanobody library for determination of MC-LR. The toxin was conjugated to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, then the conjugates were used as coated antigens for enrichment (coated MC-LR-KLH) and screening (coated MC-LR-BSA) of MC-LR phage nanobodies from an alpaca phage display nanobody library. The antigen-specific phage particles were enriched effectively with four rounds of biopanning. At the last round of enrichment, total 20 positive monoclonal phage nanobodies were obtained from the library, which were analyzed after monoclonal phage enzyme linked immunosorbent assay (ELISA), colony PCR and DNA sequencing. The most three positive nanobody genes, ANAb12, ANAb9 and ANAb7 were cloned into pET26b vector, then the nanobodies were expressed in Escherichia coli BL21 respectively. After being purified, the molecular weight (M.W.) of all nanobodies were approximate 15kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The purified nanobodies, ANAb12, ANAb9 and ANAb7 were used to establish the indirect competitive ELISA (IC-ELISA) for MC-LR, and their half-maximum inhibition concentrations (IC 50 ) were 0.87, 1.17 and 1.47μg/L, their detection limits (IC 10 ) were 0.06, 0.08 and 0.12μg/L, respectively. All of them showed strong cross-reactivity (CRs) of 82.7-116.9% for MC-RR, MC-YR and MC-WR, and weak CRs of less than 4.56% for MC-LW, less than 0.1% for MC-LY and MC-LF. It was found that all the IC-ELISAs for MC-LR spiked in tap water samples detection were with good accuracy, stability and repeatability, their recoveries were 84.0-106.5%, coefficient of variations (CVs) were 3.4-10.6%. These results showed that IC-ELISA based on the nanobodies from the alpaca phage display antibody library were promising for high sensitive determination of multiple MCs. Copyright © 2018 Elsevier Inc. All rights reserved.

Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

PubMed

Duan, Nuo; Wu, Shijia; Chen, Xiujuan; Huang, Yukun; Wang, Zhouping

2012-04-25

A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus . FAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K(d)) of 16.88 ± 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.

Method for construction of normalized cDNA libraries

DOEpatents

Soares, Marcelo B.; Efstratiadis, Argiris

1998-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

Method for construction of normalized cDNA libraries

DOEpatents

Soares, M.B.; Efstratiadis, A.

1998-11-03

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3` noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries. 19 figs.

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Martin, R G

1984-11-15

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Expression of stress-related genes in zebrawood (Astronium fraxinifolium, Anacardiaceae) seedlings following germination in microgravity

PubMed Central

Inglis, Peter W.; Ciampi, Ana Y.; Salomão, Antonieta N.; Costa, Tânia da S.A.; Azevedo, Vânia C.R.

2014-01-01

Seeds of a tropical tree species from Brazil, Astronium fraxinifolium, or zebrawood, were germinated, for the first time in microgravity, aboard the International Space Station for nine days. Following three days of subsequent growth under normal terrestrial gravitational conditions, greater root length and numbers of secondary roots was observed in the microgravity-treated seedlings compared to terrestrially germinated controls. Suppression subtractive hybridization of cDNA and EST analysis were used to detect differential gene expression in the microgravity-treated seedlings in comparison to those initially grown in normal gravity (forward subtraction). Despite their return to, and growth in normal gravity, the subtracted library derived from microgravity-treated seedlings was enriched in known microgravity stress-related ESTs, corresponding to large and small heat shock proteins, 14-3-3-like protein, polyubiquitin, and proteins involved in glutathione metabolism. In contrast, the reverse-subtracted library contained a comparatively greater variety of general metabolism-related ESTs, but was also enriched for peroxidase, possibly indicating the suppression of this protein in the microgravity-treated seedlings. Following continued growth for 30 days, higher concentrations of total chlorophyll were detected in the microgravity-exposed seedlings. PMID:24688295

Constructing and detecting a cDNA library for mites.

PubMed

Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

2015-10-01

RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

Theoretical modeling of masking DNA application in aptamer-facilitated biomarker discovery.

PubMed

Cherney, Leonid T; Obrecht, Natalia M; Krylov, Sergey N

2013-04-16

In aptamer-facilitated biomarker discovery (AptaBiD), aptamers are selected from a library of random DNA (or RNA) sequences for their ability to specifically bind cell-surface biomarkers. The library is incubated with intact cells, and cell-bound DNA molecules are separated from those unbound and amplified by the polymerase chain reaction (PCR). The partitioning/amplification cycle is repeated multiple times while alternating target cells and control cells. Efficient aptamer selection in AptaBiD relies on the inclusion of masking DNA within the cell and library mixture. Masking DNA lacks primer regions for PCR amplification and is typically taken in excess to the library. The role of masking DNA within the selection mixture is to outcompete any nonspecific binding sequences within the initial library, thus allowing specific DNA sequences (i.e., aptamers) to be selected more efficiently. Efficient AptaBiD requires an optimum ratio of masking DNA to library DNA, at which aptamers still bind specific binding sites but nonaptamers within the library do not bind nonspecific binding sites. Here, we have developed a mathematical model that describes the binding processes taking place within the equilibrium mixture of masking DNA, library DNA, and target cells. An obtained mathematical solution allows one to estimate the concentration of masking DNA that is required to outcompete the library DNA at a desirable ratio of bound masking DNA to bound library DNA. The required concentration depends on concentrations of the library and cells as well as on unknown cell characteristics. These characteristics include the concentration of total binding sites on the cell surface, N, and equilibrium dissociation constants, K(nsL) and K(nsM), for nonspecific binding of the library DNA and masking DNA, respectively. We developed a theory that allows the determination of N, K(nsL), and K(nsM) based on measurements of EC50 values for cells mixed separately with the library and masking DNA (EC50 is the concentration of fluorescently labeled DNA at which half of the maximum fluorescence signal from DNA-bound cells is reached). We also obtained expressions for signals from bound DNA (measured by flow cytometry) in terms of N, K(nsL), and K(nsM). These expressions can be used for the verification of N, K(nsL), and K(nsM) values found from EC50 measurements. The developed procedure was applied to MCF-7 breast cancer cells, and corresponding values of N, K(nsL), and K(nsM) were established for the first time. The concentration of masking DNA required for AptaBiD with MCF-7 breast cancer cells was also estimated.

DNA capture and next-generation sequencing can recover whole mitochondrial genomes from highly degraded samples for human identification

PubMed Central

2013-01-01

Background Mitochondrial DNA (mtDNA) typing can be a useful aid for identifying people from compromised samples when nuclear DNA is too damaged, degraded or below detection thresholds for routine short tandem repeat (STR)-based analysis. Standard mtDNA typing, focused on PCR amplicon sequencing of the control region (HVS I and HVS II), is limited by the resolving power of this short sequence, which misses up to 70% of the variation present in the mtDNA genome. Methods We used in-solution hybridisation-based DNA capture (using DNA capture probes prepared from modern human mtDNA) to recover mtDNA from post-mortem human remains in which the majority of DNA is both highly fragmented (<100 base pairs in length) and chemically damaged. The method ‘immortalises’ the finite quantities of DNA in valuable extracts as DNA libraries, which is followed by the targeted enrichment of endogenous mtDNA sequences and characterisation by next-generation sequencing (NGS). Results We sequenced whole mitochondrial genomes for human identification from samples where standard nuclear STR typing produced only partial profiles or demonstrably failed and/or where standard mtDNA hypervariable region sequences lacked resolving power. Multiple rounds of enrichment can substantially improve coverage and sequencing depth of mtDNA genomes from highly degraded samples. The application of this method has led to the reliable mitochondrial sequencing of human skeletal remains from unidentified World War Two (WWII) casualties approximately 70 years old and from archaeological remains (up to 2,500 years old). Conclusions This approach has potential applications in forensic science, historical human identification cases, archived medical samples, kinship analysis and population studies. In particular the methodology can be applied to any case, involving human or non-human species, where whole mitochondrial genome sequences are required to provide the highest level of maternal lineage discrimination. Multiple rounds of in-solution hybridisation-based DNA capture can retrieve whole mitochondrial genome sequences from even the most challenging samples. PMID:24289217

Mining lipolytic enzymes in community DNA from high Andean soils using a targeted approach.

PubMed

Borda-Molina, Daniel; Montaña, José Salvador; Zambrano, María Mercedes; Baena, Sandra

2017-08-01

Microbial enrichments cultures are a useful strategy to speed up the search for enzymes that can be employed in industrial processes. Lipases have gained special attention because they show unique properties such as: broad substrate specificity, enantio- and regio-selectivity and stability in organic solvents. A major goal is to identify novel lipolytic enzymes from microorganisms living in cold extreme environments such as high Andean soils, of relevance to our study being their capability be used in industrial processes. Paramo and glacier soils from the Nevados National Park in Colombia were sampled and microbial communities enriched through a fed-batch fermentation using olive oil as an inductor substrate. After 15 days of enrichment under aerobic conditions, total DNA was extracted. Subsequently, metagenomic libraries were constructed in the cosmid vector pWEB-TNC™. After functional screening, twenty and eighteen lipolytic clones were obtained from Paramo and Glacier soil enrichments, respectively. Based on lipid hydrolysis halo dimensions, the clone (Gla1) from a glacier enrichment was selected. A gene related to lipolytic activity was subcloned to evaluate enzyme properties. Phylogenetic analysis of the identified gene showed that the encoded lipase belongs to the family GDSL from a Ralstonia-like species. Interestingly, the secreted enzyme exhibited stability at high temperature and alkaline conditions, specifically the preferred conditions at 80 °C and pH 9.0. Thus, with the identification of an enzyme with non-expected properties, in this study is shown the potential of extreme cold environments to be explored for new catalytic molecules, using current molecular biology techniques, with applications in industrial processes, which demand stability under harsh conditions.

Selective recognition of N4-methylcytosine in DNA by engineered transcription-activator-like effectors.

PubMed

Rathi, Preeti; Maurer, Sara; Summerer, Daniel

2018-06-05

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N 4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'. © 2018 The Author(s).

Design and Synthesis of Biaryl DNA-Encoded Libraries.

PubMed

Ding, Yun; Franklin, G Joseph; DeLorey, Jennifer L; Centrella, Paolo A; Mataruse, Sibongile; Clark, Matthew A; Skinner, Steven R; Belyanskaya, Svetlana

2016-10-10

DNA-encoded library technology (ELT) is a powerful tool for the discovery of new small-molecule ligands to various protein targets. Here we report the design and synthesis of biaryl DNA-encoded libraries based on the scaffold of 5-formyl 3-iodobenzoic acid. Three reactions on DNA template, acylation, Suzuki-Miyaura coupling and reductive amination, were applied in the library synthesis. The three cycle library of 3.5 million diversity has delivered potent hits for phosphoinositide 3-kinase α (PI3Kα).

Normalized cDNA libraries

DOEpatents

Soares, Marcelo B.; Efstratiadis, Argiris

1997-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

Normalized cDNA libraries

DOEpatents

Soares, M.B.; Efstratiadis, A.

1997-06-10

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3{prime} noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. 4 figs.

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.

PubMed

Brammer, Leighanne A; Bolduc, Benjamin; Kass, Jessica L; Felice, Kristin M; Noren, Christopher J; Hall, Marilena Fitzsimons

2008-02-01

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.

Second-generation DNA-templated macrocycle libraries for the discovery of bioactive small molecules.

PubMed

Usanov, Dmitry L; Chan, Alix I; Maianti, Juan Pablo; Liu, David R

2018-07-01

DNA-encoded libraries have emerged as a widely used resource for the discovery of bioactive small molecules, and offer substantial advantages compared with conventional small-molecule libraries. Here, we have developed and streamlined multiple fundamental aspects of DNA-encoded and DNA-templated library synthesis methodology, including computational identification and experimental validation of a 20 × 20 × 20 × 80 set of orthogonal codons, chemical and computational tools for enhancing the structural diversity and drug-likeness of library members, a highly efficient polymerase-mediated template library assembly strategy, and library isolation and purification methods. We have integrated these improved methods to produce a second-generation DNA-templated library of 256,000 small-molecule macrocycles with improved drug-like physical properties. In vitro selection of this library for insulin-degrading enzyme affinity resulted in novel insulin-degrading enzyme inhibitors, including one of unusual potency and novel macrocycle stereochemistry (IC 50  = 40 nM). Collectively, these developments enable DNA-templated small-molecule libraries to serve as more powerful, accessible, streamlined and cost-effective tools for bioactive small-molecule discovery.

Intravenous infusion of phage-displayed antibody library in human cancer patients: enrichment and cancer-specificity of tumor-homing phage-antibodies.

PubMed

Shukla, Girja S; Krag, David N; Peletskaya, Elena N; Pero, Stephanie C; Sun, Yu-Jing; Carman, Chelsea L; McCahill, Laurence E; Roland, Thomas A

2013-08-01

Phage display is a powerful method for target discovery and selection of ligands for cancer treatment and diagnosis. Our goal was to select tumor-binding antibodies in cancer patients. Eligibility criteria included absence of preexisting anti-phage-antibodies and a Stage IV cancer status. All patients were intravenously administered 1 × 10(11) TUs/kg of an scFv library 1 to 4 h before surgical resection of their tumors. No significant adverse events related to the phage library infusion were observed. Phage were successfully recovered from all tumors. Individual clones from each patient were assessed for binding to the tumor from which clones were recovered. Multiple tumor-binding phage-antibodies were identified. Soluble scFv antibodies were produced from the phage clones showing higher tumor binding. The tumor-homing phage-antibodies and derived soluble scFvs were found to bind varying numbers (0-5) of 8 tested normal human tissues (breast, cervix, colon, kidney, liver, spleen, skin, and uterus). The clones that showed high tumor-specificity were found to bind corresponding tumors from other patients also. Clone enrichment was observed based on tumor binding and DNA sequence data. Clone sequences of multiple variable regions showed significant matches to certain cancer-related antibodies. One of the clones (07-2,355) that was found to share a 12-amino-acid-long motif with a reported IL-17A antibody was further studied for competitive binding for possible antigen target identification. We conclude that these outcomes support the safety and utility of phage display library panning in cancer patients for ligand selection and target discovery for cancer treatment and diagnosis.

A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries.

PubMed

Webster, Gordon; Watt, Lynsey C; Rinna, Joachim; Fry, John C; Evershed, Richard P; Parkes, R John; Weightman, Andrew J

2006-09-01

Marine sediment slurries enriched for anaerobic, sulfate-reducing prokaryotic communities utilizing glucose and acetate were used to provide the first comparison between stable-isotope probing (SIP) of phospholipid fatty acids (PLFA) and DNA (16S rRNA and dsrA genes) biomarkers. Different 13C-labelled substrates (glucose, acetate and pyruvate) at low concentrations (100 microM) were used over a 7-day incubation to follow and identify carbon flow into different members of the community. Limited changes in total PLFA and bacterial 16S rRNA gene DGGE profiles over 7 days suggested the presence of a stable bacterial community. A broad range of PLFA were rapidly labelled (within 12 h) in the 13C-glucose slurry but this changed with time, suggesting the presence of an active glucose-utilizing population and later development of another population able to utilize glucose metabolites. The identity of the major glucose-utilizers was unclear as 13C-enriched PLFA were common (16:0, 16:1, 18:1omega7, highest incorporation) and there was little difference between 12C- and 13C-DNA 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) profiles. Seemingly glucose, a readily utilizable substrate, resulted in widespread incorporation consistent with the higher extent of 13C-incorporation (approximately 10 times) into PLFA compared with 13C-acetate or 13C-pyruvate. 13C-PLFA in the 13C-acetate and 13C-pyruvate slurries were similar to each other and to those that developed in the 13C-glucose slurry after 4 days. These were more diagnostic, with branched odd-chain fatty acids (i15:0, a15:0 and 15:1omega6) possibly indicating the presence of Desulfococcus or Desulfosarcina sulfate-reducing bacteria (SRB) and sequences related to these SRB were in the 13C-acetate-DNA dsrA gene library. The 13C-acetate-DNA 16S rRNA gene library also contained sequences closely related to SRB, but these were the acetate-utilizing Desulfobacter sp., as well as a broad range of uncultured Bacteria. In contrast, analysis of DGGE bands from 13C-DNA demonstrated that the candidate division JS1 and Firmicutes were actively assimilating 13C-acetate. Denaturing gradient gel electrophoresis also confirmed the presence of JS1 in the 13C-DNA from the 13C-glucose slurry. These results demonstrate that JS1, originally found in deep subsurface sediments, is more widely distributed in marine sediments and provides the first indication of its metabolism; incorporation of acetate and glucose (or glucose metabolites) under anaerobic, sulfate-reducing conditions. Here we demonstrate that PLFA- and DNA-SIP can be used together in a sedimentary system, with low concentrations of 13C-substrate and overlapping incubation times (up to 7 days) to provide complementary, although not identical, information on carbon flow and the identity of active members of an anaerobic prokaryotic community.

Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

PubMed

Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

2015-01-01

Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing.

PubMed

Thoendel, Matthew; Jeraldo, Patricio R; Greenwood-Quaintance, Kerryl E; Yao, Janet Z; Chia, Nicholas; Hanssen, Arlen D; Abdel, Matthew P; Patel, Robin

2016-08-01

Metagenomic whole genome sequencing for detection of pathogens in clinical samples is an exciting new area for discovery and clinical testing. A major barrier to this approach is the overwhelming ratio of human to pathogen DNA in samples with low pathogen abundance, which is typical of most clinical specimens. Microbial DNA enrichment methods offer the potential to relieve this limitation by improving this ratio. Two commercially available enrichment kits, the NEBNext Microbiome DNA Enrichment Kit and the Molzym MolYsis Basic kit, were tested for their ability to enrich for microbial DNA from resected arthroplasty component sonicate fluids from prosthetic joint infections or uninfected sonicate fluids spiked with Staphylococcus aureus. Using spiked uninfected sonicate fluid there was a 6-fold enrichment of bacterial DNA with the NEBNext kit and 76-fold enrichment with the MolYsis kit. Metagenomic whole genome sequencing of sonicate fluid revealed 13- to 85-fold enrichment of bacterial DNA using the NEBNext enrichment kit. The MolYsis approach achieved 481- to 9580-fold enrichment, resulting in 7 to 59% of sequencing reads being from the pathogens known to be present in the samples. These results demonstrate the usefulness of these tools when testing clinical samples with low microbial burden using next generation sequencing. Copyright © 2016 Elsevier B.V. All rights reserved.

Microbeads display of proteins using emulsion PCR and cell-free protein synthesis.

PubMed

Gan, Rui; Yamanaka, Yumiko; Kojima, Takaaki; Nakano, Hideo

2008-01-01

We developed a method for coupling protein to its coding DNA on magnetic microbeads using emulsion PCR and cell-free protein synthesis in emulsion. A PCR mixture containing streptavidin-coated microbeads was compartmentalized by water-in-oil (w/o) emulsion with estimated 0.5 template molecules per droplet. The template molecules were amplified and immobilized on beads via bead-linked reverse primers and biotinylated forward primers. After amplification, the templates were sequentially labeled with streptavidin and biotinylated anti-glutathione S-transferase (GST) antibody. The pool of beads was then subjected to cell-free protein synthesis compartmentalized in another w/o emulsion, in which templates were coupled to their coding proteins. We mixed two types of DNA templates of Histidine6 tag (His6)-fused and FLAG tag-fused GST in a ratio of 1:1,000 (His6: FLAG) for use as a model DNA library. After incubation with fluorescein isothiocyanate (FITC)-labeled anti-His6 (C-term) antibody, the beads with the His6 gene were enriched 917-fold in a single-round screening by using flow cytometry. A library with a theoretical diversity of 10(6) was constructed by randomizing the middle four residues of the His6 tag. After a two-round screening, the randomized sequences were substantially converged to peptide-encoding sequences recognized by the anti-His6 antibody.

Methanotroph Diversity in Landfill Soil: Isolation of Novel Type I and Type II Methanotrophs Whose Presence Was Suggested by Culture-Independent 16S Ribosomal DNA Analysis

PubMed Central

Wise, Mark G.; McArthur, J Vaun; Shimkets, Lawrence J.

1999-01-01

The diversity of the methanotrophic community in mildly acidic landfill cover soil was assessed by three methods: two culture-independent molecular approaches and a traditional culture-based approach. For the first of the molecular studies, two primer pairs specific for the 16S rRNA gene of validly published type I (including the former type X) and type II methanotrophs were identified and tested. These primers were used to amplify directly extracted soil DNA, and the products were used to construct type I and type II clone libraries. The second molecular approach, based on denaturing gradient gel electrophoresis (DGGE), provided profiles of the methanotrophic community members as distinguished by sequence differences in variable region 3 of the 16S ribosomal DNA. For the culturing studies, an extinction-dilution technique was employed to isolate slow-growing but numerically dominant strains. The key variables of the series of enrichment conditions were initial pH (4.8 versus 6.8), air/CH4/CO2 headspace ratio (50:45:5 versus 90:9:1), and concentration of the medium (1× nitrate minimal salts [NMS] versus 0.2× NMS). Screening of the isolates showed that the nutrient-rich 1× NMS selected for type I methanotrophs, while the nutrient-poor 0.2× NMS tended to enrich for type II methanotrophs. Partial sequencing of the 16S rRNA gene from selected clones and isolates revealed some of the same novel sequence types. Phylogenetic analysis of the type I clone library suggested the presence of a new phylotype related to the Methylobacter-Methylomicrobium group, and this was confirmed by isolating two members of this cluster. The type II clone library also suggested the existence of a novel group of related species distinct from the validly published Methylosinus and Methylocystis genera, and two members of this cluster were also successfully cultured. Partial sequencing of the pmoA gene, which codes for the 27-kDa polypeptide of the particulate methane monooxygenase, reaffirmed the phylogenetic placement of the four isolates. Finally, not all of the bands separated by DGGE could be accounted for by the clones and isolates. This polyphasic assessment of community structure demonstrates that much diversity among the obligate methane oxidizers has yet to be formally described. PMID:10543800

Sequencing and analysis of 10,967 full-length cDNA clones from Xenopus laevis and Xenopus tropicalis reveals post-tetraploidization transcriptome remodeling

PubMed Central

Morin, Ryan D.; Chang, Elbert; Petrescu, Anca; Liao, Nancy; Griffith, Malachi; Kirkpatrick, Robert; Butterfield, Yaron S.; Young, Alice C.; Stott, Jeffrey; Barber, Sarah; Babakaiff, Ryan; Dickson, Mark C.; Matsuo, Corey; Wong, David; Yang, George S.; Smailus, Duane E.; Wetherby, Keith D.; Kwong, Peggy N.; Grimwood, Jane; Brinkley, Charles P.; Brown-John, Mabel; Reddix-Dugue, Natalie D.; Mayo, Michael; Schmutz, Jeremy; Beland, Jaclyn; Park, Morgan; Gibson, Susan; Olson, Teika; Bouffard, Gerard G.; Tsai, Miranda; Featherstone, Ruth; Chand, Steve; Siddiqui, Asim S.; Jang, Wonhee; Lee, Ed; Klein, Steven L.; Blakesley, Robert W.; Zeeberg, Barry R.; Narasimhan, Sudarshan; Weinstein, John N.; Pennacchio, Christa Prange; Myers, Richard M.; Green, Eric D.; Wagner, Lukas; Gerhard, Daniela S.; Marra, Marco A.; Jones, Steven J.M.; Holt, Robert A.

2006-01-01

Sequencing of full-insert clones from full-length cDNA libraries from both Xenopus laevis and Xenopus tropicalis has been ongoing as part of the Xenopus Gene Collection Initiative. Here we present 10,967 full ORF verified cDNA clones (8049 from X. laevis and 2918 from X. tropicalis) as a community resource. Because the genome of X. laevis, but not X. tropicalis, has undergone allotetraploidization, comparison of coding sequences from these two clawed (pipid) frogs provides a unique angle for exploring the molecular evolution of duplicate genes. Within our clone set, we have identified 445 gene trios, each comprised of an allotetraploidization-derived X. laevis gene pair and their shared X. tropicalis ortholog. Pairwise dN/dS, comparisons within trios show strong evidence for purifying selection acting on all three members. However, dN/dS ratios between X. laevis gene pairs are elevated relative to their X. tropicalis ortholog. This difference is highly significant and indicates an overall relaxation of selective pressures on duplicated gene pairs. We have found that the paralogs that have been lost since the tetraploidization event are enriched for several molecular functions, but have found no such enrichment in the extant paralogs. Approximately 14% of the paralogous pairs analyzed here also show differential expression indicative of subfunctionalization. PMID:16672307

Method for construction of normalized cDNA libraries

DOEpatents

Soares, Marcelo B.; Efstratiadis, Argiris

1996-01-01

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

Method for construction of normalized cDNA libraries

DOEpatents

Soares, M.B.; Efstratiadis, A.

1996-01-09

This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form. The method comprises: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3` noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. 4 figs.

Building a DNA barcode reference library for the true butterflies (Lepidoptera) of Peninsula Malaysia: what about the subspecies?

PubMed

Wilson, John-James; Sing, Kong-Wah; Sofian-Azirun, Mohd

2013-01-01

The objective of this study was to build a DNA barcode reference library for the true butterflies of Peninsula Malaysia and assess the value of attaching subspecies names to DNA barcode records. A new DNA barcode library was constructed with butterflies from the Museum of Zoology, University of Malaya collection. The library was analysed in conjunction with publicly available DNA barcodes from other Asia-Pacific localities to test the ability of the DNA barcodes to discriminate species and subspecies. Analyses confirmed the capacity of the new DNA barcode reference library to distinguish the vast majority of species (92%) and revealed that most subspecies possessed unique DNA barcodes (84%). In some cases conspecific subspecies exhibited genetic distances between their DNA barcodes that are typically seen between species, and these were often taxa that have previously been regarded as full species. Subspecies designations as shorthand for geographically and morphologically differentiated groups provide a useful heuristic for assessing how such groups correlate with clustering patterns of DNA barcodes, especially as the number of DNA barcodes per species in reference libraries increases. Our study demonstrates the value in attaching subspecies names to DNA barcode records as they can reveal a history of taxonomic concepts and expose important units of biodiversity.

Building a DNA Barcode Reference Library for the True Butterflies (Lepidoptera) of Peninsula Malaysia: What about the Subspecies?

PubMed Central

Wilson, John-James; Sing, Kong-Wah; Sofian-Azirun, Mohd

2013-01-01

The objective of this study was to build a DNA barcode reference library for the true butterflies of Peninsula Malaysia and assess the value of attaching subspecies names to DNA barcode records. A new DNA barcode library was constructed with butterflies from the Museum of Zoology, University of Malaya collection. The library was analysed in conjunction with publicly available DNA barcodes from other Asia-Pacific localities to test the ability of the DNA barcodes to discriminate species and subspecies. Analyses confirmed the capacity of the new DNA barcode reference library to distinguish the vast majority of species (92%) and revealed that most subspecies possessed unique DNA barcodes (84%). In some cases conspecific subspecies exhibited genetic distances between their DNA barcodes that are typically seen between species, and these were often taxa that have previously been regarded as full species. Subspecies designations as shorthand for geographically and morphologically differentiated groups provide a useful heuristic for assessing how such groups correlate with clustering patterns of DNA barcodes, especially as the number of DNA barcodes per species in reference libraries increases. Our study demonstrates the value in attaching subspecies names to DNA barcode records as they can reveal a history of taxonomic concepts and expose important units of biodiversity. PMID:24282514

Accelerating plant DNA barcode reference library construction using herbarium specimens: improved experimental techniques.

PubMed

Xu, Chao; Dong, Wenpan; Shi, Shuo; Cheng, Tao; Li, Changhao; Liu, Yanlei; Wu, Ping; Wu, Hongkun; Gao, Peng; Zhou, Shiliang

2015-11-01

A well-covered reference library is crucial for successful identification of species by DNA barcoding. The biggest difficulty in building such a reference library is the lack of materials of organisms. Herbarium collections are potentially an enormous resource of materials. In this study, we demonstrate that it is likely to build such reference libraries using the reconstructed (self-primed PCR amplified) DNA from the herbarium specimens. We used 179 rosaceous specimens to test the effects of DNA reconstruction, 420 randomly sampled specimens to estimate the usable percentage and another 223 specimens of true cherries (Cerasus, Rosaceae) to test the coverage of usable specimens to the species. The barcode rbcLb (the central four-sevenths of rbcL gene) and matK was each amplified in two halves and sequenced on Roche GS 454 FLX+. DNA from the herbarium specimens was typically shorter than 300 bp. DNA reconstruction enabled amplification fragments of 400-500 bp without bringing or inducing any sequence errors. About one-third of specimens in the national herbarium of China (PE) were proven usable after DNA reconstruction. The specimens in PE cover all Chinese true cherry species and 91.5% of vascular species listed in Flora of China. It is very possible to build well-covered reference libraries for DNA barcoding of vascular species in China. As exemplified in this study, DNA reconstruction and DNA-labelled next-generation sequencing can accelerate the construction of local reference libraries. By putting the local reference libraries together, a global library for DNA barcoding becomes closer to reality. © 2015 John Wiley & Sons Ltd.

Measuring Sister Chromatid Cohesion Protein Genome Occupancy in Drosophila melanogaster by ChIP-seq.

PubMed

Dorsett, Dale; Misulovin, Ziva

2017-01-01

This chapter presents methods to conduct and analyze genome-wide chromatin immunoprecipitation of the cohesin complex and the Nipped-B cohesin loading factor in Drosophila cells using high-throughput DNA sequencing (ChIP-seq). Procedures for isolation of chromatin, immunoprecipitation, and construction of sequencing libraries for the Ion Torrent Proton high throughput sequencer are detailed, and computational methods to calculate occupancy as input-normalized fold-enrichment are described. The results obtained by ChIP-seq are compared to those obtained by ChIP-chip (genomic ChIP using tiling microarrays), and the effects of sequencing depth on the accuracy are analyzed. ChIP-seq provides similar sensitivity and reproducibility as ChIP-chip, and identifies the same broad regions of occupancy. The locations of enrichment peaks, however, can differ between ChIP-chip and ChIP-seq, and low sequencing depth can splinter broad regions of occupancy into distinct peaks.

The librarian's role in an enrichment program for high school students interested in the health professions.

PubMed

Rossini, Beverly; Burnham, Judy; Wright, Andrea

2013-01-01

Librarians from the University of South Alabama Biomedical Library partnered to participate in a program that targets minority students interested in health care with instruction in information literacy. Librarians participate in the summer enrichment programs designed to encourage minority students to enter health care professions by enhancing their preparation. The curriculum developed by the Biomedical Library librarians is focused on developing information searching skills. Students indicated that the library segment helped them in their library research efforts and helped them make more effective use of available resources. Librarians involved report a sense of self-satisfaction as the program allows them to contribute to promoting greater diversity in health care professions. Participating in the summer enrichment program has been beneficial to the students and librarians.

DNA-based stable isotope probing coupled with cultivation methods implicates Methylophaga in hydrocarbon degradation

PubMed Central

Mishamandani, Sara; Gutierrez, Tony; Aitken, Michael D.

2014-01-01

Marine hydrocarbon-degrading bacteria perform a fundamental role in the oxidation and ultimate removal of crude oil and its petrochemical derivatives in coastal and open ocean environments. Those with an almost exclusive ability to utilize hydrocarbons as a sole carbon and energy source have been found confined to just a few genera. Here we used stable isotope probing (SIP), a valuable tool to link the phylogeny and function of targeted microbial groups, to investigate hydrocarbon-degrading bacteria in coastal North Carolina sea water (Beaufort Inlet, USA) with uniformly labeled [13C]n-hexadecane. The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from n-hexadecane enrichments) were identified to belong to the genus Alcanivorax, with ≤98% sequence identity to the closest type strain—thus representing a putative novel phylogenetic taxon within this genus. Unexpectedly, we also identified 13C-enriched sequences in heavy DNA fractions that were affiliated to the genus Methylophaga. This is a contentious group since, though some of its members have been proposed to degrade hydrocarbons, substantive evidence has not previously confirmed this. We used quantitative PCR primers targeting the 16S rRNA gene of the SIP-identified Alcanivorax and Methylophaga to determine their abundance in incubations amended with unlabeled n-hexadecane. Both showed substantial increases in gene copy number during the experiments. Subsequently, we isolated a strain representing the SIP-identified Methylophaga sequences (99.9% 16S rRNA gene sequence identity) and used it to show, for the first time, direct evidence of hydrocarbon degradation by a cultured Methylophaga sp. This study demonstrates the value of coupling SIP with cultivation methods to identify and expand on the known diversity of hydrocarbon-degrading bacteria in the marine environment. PMID:24578702

Strong spurious transcription likely contributes to DNA insert bias in typical metagenomic clone libraries.

PubMed

Lam, Kathy N; Charles, Trevor C

2015-01-01

Clone libraries provide researchers with a powerful resource to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, and allowed the mining of novel enzymes. Libraries are often constructed by cloning large inserts into cosmid or fosmid vectors. Recently, there have been reports of GC bias in fosmid metagenomic libraries, and it was speculated to be a result of fragmentation and loss of AT-rich sequences during cloning. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role. To explore possible mechanisms responsible for sequence bias in clone libraries, we constructed a cosmid library from a human microbiome sample and sequenced DNA from different steps during library construction: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final cosmid library, and we provide evidence that the bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after DNA is introduced into Escherichia coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for promoters and found that rpoD/σ(70) promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found the bias to be more correlated with the number of rpoD/σ(70) consensus sequences in the genome than with simple GC content. The GC bias of metagenomic libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/σ(70) consensus-like in E. coli may lead to instability, causing loss of the plasmid or loss of the insert DNA that gives rise to the transcription. Despite widespread use of E. coli to propagate foreign DNA in metagenomic libraries, the effects of in vivo transcriptional activity on clone stability are not well understood. Further work is required to tease apart the effects of transcription from those of gene product toxicity.

Application of Biocatalysis to on-DNA Carbohydrate Library Synthesis.

PubMed

Thomas, Baptiste; Lu, Xiaojie; Birmingham, William R; Huang, Kun; Both, Peter; Reyes Martinez, Juana Elizabeth; Young, Robert J; Davie, Christopher P; Flitsch, Sabine L

2017-05-04

DNA-encoded libraries are increasingly used for the discovery of bioactive lead compounds in high-throughput screening programs against specific biological targets. Although a number of libraries are now available, they cover limited chemical space due to bias in ease of synthesis and the lack of chemical reactions that are compatible with DNA tagging. For example, compound libraries rarely contain complex biomolecules such as carbohydrates with high levels of functionality, stereochemistry, and hydrophilicity. By using biocatalysis in combination with chemical methods, we aimed to significantly expand chemical space and generate generic libraries with potentially better biocompatibility. For DNA-encoded libraries, biocatalysis is particularly advantageous, as it is highly selective and can be performed in aqueous environments, which is an essential feature for this split-and-mix library technology. In this work, we demonstrated the application of biocatalysis for the on-DNA synthesis of carbohydrate-based libraries by using enzymatic oxidation and glycosylation in combination with traditional organic chemistry. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Defiant: (DMRs: easy, fast, identification and ANnoTation) identifies differentially Methylated regions from iron-deficient rat hippocampus.

PubMed

Condon, David E; Tran, Phu V; Lien, Yu-Chin; Schug, Jonathan; Georgieff, Michael K; Simmons, Rebecca A; Won, Kyoung-Jae

2018-02-05

Identification of differentially methylated regions (DMRs) is the initial step towards the study of DNA methylation-mediated gene regulation. Previous approaches to call DMRs suffer from false prediction, use extreme resources, and/or require library installation and input conversion. We developed a new approach called Defiant to identify DMRs. Employing Weighted Welch Expansion (WWE), Defiant showed superior performance to other predictors in the series of benchmarking tests on artificial and real data. Defiant was subsequently used to investigate DNA methylation changes in iron-deficient rat hippocampus. Defiant identified DMRs close to genes associated with neuronal development and plasticity, which were not identified by its competitor. Importantly, Defiant runs between 5 to 479 times faster than currently available software packages. Also, Defiant accepts 10 different input formats widely used for DNA methylation data. Defiant effectively identifies DMRs for whole-genome bisulfite sequencing (WGBS), reduced-representation bisulfite sequencing (RRBS), Tet-assisted bisulfite sequencing (TAB-seq), and HpaII tiny fragment enrichment by ligation-mediated PCR-tag (HELP) assays.

Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge.

PubMed

Cui, Zhisong; Lai, Qiliang; Dong, Chunming; Shao, Zongze

2008-08-01

The bacteria involved in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in deep sea subsurface environments are largely unknown. In order to reveal their biodiversity, sediments from 2.2 m under the bottom surface at a water depth of 3542 m were sampled on the Middle Atlantic Ridge with a gravity column sampler. The sediments were promptly enriched with either crude oil or a mixture of PAHs (naphthalene, phenanthrene and pyrene) as the sole carbon source, and further enriched with the PAH mixture mentioned above in the lab. The resulting consortia were named C2CO and C2PPN respectively. Their bacterial composition was analysed with plate cultivation, PCR-DGGE and 16S rDNA library analysis. On plates, isolates belonging to Pseudoalteromonas, Halomonas, Marinobacter, Thalassospira and Tistrella dominated the culturable populations. With PCR-DGGE, five major bands closely related to Cycloclasticus, Alteromonas, Thalassospira, Alcanivorax and Rhodospirillaceae were detected in consortium C2CO, while only one major band of Cycloclasticus was detected in consortium C2PPN. In addition, the dynamics of community structure in response to aromatic substrate alterations were examined. As a result, three ribotypes of Cycloclasticus were detected by 16S rDNA library analysis, one which played a key role in phenanthrene degradation; two Alteromonas bacteria dominated the naphthalene reselected consortium. Although bacteria of the two genera grew as the main members of the communities, none of them were isolated, probably owing to their poor cultivability. These results confirm that bacteria of Cycloclasticus are important obligate PAH degraders in marine environments, and coexist with other degrading bacteria that inhabit the deep subsurface sediment of the Atlantic. This supports the view that PAH accumulation and bioattenuation occur in remote areas consistently and continuously.

Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge

PubMed Central

Cui, Zhisong; Lai, Qiliang; Dong, Chunming; Shao, Zongze

2008-01-01

The bacteria involved in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in deep sea subsurface environments are largely unknown. In order to reveal their biodiversity, sediments from 2.2 m under the bottom surface at a water depth of 3542 m were sampled on the Middle Atlantic Ridge with a gravity column sampler. The sediments were promptly enriched with either crude oil or a mixture of PAHs (naphthalene, phenanthrene and pyrene) as the sole carbon source, and further enriched with the PAH mixture mentioned above in the lab. The resulting consortia were named C2CO and C2PPN respectively. Their bacterial composition was analysed with plate cultivation, PCR-DGGE and 16S rDNA library analysis. On plates, isolates belonging to Pseudoalteromonas, Halomonas, Marinobacter, Thalassospira and Tistrella dominated the culturable populations. With PCR-DGGE, five major bands closely related to Cycloclasticus, Alteromonas, Thalassospira, Alcanivorax and Rhodospirillaceae were detected in consortium C2CO, while only one major band of Cycloclasticus was detected in consortium C2PPN. In addition, the dynamics of community structure in response to aromatic substrate alterations were examined. As a result, three ribotypes of Cycloclasticus were detected by 16S rDNA library analysis, one which played a key role in phenanthrene degradation; two Alteromonas bacteria dominated the naphthalene reselected consortium. Although bacteria of the two genera grew as the main members of the communities, none of them were isolated, probably owing to their poor cultivability. These results confirm that bacteria of Cycloclasticus are important obligate PAH degraders in marine environments, and coexist with other degrading bacteria that inhabit the deep subsurface sediment of the Atlantic. This supports the view that PAH accumulation and bioattenuation occur in remote areas consistently and continuously. PMID:18445026

Construction of high-quality Caco-2 three-frame cDNA library and its application to yeast two-hybrid for the human astrovirus protein-protein interaction.

PubMed

Zhao, Wei; Li, Xin; Liu, Wen-Hui; Zhao, Jian; Jin, Yi-Ming; Sui, Ting-Ting

2014-09-01

Human epithelial colorectal adenocarcinoma (Caco-2) cells are widely used as an in vitro model of the human small intestinal mucosa. Caco-2 cells are host cells of the human astrovirus (HAstV) and other enteroviruses. High quality cDNA libraries are pertinent resources and critical tools for protein-protein interaction research, but are currently unavailable for Caco-2 cells. To construct a three-open reading frame, full length-expression cDNA library from the Caco-2 cell line for application to HAstV protein-protein interaction screening, total RNA was extracted from Caco-2 cells. The switching mechanism at the 5' end of the RNA transcript technique was used for cDNA synthesis. Double-stranded cDNA was digested by Sfi I and ligated to reconstruct a pGADT7-Sfi I three-frame vector. The ligation mixture was transformed into Escherichia coli HST08 premium electro cells by electroporation to construct the primary cDNA library. The library capacity was 1.0×10(6)clones. Gel electrophoresis results indicated that the fragments ranged from 0.5kb to 4.2kb. Randomly picked clones show that the recombination rate was 100%. The three-frame primary cDNA library plasmid mixture (5×10(5)cfu) was also transformed into E. coli HST08 premium electro cells, and all clones were harvested to amplify the cDNA library. To detect the sufficiency of the cDNA library, HAstV capsid protein as bait was screened and tested against the Caco-2 cDNA library by a yeast two-hybrid (Y2H) system. A total of 20 proteins were found to interact with the capsid protein. These results showed that a high-quality three-frame cDNA library from Caco-2 cells was successfully constructed. This library was efficient for the application to the Y2H system, and could be used for future research. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and Application of a Salmonid EST Database and cDNA Microarray: Data Mining and Interspecific Hybridization Characteristics

PubMed Central

Rise, Matthew L.; von Schalburg, Kristian R.; Brown, Gordon D.; Mawer, Melanie A.; Devlin, Robert H.; Kuipers, Nathanael; Busby, Maura; Beetz-Sargent, Marianne; Alberto, Roberto; Gibbs, A. Ross; Hunt, Peter; Shukin, Robert; Zeznik, Jeffrey A.; Nelson, Colleen; Jones, Simon R.M.; Smailus, Duane E.; Jones, Steven J.M.; Schein, Jacqueline E.; Marra, Marco A.; Butterfield, Yaron S.N.; Stott, Jeff M.; Ng, Siemon H.S.; Davidson, William S.; Koop, Ben F.

2004-01-01

We report 80,388 ESTs from 23 Atlantic salmon (Salmo salar) cDNA libraries (61,819 ESTs), 6 rainbow trout (Oncorhynchus mykiss) cDNA libraries (14,544 ESTs), 2 chinook salmon (Oncorhynchus tshawytscha) cDNA libraries (1317 ESTs), 2 sockeye salmon (Oncorhynchus nerka) cDNA libraries (1243 ESTs), and 2 lake whitefish (Coregonus clupeaformis) cDNA libraries (1465 ESTs). The majority of these are 3′ sequences, allowing discrimination between paralogs arising from a recent genome duplication in the salmonid lineage. Sequence assembly reveals 28,710 different S. salar, 8981 O. mykiss, 1085 O. tshawytscha, 520 O. nerka, and 1176 C. clupeaformis putative transcripts. We annotate the submitted portion of our EST database by molecular function. Higher- and lower-molecular-weight fractions of libraries are shown to contain distinct gene sets, and higher rates of gene discovery are associated with higher-molecular weight libraries. Pyloric caecum library group annotations indicate this organ may function in redox control and as a barrier against systemic uptake of xenobiotics. A microarray is described, containing 7356 salmonid elements representing 3557 different cDNAs. Analyses of cross-species hybridizations to this cDNA microarray indicate that this resource may be used for studies involving all salmonids. PMID:14962987

cDNA library construction of two human Demodexspecies.

PubMed

Niu, DongLing; Wang, RuiLing; Zhao, YaE; Yang, Rui; Hu, Li; Lei, YuYang; Dan, WeiChao

2017-06-01

The research of Demodex, a type of pathogen causing various dermatoses in animals and human beings, is lacking at RNA level. This study aims at extracting RNA and constructing cDNA library for Demodex. First, P. cuniculiand D. farinaewere mixed to establish homogenization method for RNA extraction. Second, D. folliculorumand D. breviswere collected and preserved in Trizol, which were mixed with D. farinaerespectively to extract RNA. Finally, cDNA library was constructed and its quality was assessed. The results indicated that for D. folliculorum& D. farinae, the recombination rate of cDNA library was 90.67% and the library titer was 7.50 × 104 pfu/ml. 17 of the 59 positive clones were predicted to be of D. folliculorum; For D. brevis& D. farinae, the recombination rate was 90.96% and the library titer was 7.85 x104 pfu/ml. 40 of the 59 positive clones were predicted to be of D. brevis. Further detection by specific primers demonstrated that mtDNA cox1, cox3and ATP6 detected from cDNA libraries had 96.52%-99.73% identities with the corresponding sequences in GenBank. In conclusion, the cDNA libraries constructed for Demodexmixed with D. farinaewere successful and could satisfy the requirements for functional genes detection.

Subtraction of cap-trapped full-length cDNA libraries to select rare transcripts.

PubMed

Hirozane-Kishikawa, Tomoko; Shiraki, Toshiyuki; Waki, Kazunori; Nakamura, Mari; Arakawa, Takahiro; Kawai, Jun; Fagiolini, Michela; Hensch, Takao K; Hayashizaki, Yoshihide; Carninci, Piero

2003-09-01

The normalization and subtraction of highly expressed cDNAs from relatively large tissues before cloning dramatically enhanced the gene discovery by sequencing for the mouse full-length cDNA encyclopedia, but these methods have not been suitable for limited RNA materials. To normalize and subtract full-length cDNA libraries derived from limited quantities of total RNA, here we report a method to subtract plasmid libraries excised from size-unbiased amplified lambda phage cDNA libraries that avoids heavily biasing steps such as PCR and plasmid library amplification. The proportion of full-length cDNAs and the gene discovery rate are high, and library diversity can be validated by in silico randomization.

Enriching peptide libraries for binding affinity and specificity through computationally directed library design

PubMed Central

Foight, Glenna Wink; Chen, T. Scott; Richman, Daniel; Keating, Amy E.

2017-01-01

Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model. PMID:28236241

Enriching Peptide Libraries for Binding Affinity and Specificity Through Computationally Directed Library Design.

PubMed

Foight, Glenna Wink; Chen, T Scott; Richman, Daniel; Keating, Amy E

2017-01-01

Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model.

DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries.

PubMed

Franzini, Raphael M; Neri, Dario; Scheuermann, Jörg

2014-04-15

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

IDENTIFICATION OF ACTIVE BACTERIAL COMMUNITIES IN A MODEL DRINKING WATER BIOFILM SYSTEM USING 16S RRNA-BASED CLONE LIBRARIES

EPA Science Inventory

Recent phylogenetic studies have used DNA as the target molecule for the development of environmental 16S rDNA clone libraries. As DNA may persist in the environment, DNA-based libraries cannot be used to identify metabolically active bacteria in water systems. In this study, a...

HUNT: launch of a full-length cDNA database from the Helix Research Institute.

PubMed

Yudate, H T; Suwa, M; Irie, R; Matsui, H; Nishikawa, T; Nakamura, Y; Yamaguchi, D; Peng, Z Z; Yamamoto, T; Nagai, K; Hayashi, K; Otsuki, T; Sugiyama, T; Ota, T; Suzuki, Y; Sugano, S; Isogai, T; Masuho, Y

2001-01-01

The Helix Research Institute (HRI) in Japan is releasing 4356 HUman Novel Transcripts and related information in the newly established HUNT database. The institute is a joint research project principally funded by the Japanese Ministry of International Trade and Industry, and the clones were sequenced in the governmental New Energy and Industrial Technology Development Organization (NEDO) Human cDNA Sequencing Project. The HUNT database contains an extensive amount of annotation from advanced analysis and represents an essential bioinformatics contribution towards understanding of the gene function. The HRI human cDNA clones were obtained from full-length enriched cDNA libraries constructed with the oligo-capping method and have resulted in novel full-length cDNA sequences. A large fraction has little similarity to any proteins of known function and to obtain clues about possible function we have developed original analysis procedures. Any putative function deduced here can be validated or refuted by complementary analysis results. The user can also extract information from specific categories like PROSITE patterns, PFAM domains, PSORT localization, transmembrane helices and clones with GENIUS structure assignments. The HUNT database can be accessed at http://www.hri.co.jp/HUNT.

The Application of Next-Generation Sequencing for Mutation Detection in Autosomal-Dominant Hereditary Hearing Impairment.

PubMed

Gürtler, Nicolas; Röthlisberger, Benno; Ludin, Katja; Schlegel, Christoph; Lalwani, Anil K

2017-07-01

Identification of the causative mutation using next-generation sequencing in autosomal-dominant hereditary hearing impairment, as mutation analysis in hereditary hearing impairment by classic genetic methods, is hindered by the high heterogeneity of the disease. Two Swiss families with autosomal-dominant hereditary hearing impairment. Amplified DNA libraries for next-generation sequencing were constructed from extracted genomic DNA, derived from peripheral blood, and enriched by a custom-made sequence capture library. Validated, pooled libraries were sequenced on an Illumina MiSeq instrument, 300 cycles and paired-end sequencing. Technical data analysis was performed with SeqMonk, variant analysis with GeneTalk or VariantStudio. The detection of mutations in genes related to hearing loss by next-generation sequencing was subsequently confirmed using specific polymerase-chain-reaction and Sanger sequencing. Mutation detection in hearing-loss-related genes. The first family harbored the mutation c.5383+5delGTGA in the TECTA-gene. In the second family, a novel mutation c.2614-2625delCATGGCGCCGTG in the WFS1-gene and a second mutation TCOF1-c.1028G>A were identified. Next-generation sequencing successfully identified the causative mutation in families with autosomal-dominant hereditary hearing impairment. The results helped to clarify the pathogenic role of a known mutation and led to the detection of a novel one. NGS represents a feasible approach with great potential future in the diagnostics of hereditary hearing impairment, even in smaller labs.

Discovery of cofactor-specific, bactericidal Mycobacterium tuberculosis InhA inhibitors using DNA-encoded library technology.

PubMed

Soutter, Holly H; Centrella, Paolo; Clark, Matthew A; Cuozzo, John W; Dumelin, Christoph E; Guie, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Kennedy, Kaitlyn M; Sigel, Eric A; Troast, Dawn M; Zhang, Ying; Ferguson, Andrew D; Davies, Gareth; Stead, Eleanor R; Breed, Jason; Madhavapeddi, Prashanti; Read, Jon A

2016-12-06

Millions of individuals are infected with and die from tuberculosis (TB) each year, and multidrug-resistant (MDR) strains of TB are increasingly prevalent. As such, there is an urgent need to identify novel drugs to treat TB infections. Current frontline therapies include the drug isoniazid, which inhibits the essential NADH-dependent enoyl-acyl-carrier protein (ACP) reductase, InhA. To inhibit InhA, isoniazid must be activated by the catalase-peroxidase KatG. Isoniazid resistance is linked primarily to mutations in the katG gene. Discovery of InhA inhibitors that do not require KatG activation is crucial to combat MDR TB. Multiple discovery efforts have been made against InhA in recent years. Until recently, despite achieving high potency against the enzyme, these efforts have been thwarted by lack of cellular activity. We describe here the use of DNA-encoded X-Chem (DEX) screening, combined with selection of appropriate physical properties, to identify multiple classes of InhA inhibitors with cell-based activity. The utilization of DEX screening allowed the interrogation of very large compound libraries (10 11 unique small molecules) against multiple forms of the InhA enzyme in a multiplexed format. Comparison of the enriched library members across various screening conditions allowed the identification of cofactor-specific inhibitors of InhA that do not require activation by KatG, many of which had bactericidal activity in cell-based assays.

Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool

PubMed Central

2013-01-01

Background System-wide profiling of genes and proteins in mammalian cells produce lists of differentially expressed genes/proteins that need to be further analyzed for their collective functions in order to extract new knowledge. Once unbiased lists of genes or proteins are generated from such experiments, these lists are used as input for computing enrichment with existing lists created from prior knowledge organized into gene-set libraries. While many enrichment analysis tools and gene-set libraries databases have been developed, there is still room for improvement. Results Here, we present Enrichr, an integrative web-based and mobile software application that includes new gene-set libraries, an alternative approach to rank enriched terms, and various interactive visualization approaches to display enrichment results using the JavaScript library, Data Driven Documents (D3). The software can also be embedded into any tool that performs gene list analysis. We applied Enrichr to analyze nine cancer cell lines by comparing their enrichment signatures to the enrichment signatures of matched normal tissues. We observed a common pattern of up regulation of the polycomb group PRC2 and enrichment for the histone mark H3K27me3 in many cancer cell lines, as well as alterations in Toll-like receptor and interlukin signaling in K562 cells when compared with normal myeloid CD33+ cells. Such analyses provide global visualization of critical differences between normal tissues and cancer cell lines but can be applied to many other scenarios. Conclusions Enrichr is an easy to use intuitive enrichment analysis web-based tool providing various types of visualization summaries of collective functions of gene lists. Enrichr is open source and freely available online at: http://amp.pharm.mssm.edu/Enrichr. PMID:23586463

Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool.

PubMed

Chen, Edward Y; Tan, Christopher M; Kou, Yan; Duan, Qiaonan; Wang, Zichen; Meirelles, Gabriela Vaz; Clark, Neil R; Ma'ayan, Avi

2013-04-15

System-wide profiling of genes and proteins in mammalian cells produce lists of differentially expressed genes/proteins that need to be further analyzed for their collective functions in order to extract new knowledge. Once unbiased lists of genes or proteins are generated from such experiments, these lists are used as input for computing enrichment with existing lists created from prior knowledge organized into gene-set libraries. While many enrichment analysis tools and gene-set libraries databases have been developed, there is still room for improvement. Here, we present Enrichr, an integrative web-based and mobile software application that includes new gene-set libraries, an alternative approach to rank enriched terms, and various interactive visualization approaches to display enrichment results using the JavaScript library, Data Driven Documents (D3). The software can also be embedded into any tool that performs gene list analysis. We applied Enrichr to analyze nine cancer cell lines by comparing their enrichment signatures to the enrichment signatures of matched normal tissues. We observed a common pattern of up regulation of the polycomb group PRC2 and enrichment for the histone mark H3K27me3 in many cancer cell lines, as well as alterations in Toll-like receptor and interlukin signaling in K562 cells when compared with normal myeloid CD33+ cells. Such analyses provide global visualization of critical differences between normal tissues and cancer cell lines but can be applied to many other scenarios. Enrichr is an easy to use intuitive enrichment analysis web-based tool providing various types of visualization summaries of collective functions of gene lists. Enrichr is open source and freely available online at: http://amp.pharm.mssm.edu/Enrichr.

An accurate bacterial DNA quantification assay for HTS library preparation of human biological samples.

PubMed

Seashols-Williams, Sarah; Green, Raquel; Wohlfahrt, Denise; Brand, Angela; Tan-Torres, Antonio Limjuco; Nogales, Francy; Brooks, J Paul; Singh, Baneshwar

2018-05-17

Sequencing and classification of microbial taxa within forensically relevant biological fluids has the potential for applications in the forensic science and biomedical fields. The quantity of bacterial DNA from human samples is currently estimated based on quantity of total DNA isolated. This method can miscalculate bacterial DNA quantity due to the mixed nature of the sample, and consequently library preparation is often unreliable. We developed an assay that can accurately and specifically quantify bacterial DNA within a mixed sample for reliable 16S ribosomal DNA (16S rDNA) library preparation and high throughput sequencing (HTS). A qPCR method was optimized using universal 16S rDNA primers, and a commercially available bacterial community DNA standard was used to develop a precise standard curve. Following qPCR optimization, 16S rDNA libraries from saliva, vaginal and menstrual secretions, urine, and fecal matter were amplified and evaluated at various DNA concentrations; successful HTS data were generated with as low as 20 pg of bacterial DNA. Changes in bacterial DNA quantity did not impact observed relative abundances of major bacterial taxa, but relative abundance changes of minor taxa were observed. Accurate quantification of microbial DNA resulted in consistent, successful library preparations for HTS analysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

PubMed

Booman, Marije; Borza, Tudor; Feng, Charles Y; Hori, Tiago S; Higgins, Brent; Culf, Adrian; Léger, Daniel; Chute, Ian C; Belkaid, Anissa; Rise, Marlies; Gamperl, A Kurt; Hubert, Sophie; Kimball, Jennifer; Ouellette, Rodney J; Johnson, Stewart C; Bowman, Sharen; Rise, Matthew L

2011-08-01

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

Bacterial community composition in different sediments from the Eastern Mediterranean Sea: a comparison of four 16S ribosomal DNA clone libraries.

PubMed

Polymenakou, Paraskevi N; Bertilsson, Stefan; Tselepides, Anastasios; Stephanou, Euripides G

2005-10-01

The regional variability of sediment bacterial community composition and diversity was studied by comparative analysis of four large 16S ribosomal DNA (rDNA) clone libraries from sediments in different regions of the Eastern Mediterranean Sea (Thermaikos Gulf, Cretan Sea, and South lonian Sea). Amplified rDNA restriction analysis of 664 clones from the libraries indicate that the rDNA richness and evenness was high: for example, a near-1:1 relationship among screened clones and number of unique restriction patterns when up to 190 clones were screened for each library. Phylogenetic analysis of 207 bacterial 16S rDNA sequences from the sediment libraries demonstrated that Gamma-, Delta-, and Alphaproteobacteria, Holophaga/Acidobacteria, Planctomycetales, Actinobacteria, Bacteroidetes, and Verrucomicrobia were represented in all four libraries. A few clones also grouped with the Betaproteobacteria, Nitrospirae, Spirochaetales, Chlamydiae, Firmicutes, and candidate division OPl 1. The abundance of sequences affiliated with Gammaproteobacteria was higher in libraries from shallow sediments in the Thermaikos Gulf (30 m) and the Cretan Sea (100 m) compared to the deeper South Ionian station (2790 m). Most sequences in the four sediment libraries clustered with uncultured 16S rDNA phylotypes from marine habitats, and many of the closest matches were clones from hydrocarbon seeps, benzene-mineralizing consortia, sulfate reducers, sulk oxidizers, and ammonia oxidizers. LIBSHUFF statistics of 16S rDNA gene sequences from the four libraries revealed major differences, indicating either a very high richness in the sediment bacterial communities or considerable variability in bacterial community composition among regions, or both.

Evaluation of vector-primed cDNA library production from microgram quantities of total RNA.

PubMed

Kuo, Jonathan; Inman, Jason; Brownstein, Michael; Usdin, Ted B

2004-12-15

cDNA sequences are important for defining the coding region of genes, and full-length cDNA clones have proven to be useful for investigation of the function of gene products. We produced cDNA libraries containing 3.5-5 x 10(5) primary transformants, starting with 5 mug of total RNA prepared from mouse pituitary, adrenal, thymus, and pineal tissue, using a vector-primed cDNA synthesis method. Of approximately 1000 clones sequenced, approximately 20% contained the full open reading frames (ORFs) of known transcripts, based on the presence of the initiating methionine residue codon. The libraries were complex, with 94, 91, 83 and 55% of the clones from the thymus, adrenal, pineal and pituitary libraries, respectively, represented only once. Twenty-five full-length clones, not yet represented in the Mammalian Gene Collection, were identified. Thus, we have produced useful cDNA libraries for the isolation of full-length cDNA clones that are not yet available in the public domain, and demonstrated the utility of a simple method for making high-quality libraries from small amounts of starting material.

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins.

PubMed

Dong, Dexian; Gui, Yanli; Chen, Dezhao; Li, Rongxiu

2008-01-01

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins. 2008 John Wiley & Sons, Ltd

Construction of BAC Libraries from Flow-Sorted Chromosomes.

PubMed

Šafář, Jan; Šimková, Hana; Doležel, Jaroslav

2016-01-01

Cloned DNA libraries in bacterial artificial chromosome (BAC) are the most widely used form of large-insert DNA libraries. BAC libraries are typically represented by ordered clones derived from genomic DNA of a particular organism. In the case of large eukaryotic genomes, whole-genome libraries consist of a hundred thousand to a million clones, which make their handling and screening a daunting task. The labor and cost of working with whole-genome libraries can be greatly reduced by constructing a library derived from a smaller part of the genome. Here we describe construction of BAC libraries from mitotic chromosomes purified by flow cytometric sorting. Chromosome-specific BAC libraries facilitate positional gene cloning, physical mapping, and sequencing in complex plant genomes.

Bacterial Community Response to Petroleum Hydrocarbon Amendments in Freshwater, Marine, and Hypersaline Water-Containing Microcosms

PubMed Central

Jurelevicius, Diogo; Alvarez, Vanessa Marques; Marques, Joana Montezano; de Sousa Lima, Laryssa Ribeiro Fonseca; Dias, Felipe de Almeida

2013-01-01

Hydrocarbon-degrading bacterial communities from freshwater, marine, and hypersaline Brazilian aquatic ecosystems (with water salinities corresponding to 0.2%, 4%, and 5%, respectively) were enriched with different hydrocarbons (heptadecane, naphthalene, or crude oil). Changes within the different microcosms of bacterial communities were analyzed using cultivation approaches and molecular methods (DNA and RNA extraction, followed by genetic fingerprinting and analyses of clone libraries based on the 16S rRNA-coding gene). A redundancy analysis (RDA) of the genetic fingerprint data and a principal component analysis (PCA) of the clone libraries revealed hydrocarbon-enriched bacterial communities specific for each ecosystem studied. However, within the same ecosystem, different bacterial communities were selected according to the petroleum hydrocarbon used. In general, the results demonstrated that Acinetobacter and Cloacibacterium were the dominant genera in freshwater microcosms; the Oceanospirillales order and the Marinobacter, Pseudomonas, and Cycloclasticus genera predominated in marine microcosms; and the Oceanospirillales order and the Marinobacter genus were selected in the different hydrocarbon-containing microcosms in hypersaline water. Determination of total petroleum hydrocarbons (TPHs) in all microcosms after 32 days of incubation showed a decrease in the hydrocarbon concentration compared to that for the controls. A total of 50 (41.3%) isolates from the different hydrocarbon-contaminated microcosms were associated with the dominant operational taxonomic units (OTUs) obtained from the clone libraries, and their growth in the hydrocarbon contaminating the microcosm from which they were isolated as the sole carbon source was observed. These data provide insight into the general response of bacterial communities from freshwater, marine, and hypersaline aquatic ecosystems to petroleum hydrocarbon contamination. PMID:23872573

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

PubMed

Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Song, Chen; Adalsteinsson, Viktor A; Parsons, Heather A; Lin, Nancy U; Wagle, Nikhil; Makrigiorgos, G Mike

2017-10-01

The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing. © 2017 American Association for Clinical Chemistry.

Activity-based metagenomic screening and biochemical characterization of bovine ruminal protozoan glycoside hydrolases.

PubMed

Findley, Seth D; Mormile, Melanie R; Sommer-Hurley, Andrea; Zhang, Xue-Cheng; Tipton, Peter; Arnett, Krista; Porter, James H; Kerley, Monty; Stacey, Gary

2011-11-01

The rumen, the foregut of herbivorous ruminant animals such as cattle, functions as a bioreactor to process complex plant material. Among the numerous and diverse microbes involved in ruminal digestion are the ruminal protozoans, which are single-celled, ciliated eukaryotic organisms. An activity-based screen was executed to identify genes encoding fibrolytic enzymes present in the metatranscriptome of a bovine ruminal protozoan-enriched cDNA expression library. Of the four novel genes identified, two were characterized in biochemical assays. Our results provide evidence for the effective use of functional metagenomics to retrieve novel enzymes from microbial populations that cannot be maintained in axenic cultures.

SPlinted Ligation Adapter Tagging (SPLAT), a novel library preparation method for whole genome bisulphite sequencing

PubMed Central

Manlig, Erika; Wahlberg, Per

2017-01-01

Abstract Sodium bisulphite treatment of DNA combined with next generation sequencing (NGS) is a powerful combination for the interrogation of genome-wide DNA methylation profiles. Library preparation for whole genome bisulphite sequencing (WGBS) is challenging due to side effects of the bisulphite treatment, which leads to extensive DNA damage. Recently, a new generation of methods for bisulphite sequencing library preparation have been devised. They are based on initial bisulphite treatment of the DNA, followed by adaptor tagging of single stranded DNA fragments, and enable WGBS using low quantities of input DNA. In this study, we present a novel approach for quick and cost effective WGBS library preparation that is based on splinted adaptor tagging (SPLAT) of bisulphite-converted single-stranded DNA. Moreover, we validate SPLAT against three commercially available WGBS library preparation techniques, two of which are based on bisulphite treatment prior to adaptor tagging and one is a conventional WGBS method. PMID:27899585

A Fast Solution to NGS Library Prep with Low Nanogram DNA Input

PubMed Central

Liu, Pingfang; Lohman, Gregory J.S.; Cantor, Eric; Langhorst, Bradley W.; Yigit, Erbay; Apone, Lynne M.; Munafo, Daniela B.; Stewart, Fiona J.; Evans, Thomas C.; Nichols, Nicole; Dimalanta, Eileen T.; Davis, Theodore B.; Sumner, Christine

2013-01-01

Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.

Boosting functionality of synthetic DNA circuits with tailored deactivation

PubMed Central

Montagne, Kevin; Gines, Guillaume; Fujii, Teruo; Rondelez, Yannick

2016-01-01

Molecular programming takes advantage of synthetic nucleic acid biochemistry to assemble networks of reactions, in vitro, with the double goal of better understanding cellular regulation and providing information-processing capabilities to man-made chemical systems. The function of molecular circuits is deeply related to their topological structure, but dynamical features (rate laws) also play a critical role. Here we introduce a mechanism to tune the nonlinearities associated with individual nodes of a synthetic network. This mechanism is based on programming deactivation laws using dedicated saturable pathways. We demonstrate this approach through the conversion of a single-node homoeostatic network into a bistable and reversible switch. Furthermore, we prove its generality by adding new functions to the library of reported man-made molecular devices: a system with three addressable bits of memory, and the first DNA-encoded excitable circuit. Specific saturable deactivation pathways thus greatly enrich the functional capability of a given circuit topology. PMID:27845324

[Cell-ELA-based determination of binding affinity of DNA aptamer against U87-EGFRvIII cell].

PubMed

Tan, Yan; Liang, Huiyu; Wu, Xidong; Gao, Yubo; Zhang, Xingmei

2013-05-01

A15, a DNA aptamer with binding specificity for U87 glioma cells stably overexpressing the epidermal growth factor receptor variant III (U87-EGFRvIII), was generated by cell systematic evolution of ligands by exponential enrichment (cell-SELEX) using a random nucleotide library. Subsequently, we established a cell enzyme-linked assay (cell-ELA) to detect the affinity of A15 compared to an EGFR antibody. We used A15 as a detection probe and cultured U87-EGFRvIII cells as targets. Our data indicate that the equilibrium dissociation constants (K(d)) for A15 were below 100 nmol/L and had similar affinity compared to an EGFR antibody for U87-EGFRvIII. We demonstrated that the cell-ELA was a useful method to determine the equilibrium dissociation constants (K(d)) of aptamers generated by cell-SELEX.

[Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

PubMed

Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

2017-04-12

Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

Captured metagenomics: large-scale targeting of genes based on ‘sequence capture’ reveals functional diversity in soils

PubMed Central

Manoharan, Lokeshwaran; Kushwaha, Sandeep K.; Hedlund, Katarina; Ahrén, Dag

2015-01-01

Microbial enzyme diversity is a key to understand many ecosystem processes. Whole metagenome sequencing (WMG) obtains information on functional genes, but it is costly and inefficient due to large amount of sequencing that is required. In this study, we have applied a captured metagenomics technique for functional genes in soil microorganisms, as an alternative to WMG. Large-scale targeting of functional genes, coding for enzymes related to organic matter degradation, was applied to two agricultural soil communities through captured metagenomics. Captured metagenomics uses custom-designed, hybridization-based oligonucleotide probes that enrich functional genes of interest in metagenomic libraries where only probe-bound DNA fragments are sequenced. The captured metagenomes were highly enriched with targeted genes while maintaining their target diversity and their taxonomic distribution correlated well with the traditional ribosomal sequencing. The captured metagenomes were highly enriched with genes related to organic matter degradation; at least five times more than similar, publicly available soil WMG projects. This target enrichment technique also preserves the functional representation of the soils, thereby facilitating comparative metagenomics projects. Here, we present the first study that applies the captured metagenomics approach in large scale, and this novel method allows deep investigations of central ecosystem processes by studying functional gene abundances. PMID:26490729

MTTE: an innovative strategy for the evaluation of targeted/exome enrichment efficiency

PubMed Central

Klonowska, Katarzyna; Handschuh, Luiza; Swiercz, Aleksandra; Figlerowicz, Marek; Kozlowski, Piotr

2016-01-01

Although currently available strategies for the preparation of exome-enriched libraries are well established, a final validation of the libraries in terms of exome enrichment efficiency prior to the sequencing step is of considerable importance. Here, we present a strategy for the evaluation of exome enrichment, i.e., the Multipoint Test for Targeted-enrichment Efficiency (MTTE), PCR-based approach utilizing multiplex ligation-dependent probe amplification with capillary electrophoresis separation. We used MTTE for the analysis of subsequent steps of the Illumina TruSeq Exome Enrichment procedure. The calculated values of enrichment-associated parameters (i.e., relative enrichment, relative clearance, overall clearance, and fold enrichment) and the comparison of MTTE results with the actual enrichment revealed the high reliability of our assay. Additionally, the MTTE assay enabled the determination of the sequence-associated features that may confer bias in the enrichment of different targets. Importantly, the MTTE is low cost method that can be easily adapted to the region of interest important for a particular project. Thus, the MTTE strategy is attractive for post-capture validation in a variety of targeted/exome enrichment NGS projects. PMID:27572310

MTTE: an innovative strategy for the evaluation of targeted/exome enrichment efficiency.

PubMed

Klonowska, Katarzyna; Handschuh, Luiza; Swiercz, Aleksandra; Figlerowicz, Marek; Kozlowski, Piotr

2016-10-11

Although currently available strategies for the preparation of exome-enriched libraries are well established, a final validation of the libraries in terms of exome enrichment efficiency prior to the sequencing step is of considerable importance. Here, we present a strategy for the evaluation of exome enrichment, i.e., the Multipoint Test for Targeted-enrichment Efficiency (MTTE), PCR-based approach utilizing multiplex ligation-dependent probe amplification with capillary electrophoresis separation. We used MTTE for the analysis of subsequent steps of the Illumina TruSeq Exome Enrichment procedure. The calculated values of enrichment-associated parameters (i.e., relative enrichment, relative clearance, overall clearance, and fold enrichment) and the comparison of MTTE results with the actual enrichment revealed the high reliability of our assay. Additionally, the MTTE assay enabled the determination of the sequence-associated features that may confer bias in the enrichment of different targets. Importantly, the MTTE is low cost method that can be easily adapted to the region of interest important for a particular project. Thus, the MTTE strategy is attractive for post-capture validation in a variety of targeted/exome enrichment NGS projects.

[Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

PubMed

Chen, Xiao-hong; Chen, Zhi; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan; Yao, Hang-ping

2005-03-01

To construct a cDNA library from human liver tissue of cirrhosis. The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

DNA-encoded libraries - an efficient small molecule discovery technology for the biomedical sciences.

PubMed

Kunig, Verena; Potowski, Marco; Gohla, Anne; Brunschweiger, Andreas

2018-06-27

DNA-encoded compound libraries are a highly attractive technology for the discovery of small molecule protein ligands. These compound collections consist of small molecules covalently connected to individual DNA sequences carrying readable information about the compound structure. DNA-tagging allows for efficient synthesis, handling and interrogation of vast numbers of chemically synthesized, drug-like compounds. They are screened on proteins by an efficient, generic assay based on Darwinian principles of selection. To date, selection of DNA-encoded libraries allowed for the identification of numerous bioactive compounds. Some of these compounds uncovered hitherto unknown allosteric binding sites on target proteins; several compounds proved their value as chemical biology probes unraveling complex biology; and the first examples of clinical candidates that trace their ancestry to a DNA-encoded library were reported. Thus, DNA-encoded libraries proved their value for the biomedical sciences as a generic technology for the identification of bioactive drug-like molecules numerous times. However, large scale experiments showed that even the selection of billions of compounds failed to deliver bioactive compounds for the majority of proteins in an unbiased panel of target proteins. This raises the question of compound library design.

A Glimpse into the Satellite DNA Library in Characidae Fish (Teleostei, Characiformes)

PubMed Central

Utsunomia, Ricardo; Ruiz-Ruano, Francisco J.; Silva, Duílio M. Z. A.; Serrano, Érica A.; Rosa, Ivana F.; Scudeler, Patrícia E. S.; Hashimoto, Diogo T.; Oliveira, Claudio; Camacho, Juan Pedro M.; Foresti, Fausto

2017-01-01

Satellite DNA (satDNA) is an abundant fraction of repetitive DNA in eukaryotic genomes and plays an important role in genome organization and evolution. In general, satDNA sequences follow a concerted evolutionary pattern through the intragenomic homogenization of different repeat units. In addition, the satDNA library hypothesis predicts that related species share a series of satDNA variants descended from a common ancestor species, with differential amplification of different satDNA variants. The finding of a same satDNA family in species belonging to different genera within Characidae fish provided the opportunity to test both concerted evolution and library hypotheses. For this purpose, we analyzed here sequence variation and abundance of this satDNA family in ten species, by a combination of next generation sequencing (NGS), PCR and Sanger sequencing, and fluorescence in situ hybridization (FISH). We found extensive between-species variation for the number and size of pericentromeric FISH signals. At genomic level, the analysis of 1000s of DNA sequences obtained by Illumina sequencing and PCR amplification allowed defining 150 haplotypes which were linked in a common minimum spanning tree, where different patterns of concerted evolution were apparent. This also provided a glimpse into the satDNA library of this group of species. In consistency with the library hypothesis, different variants for this satDNA showed high differences in abundance between species, from highly abundant to simply relictual variants. PMID:28855916

A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

PubMed Central

Friis, Thor Einar; Stephenson, Sally; Xiao, Yin; Whitehead, Jon

2014-01-01

The sheep (Ovis aries) is favored by many musculoskeletal tissue engineering groups as a large animal model because of its docile temperament and ease of husbandry. The size and weight of sheep are comparable to humans, which allows for the use of implants and fixation devices used in human clinical practice. The construction of a complimentary DNA (cDNA) library can capture the expression of genes in both a tissue- and time-specific manner. cDNA libraries have been a consistent source of gene discovery ever since the technology became commonplace more than three decades ago. Here, we describe the construction of a cDNA library using cells derived from sheep bones based on the pBluescript cDNA kit. Thirty clones were picked at random and sequenced. This led to the identification of a novel gene, C12orf29, which our initial experiments indicate is involved in skeletal biology. We also describe a polymerase chain reaction-based cDNA clone isolation method that allows the isolation of genes of interest from a cDNA library pool. The techniques outlined here can be applied in-house by smaller tissue engineering groups to generate tools for biomolecular research for large preclinical animal studies and highlights the power of standard cDNA library protocols to uncover novel genes. PMID:24447069

Novel selection methods for DNA-encoded chemical libraries

PubMed Central

Chan, Alix I.; McGregor, Lynn M.; Liu, David R.

2015-01-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. PMID:25723146

STK-1, the human homolog of Flk-2/Flt-3, is selectively expressed in CD34+ human bone marrow cells and is involved in the proliferation of early progenitor/stem cells.

PubMed Central

Small, D; Levenstein, M; Kim, E; Carow, C; Amin, S; Rockwell, P; Witte, L; Burrow, C; Ratajczak, M Z; Gewirtz, A M

1994-01-01

We cloned the cDNA for stem cell tyrosine kinase 1 (STK-1), the human homolog of murine Flk-2/Flt-3, from a CD34+ hematopoietic stem cell-enriched library and investigated its expression in subsets of normal human bone marrow. The cDNA encodes a protein of 993 aa with 85% identity and 92% similarity to Flk-2/Flt-3. STK-1 is a member of the type III receptor tyrosine kinase family that includes KIT (steel factor receptor), FMS (colony-stimulating factor 1R), and platelet-derived growth factor receptor. STK-1 expression in human blood and marrow is restricted to CD34+ cells, a population greatly enriched for stem/progenitor cells. Anti-STK-1 antiserum recognizes polypeptides of 160 and 130 kDa in several STK-1-expressing cell lines and in 3T3 cells transfected with a STK-1 expression vector. Antisense oligonucleotides directed against STK-1 sequences inhibited hematopoietic colony formation, most strongly in long-term bone marrow cultures. These data suggest that STK-1 may function as a growth factor receptor on hematopoietic stem and/or progenitor cells. Images Fig. 2 Fig. 3 Fig. 4 PMID:7507245

Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

PubMed

Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

1993-01-01

Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss-cDNAs. This strategy is referred to as SLIC for single-strand ligation to ss-cDNA (8).

Population-based rare variant detection via pooled exome or custom hybridization capture with or without individual indexing.

PubMed

Ramos, Enrique; Levinson, Benjamin T; Chasnoff, Sara; Hughes, Andrew; Young, Andrew L; Thornton, Katherine; Li, Allie; Vallania, Francesco L M; Province, Michael; Druley, Todd E

2012-12-06

Rare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign. We report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22-48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome. This highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sensitivity.

Procedure for normalization of cDNA libraries

DOEpatents

Bonaldo, Maria DeFatima; Soares, Marcelo Bento

1997-01-01

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

EPA Science Inventory

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based ...

PrecisePrimer: an easy-to-use web server for designing PCR primers for DNA library cloning and DNA shuffling.

PubMed

Pauthenier, Cyrille; Faulon, Jean-Loup

2014-07-01

PrecisePrimer is a web-based primer design software made to assist experimentalists in any repetitive primer design task such as preparing, cloning and shuffling DNA libraries. Unlike other popular primer design tools, it is conceived to generate primer libraries with popular PCR polymerase buffers proposed as pre-set options. PrecisePrimer is also meant to design primers in batches, such as for DNA libraries creation of DNA shuffling experiments and to have the simplest interface possible. It integrates the most up-to-date melting temperature algorithms validated with experimental data, and cross validated with other computational tools. We generated a library of primers for the extraction and cloning of 61 genes from yeast DNA genomic extract using default parameters. All primer pairs efficiently amplified their target without any optimization of the PCR conditions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Development and Synthesis of DNA-Encoded Benzimidazole Library.

PubMed

Ding, Yun; Chai, Jing; Centrella, Paolo A; Gondo, Chenaimwoyo; DeLorey, Jennifer L; Clark, Matthew A

2018-04-25

Encoded library technology (ELT) is an effective approach to the discovery of novel small-molecule ligands for biological targets. A key factor for the success of the technology is the chemical diversity of the libraries. Here we report the development of DNA-conjugated benzimidazoles. Using 4-fluoro-3-nitrobenzoic acid as a key synthon, we synthesized a 320 million-member DNA-encoded benzimidazole library using Fmoc-protected amino acids, amines and aldehydes as diversity elements. Affinity selection of the library led to the discovery of a novel, potent and specific antagonist of the NK3 receptor.

Developing new microsatellite markers in walnut (Juglans regia L.) from Juglans nigra genomic GA enriched library

Treesearch

Hayat Topcu; Nergiz Coban; Keith Woeste; Mehmet Sutyemez; Salih Kafkas

2015-01-01

We attempted to develop new polymorphic SSR primer pairs in walnut using sequences derived from Juglans nigra L. genomic enriched library with GA repeat. The designed 94 SSR primer pairs were subjected to gradient PCR in 12 walnut cultivars to determine their optimum annealing temperatures and to determine whether they produce bands. Then, the...

Novel selection methods for DNA-encoded chemical libraries.

PubMed

Chan, Alix I; McGregor, Lynn M; Liu, David R

2015-06-01

Driven by the need for new compounds to serve as biological probes and leads for therapeutic development and the growing accessibility of DNA technologies including high-throughput sequencing, many academic and industrial groups have begun to use DNA-encoded chemical libraries as a source of bioactive small molecules. In this review, we describe the technologies that have enabled the selection of compounds with desired activities from these libraries. These methods exploit the sensitivity of in vitro selection coupled with DNA amplification to overcome some of the limitations and costs associated with conventional screening methods. In addition, we highlight newer techniques with the potential to be applied to the high-throughput evaluation of DNA-encoded chemical libraries. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcriptome analysis of zebrafish embryos exposed to deltamethrin.

PubMed

Chueh, Tsung-Cheng; Hsu, Li-Sung; Kao, Chin-Ming; Hsu, Tung-Wei; Liao, Hung-Yu; Wang, Kuan-Yi; Chen, Ssu Ching

2017-05-01

Deltamethrin (DTM), a type II pyrethroid, is one of the most commonly used insecticides. The increased use of pyrethroid leads to potential adverse effects, particularly in sensitive populations such as children and pregnant women. None of the related studies was focused on the transcriptome responses in zebrafish embryos after treatment with DTM; therefore, RNA-seq, a high-throughput method, was performed to analyze the global expression of differential expressed genes (DEGs) in zebrafish embryos treated with DTM (40 and 80 μg/L) from fertilization to 48 h postfertilization (hpf) as compared with that in the control group (without DTM treatment). Two cDNA libraries were generated from treated embryos and one cDNA library from nontreated embryos, respectively. Over 92% of reads mapped to the reference in these three libraries. It was observed that many differential genes were expressed in comparison with embryos before and after DTM. The 20 most differentially expressed upregulated or downregulated genes were majorly involved in the signaling transduction. Validation of selected nine genes expression using qRT-PCR confirmed RNA-seq results. The transcriptome sequences were further subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, showing G-protein-coupled receptor signaling pathway and neuroactive ligand-receptor interaction, respectively, were most enriched. The data from this study contributed to a better understanding of the potential consequences of fish exposed to DTM, to an evaluation of the potential threat of DTM to fish populations in aquatic environments. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1548-1557, 2017. © 2016 Wiley Periodicals, Inc.

An In Vitro Translation, Selection, and Amplification System for Peptide Nucleic Acids

PubMed Central

Brudno, Yevgeny; Birnbaum, Michael E.; Kleiner, Ralph E.; Liu, David R.

2009-01-01

Methods to evolve synthetic, rather than biological, polymers could significantly expand the functional potential of polymers that emerge from in vitro evolution. Requirements for synthetic polymer evolution include: (i) sequence-specific polymerization of synthetic building blocks on an amplifiable template; (ii) display of the newly translated polymer strand in a manner that allows it to adopt folded structures; (iii) selection of synthetic polymer libraries for desired binding or catalytic properties; and (iv) amplification of template sequences surviving selection in a manner that allows subsequent translation. Here we report the development of such a system for peptide nucleic acids (PNAs) using a set of twelve PNA pentamer building blocks. We validated the system by performing six iterated cycles of translation, selection, and amplification on a library of 4.3 × 108 PNA-encoding DNA templates and observed >1,000,000-fold overall enrichment of a template encoding a biotinylated (streptavidin-binding) PNA. These results collectively provide an experimental foundation for PNA evolution in the laboratory. PMID:20081830

Procedure for normalization of cDNA libraries

DOEpatents

Bonaldo, M.D.; Soares, M.B.

1997-12-30

This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library. 1 fig.

Lab-on-a-chip platform for high throughput drug discovery with DNA-encoded chemical libraries

NASA Astrophysics Data System (ADS)

Grünzner, S.; Reddavide, F. V.; Steinfelder, C.; Cui, M.; Busek, M.; Klotzbach, U.; Zhang, Y.; Sonntag, F.

2017-02-01

The fast development of DNA-encoded chemical libraries (DECL) in the past 10 years has received great attention from pharmaceutical industries. It applies the selection approach for small molecular drug discovery. Because of the limited choices of DNA-compatible chemical reactions, most DNA-encoded chemical libraries have a narrow structural diversity and low synthetic yield. There is also a poor correlation between the ranking of compounds resulted from analyzing the sequencing data and the affinity measured through biochemical assays. By combining DECL with dynamical chemical library, the resulting DNA-encoded dynamic library (EDCCL) explores the thermodynamic equilibrium of reversible reactions as well as the advantages of DNA encoded compounds for manipulation/detection, thus leads to enhanced signal-to-noise ratio of the selection process and higher library quality. However, the library dynamics are caused by the weak interactions between the DNA strands, which also result in relatively low affinity of the bidentate interaction, as compared to a stable DNA duplex. To take advantage of both stably assembled dual-pharmacophore libraries and EDCCLs, we extended the concept of EDCCLs to heat-induced EDCCLs (hi-EDCCLs), in which the heat-induced recombination process of stable DNA duplexes and affinity capture are carried out separately. To replace the extremely laborious and repetitive manual process, a fully automated device will facilitate the use of DECL in drug discovery. Herein we describe a novel lab-on-a-chip platform for high throughput drug discovery with hi-EDCCL. A microfluidic system with integrated actuation was designed which is able to provide a continuous sample circulation by reducing the volume to a minimum. It consists of a cooled and a heated chamber for constant circulation. The system is capable to generate stable temperatures above 75 °C in the heated chamber to melt the double strands of the DNA and less than 15 °C in the cooled chamber, to reanneal the reshuffled library. In the binding chamber (the cooled chamber) specific retaining structures are integrated. These hold back beads functionalized with the target protein, while the chamber is continuously flushed with library molecules. Afterwards the whole system can be flushed with buffer to wash out unspecific bound molecules. Finally the protein-loaded beads with attached molecules can be eluted for further investigation.

Development of a fraction collection approach in capillary electrophoresis SELEX for aptamer selection.

PubMed

Luo, Zhaofeng; Zhou, Hongmin; Jiang, Hao; Ou, Huichao; Li, Xin; Zhang, Liyun

2015-04-21

Aptamers have attracted much attention due to their ability to bind to target molecules with high affinity and specificity. The development of an approach capable of efficiently generating aptamers through systematic evolution of ligands by exponential enrichment (SELEX) is particularly challenging. Herein, a fraction collection approach in capillary electrophoresis SELEX (FCE-SELEX) for the partition of a bound DNA-target complex is developed. By integrating fraction collection with a facile oil seal method for avoiding contamination while amplifying the bound DNA-target complex, in a single round of selection, a streptavidin-binding aptamer (SBA) has been generated. The affinity of aptamer SBA-36 for streptavidin (SA) is determined as 30.8 nM by surface plasmon resonance (SPR). Selectivity and biotin competition experiments demonstrate that the SBA-36 aptamer selected by FCE-SELEX is as efficient as those from other methods. Based on the ability of fraction collection in partition and collection of the aptamer-target complex from the original DNA library, FCE-SELEX can be a universal tool for the development of aptamers.

Discovery of a trans-Dichloroethene-Respiring Dehalogenimonas Species in the 1,1,2,2-Tetrachloroethane-Dechlorinating WBC-2 Consortium

PubMed Central

Manchester, Marie J.; Hug, Laura A.; Zarek, Matt; Zila, Anna

2012-01-01

The WBC-2 consortium is an organohalide-respiring anaerobic microbial enrichment culture capable of dechlorinating 1,1,2,2-tetrachloroethane (TeCA) to ethene. In the WBC-2 culture, TeCA is first transformed to trans-dichloroethene (tDCE) by dichloroelimination; tDCE is subsequently transformed to vinyl chloride (VC) and then to ethene by hydrogenolysis. Analysis of 16S rRNA gene clone libraries from culture DNA revealed sequences from three putative dechlorinating organisms belonging to Dehalococcoides, Dehalobacter, and Dehalogenimonas genera. Quantitative PCR primers were designed for each of these sequences, and their abundance was quantified in enrichment cultures over time. These data revealed that complete dechlorination of TeCA to ethene involves all three organisms. Dehalobacter spp. grew during the dihaloelimination of TeCA to tDCE, while Dehalococcoides and Dehalogenimonas spp. grew during hydrogenolysis of tDCE to ethene. This is the first time a genus other than Dehalococcoides has been implicated in dechlorination of tDCE to VC. PMID:22635995

Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay.

PubMed

Dolati, Somayeh; Ramezani, Mohammad; Nabavinia, Maryam Sadat; Soheili, Vahid; Abnous, Khalil; Taghdisi, Seyed Mohammad

2018-05-15

Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (K d  = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk. Copyright © 2018 Elsevier Inc. All rights reserved.

Identification of Isopentenol Biosynthetic Genes from Bacillus subtilis by a Screening Method Based on Isoprenoid Precursor Toxicity▿

PubMed Central

Withers, Sydnor T.; Gottlieb, Shayin S.; Lieu, Bonny; Newman, Jack D.; Keasling, Jay D.

2007-01-01

We have developed a novel method to clone terpene synthase genes. This method relies on the inherent toxicity of the prenyl diphosphate precursors to terpenes, which resulted in a reduced-growth phenotype. When these precursors were consumed by a terpene synthase, normal growth was restored. We have demonstrated that this method is capable of enriching a population of engineered Escherichia coli for those clones that express the sesquiterpene-producing amorphadiene synthase. In addition, we enriched a library of genomic DNA from the isoprene-producing bacterium Bacillus subtilis strain 6051 in E. coli engineered to produce elevated levels of isopentenyl diphosphate and dimethylallyl diphosphate. The selection resulted in the discovery of two genes (yhfR and nudF) whose protein products acted directly on the prenyl diphosphate precursors and produced isopentenol. Expression of nudF in E. coli engineered with the mevalonate-based isopentenyl pyrophosphate biosynthetic pathway resulted in the production of isopentenol. PMID:17693564

A method for high-throughput production of sequence-verified DNA libraries and strain collections.

PubMed

Smith, Justin D; Schlecht, Ulrich; Xu, Weihong; Suresh, Sundari; Horecka, Joe; Proctor, Michael J; Aiyar, Raeka S; Bennett, Richard A O; Chu, Angela; Li, Yong Fuga; Roy, Kevin; Davis, Ronald W; Steinmetz, Lars M; Hyman, Richard W; Levy, Sasha F; St Onge, Robert P

2017-02-13

The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Rapid and efficient cDNA library screening by self-ligation of inverse PCR products (SLIP).

PubMed

Hoskins, Roger A; Stapleton, Mark; George, Reed A; Yu, Charles; Wan, Kenneth H; Carlson, Joseph W; Celniker, Susan E

2005-12-02

cDNA cloning is a central technology in molecular biology. cDNA sequences are used to determine mRNA transcript structures, including splice junctions, open reading frames (ORFs) and 5'- and 3'-untranslated regions (UTRs). cDNA clones are valuable reagents for functional studies of genes and proteins. Expressed Sequence Tag (EST) sequencing is the method of choice for recovering cDNAs representing many of the transcripts encoded in a eukaryotic genome. However, EST sequencing samples a cDNA library at random, and it recovers transcripts with low expression levels inefficiently. We describe a PCR-based method for directed screening of plasmid cDNA libraries. We demonstrate its utility in a screen of libraries used in our Drosophila EST projects for 153 transcription factor genes that were not represented by full-length cDNA clones in our Drosophila Gene Collection. We recovered high-quality, full-length cDNAs for 72 genes and variously compromised clones for an additional 32 genes. The method can be used at any scale, from the isolation of cDNA clones for a particular gene of interest, to the improvement of large gene collections in model organisms and the human. Finally, we discuss the relative merits of directed cDNA library screening and RT-PCR approaches.

Hydrocarbon-degrading bacteria enriched by the Deepwater Horizon oil spill identified by cultivation and DNA-SIP

PubMed Central

Gutierrez, Tony; Singleton, David R; Berry, David; Yang, Tingting; Aitken, Michael D; Teske, Andreas

2013-01-01

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales, Cycloclasticus and Colwellia, were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly 13C-labelled hydrocarbons, we identified several aliphatic (Alcanivorax, Marinobacter)- and polycyclic aromatic hydrocarbon (Alteromonas, Cycloclasticus, Colwellia)-degrading bacteria. We also isolated several strains (Alcanivorax, Alteromonas, Cycloclasticus, Halomonas, Marinobacter and Pseudoalteromonas) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil. PMID:23788333

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens.

PubMed

Saeidi, Saman; McKain, Michael R; Kellogg, Elizabeth A

2018-03-08

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

PubMed

Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

2015-06-01

Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library*

PubMed Central

Kiefer, Jonathan D.; Srinivas, Raja R.; Lobner, Elisabeth; Tisdale, Alison W.; Mehta, Naveen K.; Yang, Nicole J.; Tidor, Bruce; Wittrup, K. Dane

2016-01-01

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (Tm of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. PMID:27582495

Construction of a genomic DNA library with a TA vector and its application in cloning of the phytoene synthase gene from the cyanobacterium Spirulina platensis M-135

NASA Astrophysics Data System (ADS)

Yoshikazu, Kawata; Shin-Ichi, Yano; Hiroyuki, Kojima

1998-03-01

An efficient and simple method for constructing a genomic DNA library using a TA cloning vector is presented. It is based on the sonicative cleavage of genomic DNA and modification of fragment ends with Taq DNA polymerase, followed by ligation using a TA vector. This method was applied for cloning of the phytoene synthase gene crt B from Spirulina platensis. This method is useful when genomic DNA cannot be efficiently digested with restriction enzymes, a problem often encountered during the construction of a genomic DNA library of cyanobacteria.

Ligation Bias in Illumina Next-Generation DNA Libraries: Implications for Sequencing Ancient Genomes

PubMed Central

Seguin-Orlando, Andaine; Schubert, Mikkel; Clary, Joel; Stagegaard, Julia; Alberdi, Maria T.; Prado, José Luis; Prieto, Alfredo; Willerslev, Eske; Orlando, Ludovic

2013-01-01

Ancient DNA extracts consist of a mixture of endogenous molecules and contaminant DNA templates, often originating from environmental microbes. These two populations of templates exhibit different chemical characteristics, with the former showing depurination and cytosine deamination by-products, resulting from post-mortem DNA damage. Such chemical modifications can interfere with the molecular tools used for building second-generation DNA libraries, and limit our ability to fully characterize the true complexity of ancient DNA extracts. In this study, we first use fresh DNA extracts to demonstrate that library preparation based on adapter ligation at AT-overhangs are biased against DNA templates starting with thymine residues, contrarily to blunt-end adapter ligation. We observe the same bias on fresh DNA extracts sheared on Bioruptor, Covaris and nebulizers. This contradicts previous reports suggesting that this bias could originate from the methods used for shearing DNA. This also suggests that AT-overhang adapter ligation efficiency is affected in a sequence-dependent manner and results in an uneven representation of different genomic contexts. We then show how this bias could affect the base composition of ancient DNA libraries prepared following AT-overhang ligation, mainly by limiting the ability to ligate DNA templates starting with thymines and therefore deaminated cytosines. This results in particular nucleotide misincorporation damage patterns, deviating from the signature generally expected for authenticating ancient sequence data. Consequently, we show that models adequate for estimating post-mortem DNA damage levels must be robust to the molecular tools used for building ancient DNA libraries. PMID:24205269

Immune-Related Transcriptome of Coptotermes formosanus Shiraki Workers: The Defense Mechanism

PubMed Central

Hussain, Abid; Li, Yi-Feng; Cheng, Yu; Liu, Yang; Chen, Chuan-Cheng; Wen, Shuo-Yang

2013-01-01

Formosan subterranean termites, Coptotermes formosanus Shiraki, live socially in microbial-rich habitats. To understand the molecular mechanism by which termites combat pathogenic microbes, a full-length normalized cDNA library and four Suppression Subtractive Hybridization (SSH) libraries were constructed from termite workers infected with entomopathogenic fungi (Metarhizium anisopliae and Beauveria bassiana), Gram-positive Bacillus thuringiensis and Gram-negative Escherichia coli, and the libraries were analyzed. From the high quality normalized cDNA library, 439 immune-related sequences were identified. These sequences were categorized as pattern recognition receptors (47 sequences), signal modulators (52 sequences), signal transducers (137 sequences), effectors (39 sequences) and others (164 sequences). From the SSH libraries, 27, 17, 22 and 15 immune-related genes were identified from each SSH library treated with M. anisopliae, B. bassiana, B. thuringiensis and E. coli, respectively. When the normalized cDNA library was compared with the SSH libraries, 37 immune-related clusters were found in common; 56 clusters were identified in the SSH libraries, and 259 were identified in the normalized cDNA library. The immune-related gene expression pattern was further investigated using quantitative real time PCR (qPCR). Important immune-related genes were characterized, and their potential functions were discussed based on the integrated analysis of the results. We suggest that normalized cDNA and SSH libraries enable us to discover functional genes transcriptome. The results remarkably expand our knowledge about immune-inducible genes in C. formosanus Shiraki and enable the future development of novel control strategies for the management of Formosan subterranean termites. PMID:23874972

Analysis of Current DNA Encoded Library Screening Data Indicates Higher False Negative Rates for Numerically Larger Libraries.

PubMed

Satz, Alexander L; Hochstrasser, Remo; Petersen, Ann C

2017-04-10

To optimize future DNA-encoded library design, we have attempted to quantify the library size at which the signal becomes undetectable. To accomplish this we (i) have calculated that percent yields of individual library members following a screen range from 0.002 to 1%, (ii) extrapolated that ∼1 million copies per library member are required at the outset of a screen, and (iii) from this extrapolation predict that false negative rates will begin to outweigh the benefit of increased diversity at library sizes >10 8 . The above analysis is based upon a large internal data set comprising multiple screens, targets, and libraries; we also augmented our internal data with all currently available literature data. In theory, high false negative rates may be overcome by employing larger amounts of library; however, we argue that using more than currently reported amounts of library (≫10 nmoles) is impractical. The above conclusions may be generally applicable to other DNA encoded library platforms, particularly those platforms that do not allow for library amplification.

Assessment of the DNA damaging potential of environmental chemicals using a quantitative high-throughput screening approach to measure p53 activation.

PubMed

Witt, Kristine L; Hsieh, Jui-Hua; Smith-Roe, Stephanie L; Xia, Menghang; Huang, Ruili; Zhao, Jinghua; Auerbach, Scott S; Hur, Junguk; Tice, Raymond R

2017-08-01

Genotoxicity potential is a critical component of any comprehensive toxicological profile. Compounds that induce DNA or chromosomal damage often activate p53, a transcription factor essential to cell cycle regulation. Thus, within the US Tox21 Program, we screened a library of ∼10,000 (∼8,300 unique) environmental compounds and drugs for activation of the p53-signaling pathway using a quantitative high-throughput screening assay employing HCT-116 cells (p53 +/+ ) containing a stably integrated β-lactamase reporter gene under control of the p53 response element (p53RE). Cells were exposed (-S9) for 16 hr at 15 concentrations (generally 1.2 nM to 92 μM) three times, independently. Excluding compounds that failed analytical chemistry analysis or were suspected of inducing assay interference, 365 (4.7%) of 7,849 unique compounds were concluded to activate p53. As part of an in-depth characterization of our results, we first compared them with results from traditional in vitro genotoxicity assays (bacterial mutation, chromosomal aberration); ∼15% of known, direct-acting genotoxicants in our library activated the p53RE. Mining the Comparative Toxicogenomics Database revealed that these p53 actives were significantly associated with increased expression of p53 downstream genes involved in DNA damage responses. Furthermore, 53 chemical substructures associated with genotoxicity were enriched in certain classes of p53 actives, for example, anthracyclines (antineoplastics) and vinca alkaloids (tubulin disruptors). Interestingly, the tubulin disruptors manifested unusual nonmonotonic concentration response curves suggesting activity through a unique p53 regulatory mechanism. Through the analysis of our results, we aim to define a role for this assay as one component of a comprehensive toxicological characterization of large compound libraries. Environ. Mol. Mutagen. 58:494-507, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Combined Use of 16S Ribosomal DNA and 16S rRNA To Study the Bacterial Community of Polychlorinated Biphenyl-Polluted Soil

PubMed Central

Nogales, Balbina; Moore, Edward R. B.; Llobet-Brossa, Enrique; Rossello-Mora, Ramon; Amann, Rudolf; Timmis, Kenneth N.

2001-01-01

The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study. PMID:11282645

Twenty-five Years of DNA-Encoded Chemical Libraries.

PubMed

Neri, Dario

2017-05-04

Reference library: The availability of DNA-encoded chemical libraries containing billions of compounds facilitates the discovery of binding molecules for pharmaceutical applications and for investigating biological processes. This Special Issue highlights the use of this library technology and some of the latest developments in the field. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optimized Reaction Conditions for Amide Bond Formation in DNA-Encoded Combinatorial Libraries.

PubMed

Li, Yizhou; Gabriele, Elena; Samain, Florent; Favalli, Nicholas; Sladojevich, Filippo; Scheuermann, Jörg; Neri, Dario

2016-08-08

DNA-encoded combinatorial libraries are increasingly being used as tools for the discovery of small organic binding molecules to proteins of biological or pharmaceutical interest. In the majority of cases, synthetic procedures for the formation of DNA-encoded combinatorial libraries incorporate at least one step of amide bond formation between amino-modified DNA and a carboxylic acid. We investigated reaction conditions and established a methodology by using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, 1-hydroxy-7-azabenzotriazole and N,N'-diisopropylethylamine (EDC/HOAt/DIPEA) in combination, which provided conversions greater than 75% for 423/543 (78%) of the carboxylic acids tested. These reaction conditions were efficient with a variety of primary and secondary amines, as well as with various types of amino-modified oligonucleotides. The reaction conditions, which also worked efficiently over a broad range of DNA concentrations and reaction scales, should facilitate the synthesis of novel DNA-encoded combinatorial libraries.

Deep Subsurface Life from North Pond: Enrichment, Isolation, Characterization and Genomes of Heterotrophic Bacteria

PubMed Central

Russell, Joseph A.; León-Zayas, Rosa; Wrighton, Kelly; Biddle, Jennifer F.

2016-01-01

Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic water-column west of the Mid-Atlantic Ridge at 22°N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. The cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface. PMID:27242705

Deep Subsurface Life from North Pond: Enrichment, Isolation, Characterization and Genomes of Heterotrophic Bacteria.

PubMed

Russell, Joseph A; León-Zayas, Rosa; Wrighton, Kelly; Biddle, Jennifer F

2016-01-01

Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic water-column west of the Mid-Atlantic Ridge at 22°N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. The cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface.

Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis.

PubMed

Danhorn, Thomas; Young, Curtis R; DeLong, Edward F

2012-11-01

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary 'next-generation' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.

Display of a maize cDNA library on baculovirus infected insect cells.

PubMed

Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

2008-08-12

Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

Preparation of fosmid libraries and functional metagenomic analysis of microbial community DNA.

PubMed

Martínez, Asunción; Osburne, Marcia S

2013-01-01

One of the most important challenges in contemporary microbial ecology is to assign a functional role to the large number of novel genes discovered through large-scale sequencing of natural microbial communities that lack similarity to genes of known function. Functional screening of metagenomic libraries, that is, screening environmental DNA clones for the ability to confer an activity of interest to a heterologous bacterial host, is a promising approach for bridging the gap between metagenomic DNA sequencing and functional characterization. Here, we describe methods for isolating environmental DNA and constructing metagenomic fosmid libraries, as well as methods for designing and implementing successful functional screens of such libraries. © 2013 Elsevier Inc. All rights reserved.

Potential for DNA-based identification of Great Lakes fauna: match and mismatch between taxa inventories and DNA barcode libraries.

PubMed

Trebitz, Anett S; Hoffman, Joel C; Grant, George W; Billehus, Tyler M; Pilgrim, Erik M

2015-07-22

DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to bioassessment and non-native species monitoring. The ability to assign species identities to DNA sequences found depends on the availability of comprehensive DNA reference libraries. Here, we compile inventories for aquatic metazoans extant in or threatening to invade the Laurentian Great Lakes and examine the availability of reference mitochondrial COI DNA sequences (barcodes) in the Barcode of Life Data System for them. We found barcode libraries largely complete for extant and threatening-to-invade vertebrates (100% of reptile, 99% of fish, and 92% of amphibian species had barcodes). In contrast, barcode libraries remain poorly developed for precisely those organisms where morphological identification is most challenging; 46% of extant invertebrates lacked reference barcodes with rates especially high among rotifers, oligochaetes, and mites. Lack of species-level identification for many aquatic invertebrates also is a barrier to matching DNA sequences with physical specimens. Attaining the potential for DNA-based identification of mixed-organism samples covering the breadth of aquatic fauna requires a concerted effort to build supporting barcode libraries and voucher collections.

Potential for DNA-based identification of Great Lakes fauna: match and mismatch between taxa inventories and DNA barcode libraries

NASA Astrophysics Data System (ADS)

Trebitz, Anett S.; Hoffman, Joel C.; Grant, George W.; Billehus, Tyler M.; Pilgrim, Erik M.

2015-07-01

DNA-based identification of mixed-organism samples offers the potential to greatly reduce the need for resource-intensive morphological identification, which would be of value both to bioassessment and non-native species monitoring. The ability to assign species identities to DNA sequences found depends on the availability of comprehensive DNA reference libraries. Here, we compile inventories for aquatic metazoans extant in or threatening to invade the Laurentian Great Lakes and examine the availability of reference mitochondrial COI DNA sequences (barcodes) in the Barcode of Life Data System for them. We found barcode libraries largely complete for extant and threatening-to-invade vertebrates (100% of reptile, 99% of fish, and 92% of amphibian species had barcodes). In contrast, barcode libraries remain poorly developed for precisely those organisms where morphological identification is most challenging; 46% of extant invertebrates lacked reference barcodes with rates especially high among rotifers, oligochaetes, and mites. Lack of species-level identification for many aquatic invertebrates also is a barrier to matching DNA sequences with physical specimens. Attaining the potential for DNA-based identification of mixed-organism samples covering the breadth of aquatic fauna requires a concerted effort to build supporting barcode libraries and voucher collections.

Easy preparation of a large-size random gene mutagenesis library in Escherichia coli.

PubMed

You, Chun; Percival Zhang, Y-H

2012-09-01

A simple and fast protocol for the preparation of a large-size mutant library for directed evolution in Escherichia coli was developed based on the DNA multimers generated by prolonged overlap extension polymerase chain reaction (POE-PCR). This protocol comprised the following: (i) a linear DNA mutant library was generated by error-prone PCR or shuffling, and a linear vector backbone was prepared by regular PCR; (ii) the DNA multimers were generated based on these two DNA templates by POE-PCR; and (iii) the one restriction enzyme-digested DNA multimers were ligated to circular plasmids, followed by transformation to E. coli. Because the ligation efficiency of one DNA fragment was several orders of magnitude higher than that of two DNA fragments for typical mutant library construction, it was very easy to generate a mutant library with a size of more than 10(7) protein mutants per 50 μl of the POE-PCR product. Via this method, four new fluorescent protein mutants were obtained based on monomeric cherry fluorescent protein. This new protocol was simple and fast because it did not require labor-intensive optimizations in restriction enzyme digestion and ligation, did not involve special plasmid design, and enabled constructing a large-size mutant library for directed enzyme evolution within 1 day. Copyright © 2012 Elsevier Inc. All rights reserved.

DNA-Compatible Nitro Reduction and Synthesis of Benzimidazoles.

PubMed

Du, Huang-Chi; Huang, Hongbing

2017-10-18

DNA-encoded chemical libraries have emerged as a cost-effective alternative to high-throughput screening (HTS) for hit identification in drug discovery. A key factor for productive DNA-encoded libraries is the chemical diversity of the small molecule moiety attached to an encoding DNA oligomer. The library structure diversity is often limited to DNA-compatible chemical reactions in aqueous media. Herein, we describe a facile process for reducing aryl nitro groups to aryl amines. The new protocol offers simple operation and circumvents the pyrophoric potential of the conventional method (Raney nickel). The reaction is performed in aqueous solution and does not compromise DNA structural integrity. The utility of this method is demonstrated by the versatile synthesis of benzimidazoles on DNA.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

PubMed

Wu, Zining; Graybill, Todd L; Zeng, Xin; Platchek, Michael; Zhang, Jean; Bodmer, Vera Q; Wisnoski, David D; Deng, Jianghe; Coppo, Frank T; Yao, Gang; Tamburino, Alex; Scavello, Genaro; Franklin, G Joseph; Mataruse, Sibongile; Bedard, Katie L; Ding, Yun; Chai, Jing; Summerfield, Jennifer; Centrella, Paolo A; Messer, Jeffrey A; Pope, Andrew J; Israel, David I

2015-12-14

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter

PubMed Central

Zhu, Bo; Mizoguchi, Takuro; Kojima, Takaaki; Nakano, Hideo

2015-01-01

The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases. PMID:25993095

Rapid isolation of novel FK506 binding proteins from multiple organisms using gDNA and cDNA T7 phage display.

PubMed

Piggott, Andrew M; Kriegel, Alison M; Willows, Robert D; Karuso, Peter

2009-10-01

Reverse chemical proteomics using T7 phage display is a powerful technique for identifying cellular receptors of biologically active small molecules. However, to date this method has generally been limited to cDNA libraries constructed from mRNA isolated from eukaryotes. In this paper, we describe the construction of the first prokaryotic T7 phage display libraries from randomly digested Pseudomonas stutzeri and Vibrio fischeri gDNA, as well as a plant cDNA library from Arabidopsis thaliana. We also describe the use of T7 phage display to identify novel proteins from environmental DNA samples using biotinylated FK506 as a model affinity probe.

Robust Sub-nanomolar Library Preparation for High Throughput Next Generation Sequencing.

PubMed

Wu, Wells W; Phue, Je-Nie; Lee, Chun-Ting; Lin, Changyi; Xu, Lai; Wang, Rong; Zhang, Yaqin; Shen, Rong-Fong

2018-05-04

Current library preparation protocols for Illumina HiSeq and MiSeq DNA sequencers require ≥2 nM initial library for subsequent loading of denatured cDNA onto flow cells. Such amounts are not always attainable from samples having a relatively low DNA or RNA input; or those for which a limited number of PCR amplification cycles is preferred (less PCR bias and/or more even coverage). A well-tested sub-nanomolar library preparation protocol for Illumina sequencers has however not been reported. The aim of this study is to provide a much needed working protocol for sub-nanomolar libraries to achieve outcomes as informative as those obtained with the higher library input (≥ 2 nM) recommended by Illumina's protocols. Extensive studies were conducted to validate a robust sub-nanomolar (initial library of 100 pM) protocol using PhiX DNA (as a control), genomic DNA (Bordetella bronchiseptica and microbial mock community B for 16S rRNA gene sequencing), messenger RNA, microRNA, and other small noncoding RNA samples. The utility of our protocol was further explored for PhiX library concentrations as low as 25 pM, which generated only slightly fewer than 50% of the reads achieved under the standard Illumina protocol starting with > 2 nM. A sub-nanomolar library preparation protocol (100 pM) could generate next generation sequencing (NGS) results as robust as the standard Illumina protocol. Following the sub-nanomolar protocol, libraries with initial concentrations as low as 25 pM could also be sequenced to yield satisfactory and reproducible sequencing results.

Utility of GenBank and the Barcode of Life Data Systems (BOLD) for the identification of forensically important Diptera from Belgium and France

PubMed Central

Sonet, Gontran; Jordaens, Kurt; Braet, Yves; Bourguignon, Luc; Dupont, Eréna; Backeljau, Thierry; De Meyer, Marc; Desmyter, Stijn

2013-01-01

Abstract Fly larvae living on dead corpses can be used to estimate post-mortem intervals. The identification of these flies is decisive in forensic casework and can be facilitated by using DNA barcodes provided that a representative and comprehensive reference library of DNA barcodes is available. We constructed a local (Belgium and France) reference library of 85 sequences of the COI DNA barcode fragment (mitochondrial cytochrome c oxidase subunit I gene), from 16 fly species of forensic interest (Calliphoridae, Muscidae, Fanniidae). This library was then used to evaluate the ability of two public libraries (GenBank and the Barcode of Life Data Systems – BOLD) to identify specimens from Belgian and French forensic cases. The public libraries indeed allow a correct identification of most specimens. Yet, some of the identifications remain ambiguous and some forensically important fly species are not, or insufficiently, represented in the reference libraries. Several search options offered by GenBank and BOLD can be used to further improve the identifications obtained from both libraries using DNA barcodes. PMID:24453564

Rapid and continuous magnetic separation in droplet microfluidic devices.

PubMed

Brouzes, Eric; Kruse, Travis; Kimmerling, Robert; Strey, Helmut H

2015-02-07

We present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization. We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries.

Rapid and continuous magnetic separation in droplet microfluidic devices

PubMed Central

Brouzes, Eric; Kruse, Travis; Kimmerling, Robert; Strey, Helmut H.

2015-01-01

We present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization. We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries. PMID:25501881

36 CFR 701.2 - Acquisition of Library material by non-purchase means.

Code of Federal Regulations, 2010 CFR

2010-07-01

... policy of the Library of Congress to foster the enrichment of its collections through gifts of materials within the terms of the Library's acquisitions policies. In implementing this policy, division chiefs and... accompanied by a signed Agreement of Deposit. (2) It is the policy of the Library of Congress to accept...

Construction of a High-Quality Yeast Two-Hybrid Library and Its Application in Identification of Interacting Proteins with Brn1 in Curvularia lunata.

PubMed

Gao, Jin-Xin; Jing, Jing; Yu, Chuan-Jin; Chen, Jie

2015-06-01

Curvularia lunata is an important maize foliar fungal pathogen that distributes widely in maize growing area in China, and several key pathogenic factors have been isolated. An yeast two-hybrid (Y2H) library is a very useful platform to further unravel novel pathogenic factors in C. lunata. To construct a high-quality full length-expression cDNA library from the C. lunata for application to pathogenesis-related protein-protein interaction screening, total RNA was extracted. The SMART (Switching Mechanism At 5' end of the RNA Transcript) technique was used for cDNA synthesis. Double-stranded cDNA was ligated into the pGADT7-Rec vector with Herring Testes Carrier DNA using homologous recombination method. The ligation mixture was transformed into competent yeast AH109 cells to construct the primary cDNA library. Eventually, a high qualitative library was successfully established according to an evaluation on quality. The transformation efficiency was about 6.39 ×10(5) transformants/3 μg pGADT7-Rec. The titer of the primary cDNA library was 2.5×10(8) cfu/mL. The numbers for the cDNA library was 2.46×10(5). Randomly picked clones show that the recombination rate was 88.24%. Gel electrophoresis results indicated that the fragments ranged from 0.4 kb to 3.0 kb. Melanin synthesis protein Brn1 (1,3,8-hydroxynaphthalene reductase) was used as a "bait" to test the sufficiency of the Y2H library. As a result, a cDNA clone encoding VelB protein that was known to be involved in the regulation of diverse cellular processes, including control of secondary metabolism containing melanin and toxin production in many filamentous fungi was identified. Further study on the exact role of the VelB gene is underway.

Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trembath-Reichert, Elizabeth; Case, David H.; Orphan, Victoria J.

Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range ofDeltaproteobacteriadiversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seepmore » sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. In addition, many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1, Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involving Epsilon-, Delta-, and Gammaproteobacteria in methane seep ecosystems.« less

Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments

DOE PAGES

Trembath-Reichert, Elizabeth; Case, David H.; Orphan, Victoria J.

2016-04-18

Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range ofDeltaproteobacteriadiversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seepmore » sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. In addition, many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1, Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involving Epsilon-, Delta-, and Gammaproteobacteria in methane seep ecosystems.« less

Characterization of microbial associations with methanotrophic archaea and sulfate-reducing bacteria through statistical comparison of nested Magneto-FISH enrichments.

PubMed

Trembath-Reichert, Elizabeth; Case, David H; Orphan, Victoria J

2016-01-01

Methane seep systems along continental margins host diverse and dynamic microbial assemblages, sustained in large part through the microbially mediated process of sulfate-coupled Anaerobic Oxidation of Methane (AOM). This methanotrophic metabolism has been linked to consortia of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria (SRB). These two groups are the focus of numerous studies; however, less is known about the wide diversity of other seep associated microorganisms. We selected a hierarchical set of FISH probes targeting a range of Deltaproteobacteria diversity. Using the Magneto-FISH enrichment technique, we then magnetically captured CARD-FISH hybridized cells and their physically associated microorganisms from a methane seep sediment incubation. DNA from nested Magneto-FISH experiments was analyzed using Illumina tag 16S rRNA gene sequencing (iTag). Enrichment success and potential bias with iTag was evaluated in the context of full-length 16S rRNA gene clone libraries, CARD-FISH, functional gene clone libraries, and iTag mock communities. We determined commonly used Earth Microbiome Project (EMP) iTAG primers introduced bias in some common methane seep microbial taxa that reduced the ability to directly compare OTU relative abundances within a sample, but comparison of relative abundances between samples (in nearly all cases) and whole community-based analyses were robust. The iTag dataset was subjected to statistical co-occurrence measures of the most abundant OTUs to determine which taxa in this dataset were most correlated across all samples. Many non-canonical microbial partnerships were statistically significant in our co-occurrence network analysis, most of which were not recovered with conventional clone library sequencing, demonstrating the utility of combining Magneto-FISH and iTag sequencing methods for hypothesis generation of associations within complex microbial communities. Network analysis pointed to many co-occurrences containing putatively heterotrophic, candidate phyla such as OD1, Atribacteria, MBG-B, and Hyd24-12 and the potential for complex sulfur cycling involving Epsilon-, Delta-, and Gammaproteobacteria in methane seep ecosystems.

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

PubMed Central

Martin, Jennifer A.; Smith, Joshua E.; Warren, Mercedes; Chávez, Jorge L.; Hagen, Joshua A.; Kelley-Loughnane, Nancy

2015-01-01

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids. PMID:25870978

Building a DNA barcode library of Alaska's non-marine arthropods.

PubMed

Sikes, Derek S; Bowser, Matthew; Morton, John M; Bickford, Casey; Meierotto, Sarah; Hildebrandt, Kyndall

2017-03-01

Climate change may result in ecological futures with novel species assemblages, trophic mismatch, and mass extinction. Alaska has a limited taxonomic workforce to address these changes. We are building a DNA barcode library to facilitate a metabarcoding approach to monitoring non-marine arthropods. Working with the Canadian Centre for DNA Barcoding, we obtained DNA barcodes from recently collected and authoritatively identified specimens in the University of Alaska Museum (UAM) Insect Collection and the Kenai National Wildlife Refuge collection. We submitted tissues from 4776 specimens, of which 81% yielded DNA barcodes representing 1662 species and 1788 Barcode Index Numbers (BINs), of primarily terrestrial, large-bodied arthropods. This represents 84% of the species available for DNA barcoding in the UAM Insect Collection. There are now 4020 Alaskan arthropod species represented by DNA barcodes, after including all records in Barcode of Life Data Systems (BOLD) of species that occur in Alaska - i.e., 48.5% of the 8277 Alaskan, non-marine-arthropod, named species have associated DNA barcodes. An assessment of the identification power of the library in its current state yielded fewer species-level identifications than expected, but the results were not discouraging. We believe we are the first to deliberately begin development of a DNA barcode library of the entire arthropod fauna for a North American state or province. Although far from complete, this library will become increasingly valuable as more species are added and costs to obtain DNA sequences fall.

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices.

PubMed

Hoople, Gordon D; Richards, Andrew; Wu, Yan; Pisano, Albert P; Zhang, Kun

2018-03-26

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

[Construction of large fragment metagenome library of natural mangrove soil].

PubMed

Jiang, Yun-Xia; Zheng, Tian-Ling

2007-11-01

Applying our optimized direct extraction method, the percentage of large fragment DNA in the total extracted mangrove soil DNA was significant increased. The large fragment metagenome library derived from natural mangrove soil over four seasons was successfully constructed by the optimized DNA extraction and electro elution purification method. All of the clones had recombinant Cosmids and each differed in their fragment profiles when Cosmid DNA was extracted from 12 randomly picked colonies and digested with BamHI. The average insert size for this library was larger than 35 kbp. This culturing-independent library at least encompassed 335 Mbp valuable genetic information of mangrove soil microbes. It allowed mining of valuable intertidal microbial resource to become a reality. It is a recommended method for those researchers who have still not circumvented the large insert environmental libraries or for those beginning research in this field, so as to avoid them attempting repetitive, fussy work.

DNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable Information.

PubMed

Neri, Dario; Lerner, Richard A

2018-06-20

The discovery of organic ligands that bind specifically to proteins is a central problem in chemistry, biology, and the biomedical sciences. The encoding of individual organic molecules with distinctive DNA tags, serving as amplifiable identification bar codes, allows the construction and screening of combinatorial libraries of unprecedented size, thus facilitating the discovery of ligands to many different protein targets. Fundamentally, one links powers of genetics and chemical synthesis. After the initial description of DNA-encoded chemical libraries in 1992, several experimental embodiments of the technology have been reduced to practice. This review provides a historical account of important milestones in the development of DNA-encoded chemical libraries, a survey of relevant ongoing research activities, and a glimpse into the future.

Partial characterization of normal and Haemophilus influenzae-infected mucosal complementary DNA libraries in chinchilla middle ear mucosa.

PubMed

Kerschner, Joseph E; Erdos, Geza; Hu, Fen Ze; Burrows, Amy; Cioffi, Joseph; Khampang, Pawjai; Dahlgren, Margaret; Hayes, Jay; Keefe, Randy; Janto, Benjamin; Post, J Christopher; Ehrlich, Garth D

2010-04-01

We sought to construct and partially characterize complementary DNA (cDNA) libraries prepared from the middle ear mucosa (MEM) of chinchillas to better understand pathogenic aspects of infection and inflammation, particularly with respect to leukotriene biogenesis and response. Chinchilla MEM was harvested from controls and after middle ear inoculation with nontypeable Haemophilus influenzae. RNA was extracted to generate cDNA libraries. Randomly selected clones were subjected to sequence analysis to characterize the libraries and to provide DNA sequence for phylogenetic analyses. Reverse transcription-polymerase chain reaction of the RNA pools was used to generate cDNA sequences corresponding to genes associated with leukotriene biosynthesis and metabolism. Sequence analysis of 921 randomly selected clones from the uninfected MEM cDNA library produced approximately 250,000 nucleotides of almost entirely novel sequence data. Searches of the GenBank database with the Basic Local Alignment Search Tool provided for identification of 515 unique genes expressed in the MEM and not previously described in chinchillas. In almost all cases, the chinchilla cDNA sequences displayed much greater homology to human or other primate genes than with rodent species. Genes associated with leukotriene metabolism were present in both normal and infected MEM. Based on both phylogenetic comparisons and gene expression similarities with humans, chinchilla MEM appears to be an excellent model for the study of middle ear inflammation and infection. The higher degree of sequence similarity between chinchillas and humans compared to chinchillas and rodents was unexpected. The cDNA libraries from normal and infected chinchilla MEM will serve as useful molecular tools in the study of otitis media and should yield important information with respect to middle ear pathogenesis.

Partial Characterization of Normal and Haemophilus influenzae–Infected Mucosal Complementary DNA Libraries in Chinchilla Middle Ear Mucosa

PubMed Central

Kerschner, Joseph E.; Erdos, Geza; Hu, Fen Ze; Burrows, Amy; Cioffi, Joseph; Khampang, Pawjai; Dahlgren, Margaret; Hayes, Jay; Keefe, Randy; Janto, Benjamin; Post, J. Christopher; Ehrlich, Garth D.

2010-01-01

Objectives We sought to construct and partially characterize complementary DNA (cDNA) libraries prepared from the middle ear mucosa (MEM) of chinchillas to better understand pathogenic aspects of infection and inflammation, particularly with respect to leukotriene biogenesis and response. Methods Chinchilla MEM was harvested from controls and after middle ear inoculation with nontypeable Haemophilus influenzae. RNA was extracted to generate cDNA libraries. Randomly selected clones were subjected to sequence analysis to characterize the libraries and to provide DNA sequence for phylogenetic analyses. Reverse transcription–polymerase chain reaction of the RNA pools was used to generate cDNA sequences corresponding to genes associated with leukotriene biosynthesis and metabolism. Results Sequence analysis of 921 randomly selected clones from the uninfected MEM cDNA library produced approximately 250,000 nucleotides of almost entirely novel sequence data. Searches of the GenBank database with the Basic Local Alignment Search Tool provided for identification of 515 unique genes expressed in the MEM and not previously described in chinchillas. In almost all cases, the chinchilla cDNA sequences displayed much greater homology to human or other primate genes than with rodent species. Genes associated with leukotriene metabolism were present in both normal and infected MEM. Conclusions Based on both phylogenetic comparisons and gene expression similarities with humans, chinchilla MEM appears to be an excellent model for the study of middle ear inflammation and infection. The higher degree of sequence similarity between chinchillas and humans compared to chinchillas and rodents was unexpected. The cDNA libraries from normal and infected chinchilla MEM will serve as useful molecular tools in the study of otitis media and should yield important information with respect to middle ear pathogenesis. PMID:20433028

Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.

PubMed

Franzini, Raphael M; Samain, Florent; Abd Elrahman, Maaly; Mikutis, Gediminas; Nauer, Angela; Zimmermann, Mauro; Scheuermann, Jörg; Hall, Jonathan; Neri, Dario

2014-08-20

DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

Laser capture microdissection: Arcturus(XT) infrared capture and UV cutting methods.

PubMed

Gallagher, Rosa I; Blakely, Steven R; Liotta, Lance A; Espina, Virginia

2012-01-01

Laser capture microdissection (LCM) is a technique that allows the precise procurement of enriched cell populations from a heterogeneous tissue under direct microscopic visualization. LCM can be used to harvest the cells of interest directly or can be used to isolate specific cells by ablating the unwanted cells, resulting in histologically enriched cell populations. The fundamental components of laser microdissection technology are (a) visualization of the cells of interest via microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. Laser energy supplied by LCM instruments can be infrared (810 nm) or ultraviolet (355 nm). Infrared lasers melt thermolabile polymers for cell capture, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes the unique features of the Arcturus(XT) laser capture microdissection instrument, which incorporates both infrared capture and ultraviolet cutting technology in one instrument, using a proteomic downstream assay as a model.

An in vivo library-versus-library selection of optimized protein-protein interactions.

PubMed

Pelletier, J N; Arndt, K M; Plückthun, A; Michnick, S W

1999-07-01

We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.

Zirconium(IV)-Catalyzed Ring Opening of on-DNA Epoxides in Water.

PubMed

Fan, Lijun; Davie, Christopher P

2017-05-04

DNA-encoded library technology (ELT) has spurred wide interest in the pharmaceutical industry as a powerful tool for hit and lead generation. In recent years a number of "DNA-compatible" chemical modifications have been published and used to synthesize vastly diverse screening libraries. Herein we report a newly developed, zirconium tetrakis(dodecyl sulfate) [Zr(DS) 4 ] catalyzed ring-opening of on-DNA epoxides in water with amines, including anilines. Subsequent cyclization of the resulting on-DNA β-amino alcohols leads to a variety of biologically interesting, nonaromatic heterocycles. Under these conditions, a library of 137 million on-DNA β-amino alcohols and their cyclization products was assembled. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

DOE PAGES

Rinke, Christian; Low, Serene; Woodcroft, Ben J.; ...

2016-09-22

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. For this study, we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diversemore » Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (~100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.« less

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rinke, Christian; Low, Serene; Woodcroft, Ben J.

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. For this study, we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diversemore » Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (~100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics.« less

Validation of picogram- and femtogram-input DNA libraries for microscale metagenomics

PubMed Central

Low, Serene; Raina, Jean-Baptiste; Skarshewski, Adam; Le, Xuyen H.; Butler, Margaret K.; Stocker, Roman; Seymour, Justin; Tyson, Gene W.

2016-01-01

High-throughput sequencing libraries are typically limited by the requirement for nanograms to micrograms of input DNA. This bottleneck impedes the microscale analysis of ecosystems and the exploration of low biomass samples. Current methods for amplifying environmental DNA to bypass this bottleneck introduce considerable bias into metagenomic profiles. Here we describe and validate a simple modification of the Illumina Nextera XT DNA library preparation kit which allows creation of shotgun libraries from sub-nanogram amounts of input DNA. Community composition was reproducible down to 100 fg of input DNA based on analysis of a mock community comprising 54 phylogenetically diverse Bacteria and Archaea. The main technical issues with the low input libraries were a greater potential for contamination, limited DNA complexity which has a direct effect on assembly and binning, and an associated higher percentage of read duplicates. We recommend a lower limit of 1 pg (∼100–1,000 microbial cells) to ensure community composition fidelity, and the inclusion of negative controls to identify reagent-specific contaminants. Applying the approach to marine surface water, pronounced differences were observed between bacterial community profiles of microliter volume samples, which we attribute to biological variation. This result is consistent with expected microscale patchiness in marine communities. We thus envision that our benchmarked, slightly modified low input DNA protocol will be beneficial for microscale and low biomass metagenomics. PMID:27688978

Methods for Selecting Phage Display Antibody Libraries.

PubMed

Jara-Acevedo, Ricardo; Diez, Paula; Gonzalez-Gonzalez, Maria; Degano, Rosa Maria; Ibarrola, Nieves; Gongora, Rafael; Orfao, Alberto; Fuentes, Manuel

2016-01-01

The selection process aims sequential enrichment of phage antibody display library in clones that recognize the target of interest or antigen as the library undergoes successive rounds of selection. In this review, selection methods most commonly used for phage display antibody libraries have been comprehensively described. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus

PubMed Central

Marques, M Carmen; Alonso-Cantabrana, Hugo; Forment, Javier; Arribas, Raquel; Alamar, Santiago; Conejero, Vicente; Perez-Amador, Miguel A

2009-01-01

Background Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. Results We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. Conclusion The new EST collection denotes an important step towards the identification of all genes in the citrus genome. Furthermore, public availability of the cDNA clones generated in this study, and not only their sequence, enables testing of the biological function of the genes represented in the collection. Expression of the citrus SEP3 homologue, CitrSEP, in Arabidopsis results in early flowering, along with other phenotypes resembling the over-expression of the Arabidopsis SEPALLATA genes. Our findings suggest that the members of the SEP gene family play similar roles in these quite distant plant species. PMID:19747386

Identification of differentially expressed genes associated with the enhancement of X-ray susceptibility by RITA in a hypopharyngeal squamous cell carcinoma cell line (FaDu).

PubMed

Luan, Jinwei; Li, Xianglan; Guo, Rutao; Liu, Shanshan; Luo, Hongyu; You, Qingshan

2016-06-01

Next generation sequencing and bio-informatic analyses were conducted to investigate the mechanism of reactivation of p53 and induction of tumor cell apoptosis (RITA)-enhancing X-ray susceptibility in FaDu cells. The cDNA was isolated from FaDu cells treated with 0 X-ray, 8 Gy X-ray, or 8 Gy X-ray + RITA. Then, cDNA libraries were created and sequenced using next generation sequencing, and each assay was repeated twice. Subsequently, differentially expressed genes (DEGs) were identified using Cuffdiff in Cufflinks and their functions were predicted by pathway enrichment analyses. Genes that were constantly up- or down-regulated in 8 Gy X-ray-treated FaDu cells and 8 Gy X-ray + RITA-treated FaDu cells were obtained as RITA genes. Afterward, the protein-protein interaction (PPI) relationships were obtained from the STRING database and a PPI network was constructed using Cytoscape. Furthermore, ClueGO was used for pathway enrichment analysis of genes in the PPI network. Total 2,040 and 297 DEGs were identified in FaDu cells treated with 8 Gy X-ray or 8 Gy X-ray + RITA, respectively. PARP3 and NEIL1 were enriched in base excision repair, and CDK1 was enriched in p53 signaling pathway. RFC2 and EZH2 were identified as RITA genes. In the PPI network, many interaction relationships were identified (e.g., RFC2-CDK1, EZH2-CDK1 and PARP3-EZH2). ClueGO analysis showed that RFC2 and EZH2 were related to cell cycle. RFC2, EZH2, CDK1, PARP3 and NEIL1 may be associated, and together enhance the susceptibility of FaDu cells treated with RITA to the deleterious effects of X-ray.

Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections.

PubMed

Hammoumi, Saliha; Vallaeys, Tatiana; Santika, Ayi; Leleux, Philippe; Borzym, Ewa; Klopp, Christophe; Avarre, Jean-Christophe

2016-01-01

Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp, Cyprinus carpio L. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×10 7 . The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3.

Targeted genomic enrichment and sequencing of CyHV-3 from carp tissues confirms low nucleotide diversity and mixed genotype infections

PubMed Central

Hammoumi, Saliha; Vallaeys, Tatiana; Santika, Ayi; Leleux, Philippe; Borzym, Ewa; Klopp, Christophe

2016-01-01

Koi herpesvirus disease (KHVD) is an emerging disease that causes mass mortality in koi and common carp, Cyprinus carpio L. Its causative agent is Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV). Although data on the pathogenesis of this deadly virus is relatively abundant in the literature, still little is known about its genomic diversity and about the molecular mechanisms that lead to such a high virulence. In this context, we developed a new strategy for sequencing full-length CyHV-3 genomes directly from infected fish tissues. Total genomic DNA extracted from carp gill tissue was specifically enriched with CyHV-3 sequences through hybridization to a set of nearly 2 million overlapping probes designed to cover the entire genome length, using KHV-J sequence (GenBank accession number AP008984) as reference. Applied to 7 CyHV-3 specimens from Poland and Indonesia, this targeted genomic enrichment enabled recovery of the full genomes with >99.9% reference coverage. The enrichment rate was directly correlated to the estimated number of viral copies contained in the DNA extracts used for library preparation, which varied between ∼5000 and ∼2×107. The average sequencing depth was >200 for all samples, thus allowing the search for variants with high confidence. Sequence analyses highlighted a significant proportion of intra-specimen sequence heterogeneity, suggesting the presence of mixed infections in all investigated fish. They also showed that inter-specimen genetic diversity at the genome scale was very low (>99.95% of sequence identity). By enabling full genome comparisons directly from infected fish tissues, this new method will be valuable to trace outbreaks rapidly and at a reasonable cost, and in turn to understand the transmission routes of CyHV-3. PMID:27703859

A new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system.

PubMed

Oba, Mami; Tsuchiaka, Shinobu; Omatsu, Tsutomu; Katayama, Yukie; Otomaru, Konosuke; Hirata, Teppei; Aoki, Hiroshi; Murata, Yoshiteru; Makino, Shinji; Nagai, Makoto; Mizutani, Tetsuya

2018-01-08

We tested usefulness of a target enrichment system SureSelect, a comprehensive viral nucleic acid detection method, for rapid identification of viral pathogens in feces samples of cattle, pigs and goats. This system enriches nucleic acids of target viruses in clinical/field samples by using a library of biotinylated RNAs with sequences complementary to the target viruses. The enriched nucleic acids are amplified by PCR and subjected to next generation sequencing to identify the target viruses. In many samples, SureSelect target enrichment method increased efficiencies for detection of the viruses listed in the biotinylated RNA library. Furthermore, this method enabled us to determine nearly full-length genome sequence of porcine parainfluenza virus 1 and greatly increased Breadth, a value indicating the ratio of the mapping consensus length in the reference genome, in pig samples. Our data showed usefulness of SureSelect target enrichment system for comprehensive analysis of genomic information of various viruses in field samples. Copyright © 2017 Elsevier Inc. All rights reserved.

Multi-Threaded DNA Tag/Anti-Tag Library Generator for Multi-Core Platforms

DTIC Science & Technology

2009-05-01

base pair)  Watson ‐ Crick  strand pairs that bind perfectly within pairs, but poorly across pairs. A variety  of  DNA  strand hybridization metrics...AFRL-RI-RS-TR-2009-131 Final Technical Report May 2009 MULTI-THREADED DNA TAG/ANTI-TAG LIBRARY GENERATOR FOR MULTI-CORE PLATFORMS...TYPE Final 3. DATES COVERED (From - To) Jun 08 – Feb 09 4. TITLE AND SUBTITLE MULTI-THREADED DNA TAG/ANTI-TAG LIBRARY GENERATOR FOR MULTI-CORE

Isolation and characterization of novel microsatellite markers from the sika deer (Cervus nippon) genome.

PubMed

Li, Y M; Bai, C Y; Niu, W P; Yu, H; Yang, R J; Yan, S Q; Zhang, J Y; Zhang, M J; Zhao, Z H

2015-09-28

Microsatellite markers are widely and evenly distributed, and are highly polymorphic. Rapid and convenient detection through automated analysis means that microsatellite markers are widely used in the construction of plant and animal genetic maps, in quantitative trait loci localization, marker-assisted selection, identification of genetic relationships, and genetic diversity and phylogenetic tree construction. However, few microsatellite markers remain to be isolated. We used streptavidin magnetic beads to affinity-capture and construct a (CA)n microsatellite DNA-enriched library from sika deer. We selected sequences containing more than six repeats to design primers. Clear bands were selected, which were amplified using non-specific primers following PCR amplification to screen polymorphisms in a group of 65 unrelated sika deer. The positive clone rate reached 82.9% by constructing the enriched library, and we then selected positive clones for sequencing. There were 395 sequences with CA repeats, and the CA repeat number was 4-105. We selected sequences containing more than six repeats to design primers, of which 297 pairs were designed. We next selected clear bands and used non-specific primers to amplify following PCR amplification. In total, 245 pairs of primers were screened. We then selected 50 pairs of primers to randomly screen for polymorphisms. We detected 47 polymorphic and 3 monomorphic loci in 65 unrelated sika deer. These newly isolated and characterized microsatellite loci can be used to construct genetic maps and for lineage testing in deer. In addition, they can be used for comparative genomics between Cervidae species.

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

PubMed

Bidlingmaier, Scott; Su, Yang; Liu, Bin

2015-01-01

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

A novel technology for the detection, enrichment, and separation of trace amounts of target DNA based on amino-modified fluorescent magnetic composite nanoparticles.

PubMed

Wang, Guannan; Su, Xingguang

2010-06-01

A novel, highly sensitive technology for the detection, enrichment, and separation of trace amounts of target DNA was developed on the basis of amino-modified fluorescent magnetic composite nanoparticles (AFMN). In this study, the positively charged amino-modified composite nanoparticles conjugate with the negatively charged capture DNA through electrostatic binding. The optimal combination of AFMN and capture DNA was measured by dynamic light scattering (DLS) and UV-vis absorption spectroscopy. The highly sensitive detection of trace amounts of target DNA was achieved through enrichment by means of AFMN. The detection limit for target DNA is 0.4 pM, which could be further improved by using a more powerful magnet. Because of their different melting temperatures, single-base mismatched target DNA could be separated from perfectly complementary target DNA. In addition, the photoluminescence (PL) signals of perfectly complementary target DNA and single-base mismatched DNA as well as the hybridization kinetics of different concentrations of target DNA at different reaction times have also been studied. Most importantly, the detection, enrichment, and separation ability of AFMN was further verified with milk. Simple and satisfactory results were obtained, which show the great potential in the fields of mutation identification and clinical diagnosis.

Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags

PubMed Central

Litovchick, Alexander; Clark, Matthew A; Keefe, Anthony D

2014-01-01

The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method “relay primer-dependent bypass” utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3′-terminus prior to ligation to adjacent oligonucleotides. The second reading method “repeat-dependent bypass” utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3′-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries. PMID:25483841

Overexpression of DEMETER, a DNA demethylase, promotes early apical bud maturation in poplar.

PubMed

Conde, Daniel; Moreno-Cortés, Alicia; Dervinis, Christopher; Ramos-Sánchez, José M; Kirst, Matias; Perales, Mariano; González-Melendi, Pablo; Allona, Isabel

2017-11-01

The transition from active growth to dormancy is critical for the survival of perennial plants. We identified a DEMETER-like (CsDML) cDNA from a winter-enriched cDNA subtractive library in chestnut (Castanea sativa Mill.), an economically and ecologically important species. Next, we characterized this DNA demethylase and its putative ortholog in the more experimentally tractable hybrid poplar (Populus tremula × alba), under the signals that trigger bud dormancy in trees. We performed phylogenetic and protein sequence analysis, gene expression profiling, and 5-methyl-cytosine methylation immunodetection studies to evaluate the role of CsDML and its homolog in poplar, PtaDML6. Transgenic hybrid poplars overexpressing CsDML were produced and analysed. Short days and cold temperatures induced CsDML and PtaDML6. Overexpression of CsDML accelerated short-day-induced bud formation, specifically from Stages 1 to 0. Buds acquired a red-brown coloration earlier than wild-type plants, alongside with the up-regulation of flavonoid biosynthesis enzymes and accumulation of flavonoids in the shoot apical meristem and bud scales. Our data show that the CsDML gene induces bud formation needed for the survival of the apical meristem under the harsh conditions of winter. © 2017 John Wiley & Sons Ltd.

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application.

PubMed

Zhong, Jian; Ye, Zhenqing; Lenz, Samuel W; Clark, Chad R; Bharucha, Adil; Farrugia, Gianrico; Robertson, Keith D; Zhang, Zhiguo; Ordog, Tamas; Lee, Jeong-Heon

2017-12-21

Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Development and characterization of microsatellite markers for Hibiscus glaber Matsum. ex Nakai, an endemic tree species of the oceanic Bonin Islands, Japan.

PubMed

Ohtani, Masato; Tani, Naoki; Yoshimaru, Hiroshi

2008-11-01

Polymorphic microsatellite markers were developed for Hibiscus glaber, an endemic tree of the Bonin Islands. Eighty-seven of the 208 sequences from an enriched library were unique and containing microsatellites. Ten loci were proved to be highly polymorphic among 78 individuals from the Nishi-jima Island. Total exclusionary powers for the first and the second parents were 99.989% and 99.999%, respectively. Nine loci also amplified single fragment from genomic DNA of H. tiliaceus, a related and widespread congener. Our markers can be reliably used for the estimation of current gene flow within/among populations of the two woody Hibiscus species. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

Design and screening of M13 phage display cDNA libraries.

PubMed

Georgieva, Yuliya; Konthur, Zoltán

2011-02-17

The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF) libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS) will be presented.

Enriching User-Oriented Class Associations for Library Classification Schemes.

ERIC Educational Resources Information Center

Pu, Hsiao-Tieh; Yang, Chyan

2003-01-01

Explores the possibility of adding user-oriented class associations to hierarchical library classification schemes. Analyses a log of book circulation records from a university library in Taiwan and shows that classification schemes can be made more adaptable by analyzing circulation patterns of similar users. (Author/LRW)

Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge.

PubMed

Brulle, Franck; Jeffroy, Fanny; Madec, Stéphanie; Nicolas, Jean-Louis; Paillard, Christine

2012-10-01

The Manila clam, Ruditapes philippinarum, is an economically-important, commercial shellfish; harvests are diminished in some European waters by a pathogenic bacterium, Vibrio tapetis, that causes Brown Ring disease. To identify molecular characteristics associated with susceptibility or resistance to Brown Ring disease, Suppression Subtractive Hybridization (SSH) analyzes were performed to construct cDNA libraries enriched in up- or down-regulated transcripts from clam immune cells, hemocytes, after a 3-h in vitro challenge with cultured V. tapetis. Nine hundred and ninety eight sequences from the two libraries were sequenced, and an in silico analysis identified 235 unique genes. BLAST and "Gene ontology" classification analyzes revealed that 60.4% of the Expressed Sequence Tags (ESTs) have high similarities with genes involved in various physiological functions, such as immunity, apoptosis and cytoskeleton organization; whereas, 39.6% remain unidentified. From the 235 unique genes, we selected 22 candidates based upon physiological function and redundancy in the libraries. Then, Real-Time PCR analysis identified 3 genes related to cytoskeleton organization showing significant variation in expression attributable to V. tapetis exposure. Disruption in regulation of these genes is consistent with the etiologic agent of Brown Ring disease in Manila clams. Copyright © 2012 Elsevier Ltd. All rights reserved.

In vitro selection and characterization of single stranded DNA aptamers for luteolin: A possible recognition tool.

PubMed

Tuma Sabah, Jinan; Zulkifli, Razauden Mohamed; Shahir, Shafinaz; Ahmed, Farediah; Abdul Kadir, Mohammed Rafiq; Zakaria, Zarita

2018-05-15

Distinctive bioactivities possessed by luteolin (3', 4', 5, 7-tetrahydroxy-flavone) are advantageous for sundry practical applications. This paper reports the in vitro selection and characterization of single stranded-DNA (ssDNA) aptamers, specific for luteolin (LUT). 76-mer library containing 1015 randomized ssDNA were screened via systematic evolution of ligands by exponential enrichment (SELEX). The recovered ssDNA pool from the 8th round was amplified with unlabeled primers and cloned into PSTBlue-1 vector prior to sequencing. 22 of LUT-binding aptamer variants were further classified into one of the seven groups based on their N40 random sequence regions, wherein one representative from each group was characterized. The dissociation constant of aptamers designated as LUT#28, LUT#20 and LUT#3 was discerned to be 107, 214 and 109 nM, respectively with high binding affinity towards LUT. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Furthermore, LUT#3 displayed higher specificity with insignificant binding toward kaempferol and quercetin despite its structural and functional similarity compared to LUT#28 and LUT#20. Further LUT#3 can detect free luteolin within 0.2-1 mM in solution. It was suggested that LUT#3 aptamer were the most suitable for LUT recognition tool at laboratory scale based on the condition tested. Copyright © 2018 Elsevier Inc. All rights reserved.

Corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures.

PubMed

Thomas, William D; Golomb, Miriam; Smith, George P

2010-12-15

Phage display is used to discover peptides or proteins with a desired target property-most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phages or peptides (TUPs), that lack the target behavior. Many TUPs are propagation related; they have mutations conferring a growth advantage and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus-strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus-strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus-strand origin. The founder's infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. Copyright © 2010 Elsevier Inc. All rights reserved.

Corruption of phage-display libraries by target-unrelated clones: Diagnosis and countermeasures

PubMed Central

Thomas, William D.; Golomb, Miriam; Smith, George P.

2010-01-01

Phage display is used to discover peptides or proteins with a desired target property—most often, affinity for a target selector molecule. Libraries of phage clones displaying diverse surface peptides are subject to a selection process designed to enrich for the target behavior, and subsequently propagated to restore phage numbers. A recurrent problem is enrichment of clones, called target-unrelated phage (TUPs), that lack the target behavior. Many TUPs are propagation-related; they have mutations conferring a growth advantage, and are enriched during the propagations accompanying selection. Unlike other filamentous phage libraries, fd-tet-based libraries are relatively resistant to propagation-related TUP corruption. Their minus strand origin is disrupted by a large cassette that simultaneously confers resistance to tetracycline and imposes a rate-limiting growth defect that cannot be bypassed with simple mutations. Nonetheless, a new type of propagation-related TUP emerged in the output of in vivo selections from an fd-tet library. The founding clone had a complex rearrangement that restored the minus strand origin while retaining tetracycline resistance. The rearrangement involved two recombination events, one with a contaminant having a wild-type minus strand origin. The founder’s infectivity advantage spread by simple recombination to clones displaying different peptides. We propose measures for minimizing TUP corruption. PMID:20692225

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

PubMed

Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

Identification of Genes Enriched in GnRH Neurons by Translating Ribosome Affinity Purification and RNAseq in Mice.

PubMed

Burger, Laura L; Vanacker, Charlotte; Phumsatitpong, Chayarndorn; Wagenmaker, Elizabeth R; Wang, Luhong; Olson, David P; Moenter, Suzanne M

2018-04-01

Gonadotropin-releasing hormone (GnRH) neurons are a nexus of fertility regulation. We used translating ribosome affinity purification coupled with RNA sequencing to examine messenger RNAs of GnRH neurons in adult intact and gonadectomized (GDX) male and female mice. GnRH neuron ribosomes were tagged with green fluorescent protein (GFP) and GFP-labeled polysomes isolated by immunoprecipitation, producing one RNA fraction enhanced for GnRH neuron transcripts and one RNA fraction depleted. Complementary DNA libraries were created from each fraction and 50-base, paired-end sequencing done and differential expression (enhanced fraction/depleted fraction) determined with a threshold of >1.5- or <0.66-fold (false discovery rate P ≤ 0.05). A core of ∼840 genes was differentially expressed in GnRH neurons in all treatments, including enrichment for Gnrh1 (∼40-fold), and genes critical for GnRH neuron and/or gonadotrope development. In contrast, non-neuronal transcripts were not enriched or were de-enriched. Several epithelial markers were also enriched, consistent with the olfactory epithelial origins of GnRH neurons. Interestingly, many synaptic transmission pathways were de-enriched, in accordance with relatively low innervation of GnRH neurons. The most striking difference between intact and GDX mice of both sexes was a marked downregulation of genes associated with oxidative phosphorylation and upregulation of glucose transporters in GnRH neurons from GDX mice. This may suggest that GnRH neurons switch to an alternate fuel to increase adenosine triphosphate production in the absence of negative feedback when GnRH release is elevated. Knowledge of the GnRH neuron translatome and its regulation can guide functional studies and can be extended to disease states, such as polycystic ovary syndrome.

Flow cytometry for enrichment and titration in massively parallel DNA sequencing

PubMed Central

Sandberg, Julia; Ståhl, Patrik L.; Ahmadian, Afshin; Bjursell, Magnus K.; Lundeberg, Joakim

2009-01-01

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols. PMID:19304748

Deep subsurface life from North Pond: Enrichment, isolation, characterization and genomes of heterotrophic bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russell, Joseph A.; Leon-Zayas, Rosa; Wrighton, Kelly

Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic watercolumn west of the Mid-Atlantic Ridge at 22° N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sedimentmore » column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. Furthermore, the cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface.« less

Deep subsurface life from North Pond: Enrichment, isolation, characterization and genomes of heterotrophic bacteria

DOE PAGES

Russell, Joseph A.; Leon-Zayas, Rosa; Wrighton, Kelly; ...

2016-05-10

Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic watercolumn west of the Mid-Atlantic Ridge at 22° N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sedimentmore » column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. Furthermore, the cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface.« less

Enriching Critical Thinking and Language Learning with Educational Digital Libraries

ERIC Educational Resources Information Center

Lu, Hsin-lin

2012-01-01

As the amount of information available in online digital libraries increases exponentially, questions arise concerning the most productive way to use that information to advance learning. Applying the earlier information seeking theories advocated by Kelly (1963), Taylor (1968), and Belkin (1980) to the digital libraries experience, Carol Kuhlthau…

DNA polymerase preference determines PCR priming efficiency.

PubMed

Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

2014-01-30

Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially available DNA polymerases. The results suggest that the interaction of the DNA polymerase with the primer:template junction during the initiation of DNA polymerization is very important in terms of overall amplification bias and has broader implications for both the primer design process and multiplex PCR.

Open resource metagenomics: a model for sharing metagenomic libraries.

PubMed

Neufeld, J D; Engel, K; Cheng, J; Moreno-Hagelsieb, G; Rose, D R; Charles, T C

2011-11-30

Both sequence-based and activity-based exploitation of environmental DNA have provided unprecedented access to the genomic content of cultivated and uncultivated microorganisms. Although researchers deposit microbial strains in culture collections and DNA sequences in databases, activity-based metagenomic studies typically only publish sequences from the hits retrieved from specific screens. Physical metagenomic libraries, conceptually similar to entire sequence datasets, are usually not straightforward to obtain by interested parties subsequent to publication. In order to facilitate unrestricted distribution of metagenomic libraries, we propose the adoption of open resource metagenomics, in line with the trend towards open access publishing, and similar to culture- and mutant-strain collections that have been the backbone of traditional microbiology and microbial genetics. The concept of open resource metagenomics includes preparation of physical DNA libraries, preferably in versatile vectors that facilitate screening in a diversity of host organisms, and pooling of clones so that single aliquots containing complete libraries can be easily distributed upon request. Database deposition of associated metadata and sequence data for each library provides researchers with information to select the most appropriate libraries for further research projects. As a starting point, we have established the Canadian MetaMicroBiome Library (CM(2)BL [1]). The CM(2)BL is a publicly accessible collection of cosmid libraries containing environmental DNA from soils collected from across Canada, spanning multiple biomes. The libraries were constructed such that the cloned DNA can be easily transferred to Gateway® compliant vectors, facilitating functional screening in virtually any surrogate microbial host for which there are available plasmid vectors. The libraries, which we are placing in the public domain, will be distributed upon request without restriction to members of both the academic research community and industry. This article invites the scientific community to adopt this philosophy of open resource metagenomics to extend the utility of functional metagenomics beyond initial publication, circumventing the need to start from scratch with each new research project.

Open resource metagenomics: a model for sharing metagenomic libraries

PubMed Central

Neufeld, J.D.; Engel, K.; Cheng, J.; Moreno-Hagelsieb, G.; Rose, D.R.; Charles, T.C.

2011-01-01

Both sequence-based and activity-based exploitation of environmental DNA have provided unprecedented access to the genomic content of cultivated and uncultivated microorganisms. Although researchers deposit microbial strains in culture collections and DNA sequences in databases, activity-based metagenomic studies typically only publish sequences from the hits retrieved from specific screens. Physical metagenomic libraries, conceptually similar to entire sequence datasets, are usually not straightforward to obtain by interested parties subsequent to publication. In order to facilitate unrestricted distribution of metagenomic libraries, we propose the adoption of open resource metagenomics, in line with the trend towards open access publishing, and similar to culture- and mutant-strain collections that have been the backbone of traditional microbiology and microbial genetics. The concept of open resource metagenomics includes preparation of physical DNA libraries, preferably in versatile vectors that facilitate screening in a diversity of host organisms, and pooling of clones so that single aliquots containing complete libraries can be easily distributed upon request. Database deposition of associated metadata and sequence data for each library provides researchers with information to select the most appropriate libraries for further research projects. As a starting point, we have established the Canadian MetaMicroBiome Library (CM2BL [1]). The CM2BL is a publicly accessible collection of cosmid libraries containing environmental DNA from soils collected from across Canada, spanning multiple biomes. The libraries were constructed such that the cloned DNA can be easily transferred to Gateway® compliant vectors, facilitating functional screening in virtually any surrogate microbial host for which there are available plasmid vectors. The libraries, which we are placing in the public domain, will be distributed upon request without restriction to members of both the academic research community and industry. This article invites the scientific community to adopt this philosophy of open resource metagenomics to extend the utility of functional metagenomics beyond initial publication, circumventing the need to start from scratch with each new research project. PMID:22180823

Coral life history and symbiosis: Functional genomic resources for two reef building Caribbean corals, Acropora palmata and Montastraea faveolata

PubMed Central

Schwarz, Jodi A; Brokstein, Peter B; Voolstra, Christian; Terry, Astrid Y; Miller, David J; Szmant, Alina M; Coffroth, Mary Alice; Medina, Mónica

2008-01-01

Background Scleractinian corals are the foundation of reef ecosystems in tropical marine environments. Their great success is due to interactions with endosymbiotic dinoflagellates (Symbiodinium spp.), with which they are obligately symbiotic. To develop a foundation for studying coral biology and coral symbiosis, we have constructed a set of cDNA libraries and generated and annotated ESTs from two species of corals, Acropora palmata and Montastraea faveolata. Results We generated 14,588 (Ap) and 3,854 (Mf) high quality ESTs from five life history/symbiosis stages (spawned eggs, early-stage planula larvae, late-stage planula larvae either infected with symbionts or uninfected, and adult coral). The ESTs assembled into a set of primarily stage-specific clusters, producing 4,980 (Ap), and 1,732 (Mf) unigenes. The egg stage library, relative to the other developmental stages, was enriched in genes functioning in cell division and proliferation, transcription, signal transduction, and regulation of protein function. Fifteen unigenes were identified as candidate symbiosis-related genes as they were expressed in all libraries constructed from the symbiotic stages and were absent from all of the non symbiotic stages. These include several DNA interacting proteins, and one highly expressed unigene (containing 17 cDNAs) with no significant protein-coding region. A significant number of unigenes (25) encode potential pattern recognition receptors (lectins, scavenger receptors, and others), as well as genes that may function in signaling pathways involved in innate immune responses (toll-like signaling, NFkB p105, and MAP kinases). Comparison between the A. palmata and an A. millepora EST dataset identified ferritin as a highly expressed gene in both datasets that appears to be undergoing adaptive evolution. Five unigenes appear to be restricted to the Scleractinia, as they had no homology to any sequences in the nr databases nor to the non-scleractinian cnidarians Nematostella vectensis and Hydra magnipapillata. Conclusion Partial sequencing of 5 cDNA libraries each for A. palmata and M. faveolata has produced a rich set of candidate genes (4,980 genes from A. palmata, and 1,732 genes from M. faveolata) that we can use as a starting point for examining the life history and symbiosis of these two species, as well as to further expand the dataset of cnidarian genes for comparative genomics and evolutionary studies. PMID:18298846

Coral Life History and Symbiosis: functional genomic resources for two reef building Caribbean corals, Acropora palmata and Montastraea faveolata

DOE PAGES

Schwarz, Jodi A.; Brokstein, Peter B.; Voolstra, Christian R.; ...

2008-02-25

Scleractinian corals are the foundation of reef ecosystems in tropical marine environments. Their great success is due to interactions with endosymbiotic dinoflagellates (Symbiodinium spp.), with which they are obligately symbiotic. To develop a foundation for studying coral biology and coral symbiosis, we have constructed a set of cDNA libraries and generated and annotated ESTs from two species of corals, Acropora palmata and Montastraea faveolata. Here we generated 14,588 (Ap) and 3,854 (Mf) high quality ESTs from five life history/symbiosis stages (spawned eggs, early-stage planula larvae, late-stage planula larvae either infected with symbionts or uninfected, and adult coral). The ESTs assembledmore » into a set of primarily stage-specific clusters, producing 4,980 (Ap), and 1,732 (Mf) unigenes. The egg stage library, relative to the other developmental stages, was enriched in genes functioning in cell division and proliferation, transcription, signal transduction, and regulation of protein function. Fifteen unigenes were identified as candidate symbiosis-related genes as they were expressed in all libraries constructed from the symbiotic stages and were absent from all of the non symbiotic stages. These include several DNA interacting proteins, and one highly expressed unigene (containing 17 cDNAs) with no significant protein-coding region. A significant number of unigenes (25) encode potential pattern recognition receptors (lectins, scavenger receptors, and others), as well as genes that may function in signaling pathways involved in innate immune responses (toll-like signaling, NFkB p105, and MAP kinases). Comparison between the A. palmata and an A. millepora EST dataset identified ferritin as a highly expressed gene in both datasets that appears to be undergoing adaptive evolution. Five unigenes appear to be restricted to the Scleractinia, as they had no homology to any sequences in the nr databases nor to the non-scleractinian cnidarians Nematostella vectensis and Hydra magnipapillata. In conclusion, partial sequencing of 5 cDNA libraries each for A. palmata and M. faveolata has produced a rich set of candidate genes (4,980 genes from A. palmata, and 1,732 genes from M. faveolata) that we can use as a starting point for examining the life history and symbiosis of these two species, as well as to further expand the dataset of cnidarian genes for comparative genomics and evolutionary studies.« less

Coral Life History and Symbiosis: functional genomic resources for two reef building Caribbean corals, Acropora palmata and Montastraea faveolata

DOE Office of Scientific and Technical Information (OSTI.GOV)

Schwarz, Jodi A.; Brokstein, Peter B.; Voolstra, Christian R.

Scleractinian corals are the foundation of reef ecosystems in tropical marine environments. Their great success is due to interactions with endosymbiotic dinoflagellates (Symbiodinium spp.), with which they are obligately symbiotic. To develop a foundation for studying coral biology and coral symbiosis, we have constructed a set of cDNA libraries and generated and annotated ESTs from two species of corals, Acropora palmata and Montastraea faveolata. Here we generated 14,588 (Ap) and 3,854 (Mf) high quality ESTs from five life history/symbiosis stages (spawned eggs, early-stage planula larvae, late-stage planula larvae either infected with symbionts or uninfected, and adult coral). The ESTs assembledmore » into a set of primarily stage-specific clusters, producing 4,980 (Ap), and 1,732 (Mf) unigenes. The egg stage library, relative to the other developmental stages, was enriched in genes functioning in cell division and proliferation, transcription, signal transduction, and regulation of protein function. Fifteen unigenes were identified as candidate symbiosis-related genes as they were expressed in all libraries constructed from the symbiotic stages and were absent from all of the non symbiotic stages. These include several DNA interacting proteins, and one highly expressed unigene (containing 17 cDNAs) with no significant protein-coding region. A significant number of unigenes (25) encode potential pattern recognition receptors (lectins, scavenger receptors, and others), as well as genes that may function in signaling pathways involved in innate immune responses (toll-like signaling, NFkB p105, and MAP kinases). Comparison between the A. palmata and an A. millepora EST dataset identified ferritin as a highly expressed gene in both datasets that appears to be undergoing adaptive evolution. Five unigenes appear to be restricted to the Scleractinia, as they had no homology to any sequences in the nr databases nor to the non-scleractinian cnidarians Nematostella vectensis and Hydra magnipapillata. In conclusion, partial sequencing of 5 cDNA libraries each for A. palmata and M. faveolata has produced a rich set of candidate genes (4,980 genes from A. palmata, and 1,732 genes from M. faveolata) that we can use as a starting point for examining the life history and symbiosis of these two species, as well as to further expand the dataset of cnidarian genes for comparative genomics and evolutionary studies.« less

Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

PubMed Central

Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

1998-01-01

Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton δ-cadinene synthase (50% identity). PMID:9482865

PuLSE: Quality control and quantification of peptide sequences explored by phage display libraries.

PubMed

Shave, Steven; Mann, Stefan; Koszela, Joanna; Kerr, Alastair; Auer, Manfred

2018-01-01

The design of highly diverse phage display libraries is based on assumption that DNA bases are incorporated at similar rates within the randomized sequence. As library complexity increases and expected copy numbers of unique sequences decrease, the exploration of library space becomes sparser and the presence of truly random sequences becomes critical. We present the program PuLSE (Phage Library Sequence Evaluation) as a tool for assessing randomness and therefore diversity of phage display libraries. PuLSE runs on a collection of sequence reads in the fastq file format and generates tables profiling the library in terms of unique DNA sequence counts and positions, translated peptide sequences, and normalized 'expected' occurrences from base to residue codon frequencies. The output allows at-a-glance quantitative quality control of a phage library in terms of sequence coverage both at the DNA base and translated protein residue level, which has been missing from toolsets and literature. The open source program PuLSE is available in two formats, a C++ source code package for compilation and integration into existing bioinformatics pipelines and precompiled binaries for ease of use.

The use of high throughput DNA sequence analysis to assess the endophytic microbiome of date palm roots grown under different levels of salt stress.

PubMed

Yaish, Mahmoud W; Al-Harrasi, Ibtisam; Alansari, Aliya S; Al-Yahyai, Rashid; Glick, Bernard R

2016-09-01

Date palms are able to grow under diverse abiotic stress conditions including in saline soils, where microbial communities may be help in the plant's salinity tolerance. These communities able to produce specific growth promoting substances can enhance date palm growth in a saline environment. However, these communities are poorly defined. In the work reported here, the date palm endophytic bacterial and fungal communities were identified using the pyrosequencing method, and the microbial differential abundance in the root upon exposure to salinity stress was estimated. Approximately 150,061 reads were produced from the analysis of six ribosomal DNA libraries, which were prepared from endophytic microorganisms colonizing date palm root tissues. DNA sequence analysis of these libraries predicted the presence of a variety of bacterial and fungal endophytic species, some known and others unknown. The microbial community compositions of 30% and 8% of the bacterial and fungal species, respectively, were significantly (p ≤ 0.05) altered in response to salinity stress. Differential enrichment analysis showed that microbe diversity indicated by the Chao, Shannon and Simpson indices were slightly reduced, however, the overall microbial community structures were not significantly affected as a consequence of salinity. This may reflect a buffering effect by the host plant on the internal environments that these communities are colonizing. Some of the endophytes identified in this study were strains that were previously isolated from saline and marine environments. This suggests possible interactions with the plant that are favorable to salinity tolerance in date palm. [Int Microbiol 19(3):143-155 (2016)]. Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.

Development of goose- and duck-specific DNA markers to determine sources of Escherichia coli in waterways.

PubMed

Hamilton, Matthew J; Yan, Tao; Sadowsky, Michael J

2006-06-01

The contamination of waterways with fecal material is a persistent threat to public health. Identification of the sources of fecal contamination is a vital component for abatement strategies and for determination of total maximum daily loads. While phenotypic and genotypic techniques have been used to determine potential sources of fecal bacteria in surface waters, most methods require construction of large known-source libraries, and they often fail to adequately differentiate among environmental isolates originating from different animal sources. In this study, we used pooled genomic tester and driver DNAs in suppression subtractive hybridizations to enrich for host source-specific DNA markers for Escherichia coli originating from locally isolated geese. Seven markers were identified. When used as probes in colony hybridization studies, the combined marker DNAs identified 76% of the goose isolates tested and cross-hybridized, on average, with 5% of the human E. coli strains and with less than 10% of the strains obtained from other animal hosts. In addition, the combined probes identified 73% of the duck isolates examined, suggesting that they may be useful for determining the contribution of waterfowl to fecal contamination. However, the hybridization probes reacted mainly with E. coli isolates obtained from geese in the upper midwestern United States, indicating that there is regional specificity of the markers identified. Coupled with high-throughput, automated macro- and microarray screening, these markers may provide a quantitative, cost-effective, and accurate library-independent method for determining the sources of genetically diverse E. coli strains for use in source-tracking studies. However, future efforts to generate DNA markers specific for E. coli must include isolates obtained from geographically diverse animal hosts.

Sequencing on the SOLiD 5500xl System - in-depth characterization of the GC bias.

PubMed

Roeh, Simone; Weber, Peter; Rex-Haffner, Monika; Deussing, Jan M; Binder, Elisabeth B; Jakovcevski, Mira

2017-07-04

Different types of sequencing biases have been described and subsequently improved for a variety of sequencing systems, mostly focusing on the widely used Illumina systems. Similar studies are missing for the SOLiD 5500xl system, a sequencer which produced many data sets available to researchers today. Describing and understanding the bias is important to accurately interpret and integrate these published data in various ongoing research projects. We report a particularly strong GC bias for this sequencing system when analyzing a defined gDNA mix of 5 microbes with a wide range of different GC contents (20-72%) when comparing to the expected distribution and Illumina MiSeq data from the same DNA pool. Since we observed this bias already under PCR-free conditions, changing the PCR conditions during library preparation - a common strategy to handle bias in the Illumina system - was not relevant. Source of the bias appeared to be an uneven heat distribution during the SOLiD emulsion PCR (ePCR) - for enrichment of libraries prior loading - since ePCR in either small pouches or in 96-well plates improved the GC bias. Sequencing of chromatin immunoprecipitated DNA (ChIP-seq) is a common approach in epigenetics. ChIP-seq of the mixed source histone mark H3K9ac (acetyl Histone H3 lysine 9), typically found on promoter regions and on gene bodies, including CpG islands, performed on a SOLiD 5500xl machine, resulted in major loss of reads at GC rich loci (GC content ≥ 62%), not explained by low sequencing depth. This was improved with adaptations of the ePCR.

Phylogenetic characterization of a biogas plant microbial community integrating clone library 16S-rDNA sequences and metagenome sequence data obtained by 454-pyrosequencing.

PubMed

Kröber, Magdalena; Bekel, Thomas; Diaz, Naryttza N; Goesmann, Alexander; Jaenicke, Sebastian; Krause, Lutz; Miller, Dimitri; Runte, Kai J; Viehöver, Prisca; Pühler, Alfred; Schlüter, Andreas

2009-06-01

The phylogenetic structure of the microbial community residing in a fermentation sample from a production-scale biogas plant fed with maize silage, green rye and liquid manure was analysed by an integrated approach using clone library sequences and metagenome sequence data obtained by 454-pyrosequencing. Sequencing of 109 clones from a bacterial and an archaeal 16S-rDNA amplicon library revealed that the obtained nucleotide sequences are similar but not identical to 16S-rDNA database sequences derived from different anaerobic environments including digestors and bioreactors. Most of the bacterial 16S-rDNA sequences could be assigned to the phylum Firmicutes with the most abundant class Clostridia and to the class Bacteroidetes, whereas most archaeal 16S-rDNA sequences cluster close to the methanogen Methanoculleus bourgensis. Further sequences of the archaeal library most probably represent so far non-characterised species within the genus Methanoculleus. A similar result derived from phylogenetic analysis of mcrA clone sequences. The mcrA gene product encodes the alpha-subunit of methyl-coenzyme-M reductase involved in the final step of methanogenesis. BLASTn analysis applying stringent settings resulted in assignment of 16S-rDNA metagenome sequence reads to 62 16S-rDNA amplicon sequences thus enabling frequency of abundance estimations for 16S-rDNA clone library sequences. Ribosomal Database Project (RDP) Classifier processing of metagenome 16S-rDNA reads revealed abundance of the phyla Firmicutes, Bacteroidetes and Euryarchaeota and the orders Clostridiales, Bacteroidales and Methanomicrobiales. Moreover, a large fraction of 16S-rDNA metagenome reads could not be assigned to lower taxonomic ranks, demonstrating that numerous microorganisms in the analysed fermentation sample of the biogas plant are still unclassified or unknown.

Computational design of chimeric protein libraries for directed evolution.

PubMed

Silberg, Jonathan J; Nguyen, Peter Q; Stevenson, Taylor

2010-01-01

The best approach for creating libraries of functional proteins with large numbers of nondisruptive amino acid substitutions is protein recombination, in which structurally related polypeptides are swapped among homologous proteins. Unfortunately, as more distantly related proteins are recombined, the fraction of variants having a disrupted structure increases. One way to enrich the fraction of folded and potentially interesting chimeras in these libraries is to use computational algorithms to anticipate which structural elements can be swapped without disturbing the integrity of a protein's structure. Herein, we describe how the algorithm Schema uses the sequences and structures of the parent proteins recombined to predict the structural disruption of chimeras, and we outline how dynamic programming can be used to find libraries with a range of amino acid substitution levels that are enriched in variants with low Schema disruption.

Winnowing DNA for Rare Sequences: Highly Specific Sequence and Methylation Based Enrichment

PubMed Central

Thompson, Jason D.; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

2012-01-01

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue. PMID:22355378

[Current applications of high-throughput DNA sequencing technology in antibody drug research].

PubMed

Yu, Xin; Liu, Qi-Gang; Wang, Ming-Rong

2012-03-01

Since the publication of a high-throughput DNA sequencing technology based on PCR reaction was carried out in oil emulsions in 2005, high-throughput DNA sequencing platforms have been evolved to a robust technology in sequencing genomes and diverse DNA libraries. Antibody libraries with vast numbers of members currently serve as a foundation of discovering novel antibody drugs, and high-throughput DNA sequencing technology makes it possible to rapidly identify functional antibody variants with desired properties. Herein we present a review of current applications of high-throughput DNA sequencing technology in the analysis of antibody library diversity, sequencing of CDR3 regions, identification of potent antibodies based on sequence frequency, discovery of functional genes, and combination with various display technologies, so as to provide an alternative approach of discovery and development of antibody drugs.

Construction of a metagenomic DNA library of sponge symbionts and screening of antibacterial metabolites

NASA Astrophysics Data System (ADS)

Chen, Juan; Zhu, Tianjiao; Li, Dehai; Cui, Chengbin; Fang, Yuchun; Liu, Hongbing; Liu, Peipei; Gu, Qianqun; Zhu, Weiming

2006-04-01

To study the bioactive metabolites produced by sponge-derived uncultured symbionts, a metagenomic DNA library of the symbionts of sponge Gelliodes gracilis was constructed. The average size of DNA inserts in the library was 20 kb. This library was screened for antibiotic activity using paper dise assaying. Two clones displayed the antibacterial activity against Micrococcus tetragenus. The metabolites of these two clones were analyzed through HPLC. The result showed that their metabolites were quite different from those of the host E. coli DH5α and the host containing vector pHZ132. This study may present a new approach to exploring bioactive metabolites of sponge symbionts.

Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

PubMed Central

LeProust, Emily M.; Peck, Bill J.; Spirin, Konstantin; McCuen, Heather Brummel; Moore, Bridget; Namsaraev, Eugeni; Caruthers, Marvin H.

2010-01-01

We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies’ SurePrint® DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology). PMID:20308161

Rapid and continuous magnetic separation in droplet microfluidic devices

DOE PAGES

Brouzes, Eric; Kruse, Travis; Kimmerling, Robert; ...

2014-12-03

Here, we present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization.more » We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries.« less

Characterization of Bleomycin-Mediated Cleavage of a Hairpin DNA Library

PubMed Central

Segerman, Zachary J.; Roy, Basab; Hecht, Sidney M.

2013-01-01

A study of BLM A5 was conducted using a previously isolated library of hairpin DNAs found to bind strongly to metal free BLM. The ability of Fe(II)•BLM to effect cleavage on both the 3' and 5'-arms of the hairpin DNAs was characterized. The strongly bound DNAs were found to be efficient substrates for Fe•BLM A5-mediated hairpin DNA cleavage. Surprisingly, the most prevalent site of BLM-mediated cleavage was found to be the 5′-AT-3′ dinucleotide sequence. This dinucleotide sequence, and other sequences generally not cleaved well by BLM when examined using arbitrarily chosen DNA substrates, were apparent when examining the library of ten hairpin DNAs. In total, 132 sites of DNA cleavage were produced by exposure of the hairpin DNA library to Fe•BLM A5. The existence of multiple sites of cleavage on both the 3′- and 5′-arms of the hairpin DNAs suggested that some of these might be double-strand cleavage events. Accordingly, an assay was developed with which to test the propensity of the hairpin DNAs to undergo double-strand DNA damage. One hairpin DNA was characterized using this method, and gave results consistent with earlier reports of double-strand DNA cleavage, but with a sequence selectivity different from those reported previously. PMID:23834496

The Porcelain Crab Transcriptome and PCAD, the Porcelain Crab Microarray and Sequence Database

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tagmount, Abderrahmane; Wang, Mei; Lindquist, Erika

2010-01-27

Background: With the emergence of a completed genome sequence of the freshwater crustacean Daphnia pulex, construction of genomic-scale sequence databases for additional crustacean sequences are important for comparative genomics and annotation. Porcelain crabs, genus Petrolisthes, have been powerful crustacean models for environmental and evolutionary physiology with respect to thermal adaptation and understanding responses of marine organisms to climate change. Here, we present a large-scale EST sequencing and cDNA microarray database project for the porcelain crab Petrolisthes cinctipes. Methodology/Principal Findings: A set of ~;;30K unique sequences (UniSeqs) representing ~;;19K clusters were generated from ~;;98K high quality ESTs from a set ofmore » tissue specific non-normalized and mixed-tissue normalized cDNA libraries from the porcelain crab Petrolisthes cinctipes. Homology for each UniSeq was assessed using BLAST, InterProScan, GO and KEGG database searches. Approximately 66percent of the UniSeqs had homology in at least one of the databases. All EST and UniSeq sequences along with annotation results and coordinated cDNA microarray datasets have been made publicly accessible at the Porcelain Crab Array Database (PCAD), a feature-enriched version of the Stanford and Longhorn Array Databases.Conclusions/Significance: The EST project presented here represents the third largest sequencing effort for any crustacean, and the largest effort for any crab species. Our assembly and clustering results suggest that our porcelain crab EST data set is equally diverse to the much larger EST set generated in the Daphnia pulex genome sequencing project, and thus will be an important resource to the Daphnia research community. Our homology results support the pancrustacea hypothesis and suggest that Malacostraca may be ancestral to Branchiopoda and Hexapoda. Our results also suggest that our cDNA microarrays cover as much of the transcriptome as can reasonably be captured in EST library sequencing approaches, and thus represent a rich resource for studies of environmental genomics.« less

High-resolution mapping and sequence analysis of 597 cDNA clones transcribed from the 1 Mb region in human chromosome 4q16.3 containing Huntington disease gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hadano, S.; Ishida, Y.; Tomiyasu, H.

1994-09-01

To complete a transcription map of the 1 Mb region in human chromosome 4p16.3 containing the Huntington disease (HD) gene, the isolation of cDNA clones are being performed throughout. Our method relies on a direct screening of the cDNA libraries probed with single copy microclones from 3 YAC clones spanning 1 Mbp of the HD gene region. AC-DNAs were isolated by a preparative pulsed-field gel electrophoresis, amplified by both a single unique primer (SUP)-PCR and a linker ligation PCR, and 6 microclone-DNA libraries were generated. Then, 8,640 microclones from these libraries were independently amplified by PCR, and arrayed onto themore » membranes. 800-900 microclones that were not cross-hybridized with total human and yeast genomic DNA, TAC vector DNA, and ribosomal cDNA on a dot hybridization (putatively carrying single copy sequences) were pooled to make 9 probe pools. A total of {approximately}1.8x10{sup 7} plaques from the human brain cDNA libraries was screened with 9 pool-probes, and then 672 positive cDNA clones were obtained. So far, 597 cDNA clones were defined and arrayed onto a map of the 1 Mbp of the HD gene region by hybridization with HD region-specific cosmid contigs and YAC clones. Further characterization including a DNA sequencing and Northern blot analysis is currently underway.« less

Discovery of User-Oriented Class Associations for Enriching Library Classification Schemes.

ERIC Educational Resources Information Center

Pu, Hsiao-Tieh

2002-01-01

Presents a user-based approach to exploring the possibility of adding user-oriented class associations to hierarchical library classification schemes. Classes not grouped in the same subject hierarchies yet relevant to users' knowledge are obtained by analyzing a log book of a university library's circulation records, using collaborative filtering…

Libraries Alive: Promoting Libraries and Literature--Practical Applications for the Teacher-Librarian.

ERIC Educational Resources Information Center

Boyd, Suzette

Enthusiasm for and a commitment to literature are the essential tools needed to successfully promote the enriching, challenging, and thought-provoking world of books. This paper focuses on the promotion of literature inside the classroom, inside the library, and in the wider community. Programs at Methodist Ladies' College (Australia), a boarding…

Recent advances on the encoding and selection methods of DNA-encoded chemical library.

PubMed

Shi, Bingbing; Zhou, Yu; Huang, Yiran; Zhang, Jianfu; Li, Xiaoyu

2017-02-01

DNA-encoded chemical library (DEL) has emerged as a powerful and versatile tool for ligand discovery in chemical biology research and in drug discovery. Encoding and selection methods are two of the most important technological aspects of DEL that can dictate the performance and utilities of DELs. In this digest, we have summarized recent advances on the encoding and selection strategies of DEL and also discussed the latest developments on DNA-encoded dynamic library, a new frontier in DEL research. Copyright © 2016 Elsevier Ltd. All rights reserved.

DNA Barcode Authentication and Library Development for the Wood of Six Commercial Pterocarpus Species: the Critical Role of Xylarium Specimens.

PubMed

Jiao, Lichao; Yu, Min; Wiedenhoeft, Alex C; He, Tuo; Li, Jianing; Liu, Bo; Jiang, Xiaomei; Yin, Yafang

2018-01-31

DNA barcoding has been proposed as a useful tool for forensic wood identification and development of a reliable DNA reference library is an essential first step. Xylaria (wood collections) are potentially enormous data repositories if DNA information could be extracted from wood specimens. In this study, 31 xylarium wood specimens and 8 leaf specimens of six important commercial species of Pterocarpus were selected to investigate the reliability of DNA barcodes for authentication at the species level and to determine the feasibility of building wood DNA barcode reference libraries from xylarium specimens. Four DNA barcodes (ITS2, matK, ndhF-rpl32 and rbcL) and their combination were tested to evaluate their discrimination ability for Pterocarpus species with both TaxonDNA and tree-based analytical methods. The results indicated that the combination barcode of matK + ndhF-rpl32 + ITS2 yielded the best discrimination for the Pterocarpus species studied. The mini-barcode ndhF-rpl32 (167-173 bps) performed well distinguishing P. santalinus from its wood anatomically inseparable species P. tinctorius. Results from this study verified not only the feasibility of building DNA barcode libraries using xylarium wood specimens, but the importance of using wood rather than leaves as the source tissue, when wood is the botanical material to be identified.

Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin

PubMed Central

Eduardoff, Mayra; Xavier, Catarina; Strobl, Christina; Casas-Vargas, Andrea; Parson, Walther

2017-01-01

The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for final implementation in forensic genetic laboratories. PMID:28934125

Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library

NASA Astrophysics Data System (ADS)

Litovchick, Alexander; Dumelin, Christoph E.; Habeshian, Sevan; Gikunju, Diana; Guié, Marie-Aude; Centrella, Paolo; Zhang, Ying; Sigel, Eric A.; Cuozzo, John W.; Keefe, Anthony D.; Clark, Matthew A.

2015-06-01

A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase.

Effect of organic phosphorus and nitrogen enrichment of mesotrophic lake water on dynamics and diversity of planktonic microbial communities--DNA and protein case studies (mesocosm experiments).

PubMed

Chróst, Ryszard J; Adamczewski, Tomasz; Kalinowska, Krystyna; Skowrońska, Agnieszka

2009-01-01

Effects of mesotrophic lake water enrichment with organic phosphorus and nitrogen substrates (DNA and model protein, bovine serum albumin--BSA) on dynamics and diversity of natural microbial communities (bacteria, heterotrophic nanoflagellates, ciliates) were studied in mesocosm experiments. Simultaneous enrichment with DNA and BSA strongly increased the abundance and biomass of all studied groups of microorganisms and induced changes in their morphological and taxonomic structure. The increased participation of large heterotrophic nanoflagellates cells (larger than 10 microm) in their total numbers and shifts in taxonomic and trophic structure of the ciliates, from algivorous to small bacterivorous, species were observed. Grazing caused changes in bacterial size distribution in all enriched mesocosms. Large (10-50 microm) filamentous bacteria significantly contributed to the total bacterial numbers and biomass. Pronounced increase in populations of beta- and gamma-Proteobacteria was found in lake water enriched with organic P and N sources, whereas alpha-Proteobacteria did not change markedly in the studied mesocosms. DNA additions stimulated the rates of bacterial secondary production. BSA shortened the rates of bacterial biomass turnover in lake water. Relatively high and constant (approximately 30%) percentage contribution of active bacteria (MEM+) in two mesocosms enriched with DNA and DNA+BSA suggested the important role of nucleic acids as a source of phosphorus for bacterial growth, activity and production. Numerous and statistically significant correlations between bacteria and protists indicated the direct and selective predator-prey relationship.

Physical mapping of complex genomes

DOEpatents

Evans, G.A.

1993-06-15

A method for the simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts in the pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert in the common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed.

A Mini-Library of Sequenced Human DNA Fragments: Linking Bench Experiments with Informatics

ERIC Educational Resources Information Center

Dalgleish, Raymond; Shanks, Morag E.; Monger, Karen; Butler, Nicola J.

2012-01-01

We describe the development of a mini-library of human DNA fragments for use in an enquiry-based learning (EBL) undergraduate practical incorporating "wet-lab" and bioinformatics tasks. In spite of the widespread emergence of the polymerase chain reaction (PCR), the cloning and analysis of DNA fragments in "Escherichia coli"…

Shuffle Optimizer: A Program to Optimize DNA Shuffling for Protein Engineering.

PubMed

Milligan, John N; Garry, Daniel J

2017-01-01

DNA shuffling is a powerful tool to develop libraries of variants for protein engineering. Here, we present a protocol to use our freely available and easy-to-use computer program, Shuffle Optimizer. Shuffle Optimizer is written in the Python computer language and increases the nucleotide homology between two pieces of DNA desired to be shuffled together without changing the amino acid sequence. In addition we also include sections on optimal primer design for DNA shuffling and library construction, a small-volume ultrasonicator method to create sheared DNA, and finally a method to reassemble the sheared fragments and recover and clone the library. The Shuffle Optimizer program and these protocols will be useful to anyone desiring to perform any of the nucleotide homology-dependent shuffling methods.

BiNChE: a web tool and library for chemical enrichment analysis based on the ChEBI ontology.

PubMed

Moreno, Pablo; Beisken, Stephan; Harsha, Bhavana; Muthukrishnan, Venkatesh; Tudose, Ilinca; Dekker, Adriano; Dornfeldt, Stefanie; Taruttis, Franziska; Grosse, Ivo; Hastings, Janna; Neumann, Steffen; Steinbeck, Christoph

2015-02-21

Ontology-based enrichment analysis aids in the interpretation and understanding of large-scale biological data. Ontologies are hierarchies of biologically relevant groupings. Using ontology annotations, which link ontology classes to biological entities, enrichment analysis methods assess whether there is a significant over or under representation of entities for ontology classes. While many tools exist that run enrichment analysis for protein sets annotated with the Gene Ontology, there are only a few that can be used for small molecules enrichment analysis. We describe BiNChE, an enrichment analysis tool for small molecules based on the ChEBI Ontology. BiNChE displays an interactive graph that can be exported as a high-resolution image or in network formats. The tool provides plain, weighted and fragment analysis based on either the ChEBI Role Ontology or the ChEBI Structural Ontology. BiNChE aids in the exploration of large sets of small molecules produced within Metabolomics or other Systems Biology research contexts. The open-source tool provides easy and highly interactive web access to enrichment analysis with the ChEBI ontology tool and is additionally available as a standalone library.

Chemical Space of DNA-Encoded Libraries.

PubMed

Franzini, Raphael M; Randolph, Cassie

2016-07-28

In recent years, DNA-encoded chemical libraries (DECLs) have attracted considerable attention as a potential discovery tool in drug development. Screening encoded libraries may offer advantages over conventional hit discovery approaches and has the potential to complement such methods in pharmaceutical research. As a result of the increased application of encoded libraries in drug discovery, a growing number of hit compounds are emerging in scientific literature. In this review we evaluate reported encoded library-derived structures and identify general trends of these compounds in relation to library design parameters. We in particular emphasize the combinatorial nature of these libraries. Generally, the reported molecules demonstrate the ability of this technology to afford hits suitable for further lead development, and on the basis of them, we derive guidelines for DECL design.

Transcriptomic and physiological analyses of Medicago sativa L. roots in response to lead stress

PubMed Central

Zhang, Shichao; Guo, Qiang; Jin, Yan; Chen, Jingjing; Ma, Hongxia

2017-01-01

Lead (Pb) is one of the nonessential and toxic metals that threaten the environment and human health. Medicago sativa L. is a legume with high salt tolerance and high biomass production. It is not only a globally important forage crop but is also an ideal plant for phytoremediation. However, the biological and molecular mechanisms that respond to heavy metals are still not well defined in M. sativa. In this study, de novo and strand-specific RNA-sequencing was performed to identify genes involved in the Pb stress response in M. sativa roots. A total of 415,350 unigenes were obtained from the assembled cDNA libraries, among which 5,416 were identified as significantly differentially expressed genes (DEGs) (false discovery rate < 0.005) between cDNA libraries from control and Pb-treated plants. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DEGs showed they mainly clustered with terms associated with binding, transport, membranes, and the pathways related to signal and energy metabolism. Moreover, a number of candidate genes included antioxidant enzymes, metal transporters, and transcription factors involved in heavy metal response were upregulated under Pb stress. Quantitative real-time PCR(qRT-PCR) validation of the expression patterns of 10 randomly selected candidate DEGs were consistent with the transcriptome analysis results. Thus, this study offers new information towards the investigation of biological changes and molecular mechanisms related to Pb stress response in plants. PMID:28388670

Sialome of a Generalist Lepidopteran Herbivore: Identification of Transcripts and Proteins from Helicoverpa armigera Labial Salivary Glands

PubMed Central

Celorio-Mancera, Maria de la Paz; Courtiade, Juliette; Muck, Alexander; Heckel, David G.; Musser, Richard O.; Vogel, Heiko

2011-01-01

Although the importance of insect saliva in insect-host plant interactions has been acknowledged, there is very limited information on the nature and complexity of the salivary proteome in lepidopteran herbivores. We inspected the labial salivary transcriptome and proteome of Helicoverpa armigera, an important polyphagous pest species. To identify the majority of the salivary proteins we have randomly sequenced 19,389 expressed sequence tags (ESTs) from a normalized cDNA library of salivary glands. In parallel, a non-cytosolic enriched protein fraction was obtained from labial salivary glands and subjected to two-dimensional gel electrophoresis (2-DE) and de novo peptide sequencing. This procedure allowed comparison of peptides and EST sequences and enabled us to identify 65 protein spots from the secreted labial saliva 2DE proteome. The mass spectrometry analysis revealed ecdysone, glucose oxidase, fructosidase, carboxyl/cholinesterase and an uncharacterized protein previously detected in H. armigera midgut proteome. Consistently, their corresponding transcripts are among the most abundant in our cDNA library. We did find redundancy of sequence identification of saliva-secreted proteins suggesting multiple isoforms. As expected, we found several enzymes responsible for digestion and plant offense. In addition, we identified non-digestive proteins such as an arginine kinase and abundant proteins of unknown function. This identification of secreted salivary gland proteins allows a more comprehensive understanding of insect feeding and poses new challenges for the elucidation of protein function. PMID:22046331

Single Cell Transcriptomics of Hypothalamic Warm Sensitive Neurons that Control Core Body Temperature and Fever Response

PubMed Central

Eberwine, James; Bartfai, Tamas

2011-01-01

We report on an ‘unbiased’ molecular characterization of individual, adult neurons, active in a central, anterior hypothalamic neuronal circuit, by establishing cDNA libraries from each individual, electrophysiologically identified warm sensitive neuron (WSN). The cDNA libraries were analyzed by Affymetrix microarray. The presence and frequency of cDNAs was confirmed and enhanced with Illumina sequencing of each single cell cDNA library. cDNAs encoding the GABA biosynthetic enzyme. GAD1 and of adrenomedullin, galanin, prodynorphin, somatostatin, and tachykinin were found in the WSNs. The functional cellular and in vivo studies on dozens of the more than 500 neurotransmitter -, hormone- receptors and ion channels, whose cDNA was identified and sequence confirmed, suggest little or no discrepancy between the transcriptional and functional data in WSNs; whenever agonists were available for a receptor whose cDNA was identified, a functional response was found.. Sequencing single neuron libraries permitted identification of rarely expressed receptors like the insulin receptor, adiponectin receptor2 and of receptor heterodimers; information that is lost when pooling cells leads to dilution of signals and mixing signals. Despite the common electrophysiological phenotype and uniform GAD1 expression, WSN- transcriptomes show heterogenity, suggesting strong epigenetic influence on the transcriptome. Our study suggests that it is well-worth interrogating the cDNA libraries of single neurons by sequencing and chipping. PMID:20970451

An optimized methodology for whole genome sequencing of RNA respiratory viruses from nasopharyngeal aspirates.

PubMed

Goya, Stephanie; Valinotto, Laura E; Tittarelli, Estefania; Rojo, Gabriel L; Nabaes Jodar, Mercedes S; Greninger, Alexander L; Zaiat, Jonathan J; Marti, Marcelo A; Mistchenko, Alicia S; Viegas, Mariana

2018-01-01

Over the last decade, the number of viral genome sequences deposited in available databases has grown exponentially. However, sequencing methodology vary widely and many published works have relied on viral enrichment by viral culture or nucleic acid amplification with specific primers rather than through unbiased techniques such as metagenomics. The genome of RNA viruses is highly variable and these enrichment methodologies may be difficult to achieve or may bias the results. In order to obtain genomic sequences of human respiratory syncytial virus (HRSV) from positive nasopharyngeal aspirates diverse methodologies were evaluated and compared. A total of 29 nearly complete and complete viral genomes were obtained. The best performance was achieved with a DNase I treatment to the RNA directly extracted from the nasopharyngeal aspirate (NPA), sequence-independent single-primer amplification (SISPA) and library preparation performed with Nextera XT DNA Library Prep Kit with manual normalization. An average of 633,789 and 1,674,845 filtered reads per library were obtained with MiSeq and NextSeq 500 platforms, respectively. The higher output of NextSeq 500 was accompanied by the increasing of duplicated reads percentage generated during SISPA (from an average of 1.5% duplicated viral reads in MiSeq to an average of 74% in NextSeq 500). HRSV genome recovery was not affected by the presence or absence of duplicated reads but the computational demand during the analysis was increased. Considering that only samples with viral load ≥ E+06 copies/ml NPA were tested, no correlation between sample viral loads and number of total filtered reads was observed, nor with the mapped viral reads. The HRSV genomes showed a mean coverage of 98.46% with the best methodology. In addition, genomes of human metapneumovirus (HMPV), human rhinovirus (HRV) and human parainfluenza virus types 1-3 (HPIV1-3) were also obtained with the selected optimal methodology.

Laser Capture Microdissection for Protein and NanoString RNA analysis

PubMed Central

Golubeva, Yelena; Salcedo, Rosalba; Mueller, Claudius; Liotta, Lance A.; Espina, Virginia

2013-01-01

Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly, or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are a) visualization of cells via light microscopy, b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810nm), while in the cutting mode the laser is ultraviolet (355nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes laser capture microdissection using an ArcturusXT instrument for protein LCM sample analysis, and using a mmi CellCut Plus® instrument for RNA analysis via NanoString technology. PMID:23027006

Ultra-deep sequencing enables high-fidelity recovery of biodiversity for bulk arthropod samples without PCR amplification

PubMed Central

2013-01-01

Background Next-generation-sequencing (NGS) technologies combined with a classic DNA barcoding approach have enabled fast and credible measurement for biodiversity of mixed environmental samples. However, the PCR amplification involved in nearly all existing NGS protocols inevitably introduces taxonomic biases. In the present study, we developed new Illumina pipelines without PCR amplifications to analyze terrestrial arthropod communities. Results Mitochondrial enrichment directly followed by Illumina shotgun sequencing, at an ultra-high sequence volume, enabled the recovery of Cytochrome c Oxidase subunit 1 (COI) barcode sequences, which allowed for the estimation of species composition at high fidelity for a terrestrial insect community. With 15.5 Gbp Illumina data, approximately 97% and 92% were detected out of the 37 input Operational Taxonomic Units (OTUs), whether the reference barcode library was used or not, respectively, while only 1 novel OTU was found for the latter. Additionally, relatively strong correlation between the sequencing volume and the total biomass was observed for species from the bulk sample, suggesting a potential solution to reveal relative abundance. Conclusions The ability of the new Illumina PCR-free pipeline for DNA metabarcoding to detect small arthropod specimens and its tendency to avoid most, if not all, false positives suggests its great potential in biodiversity-related surveillance, such as in biomonitoring programs. However, further improvement for mitochondrial enrichment is likely needed for the application of the new pipeline in analyzing arthropod communities at higher diversity. PMID:23587339

Methylation-sensitive enrichment of minor DNA alleles using a double-strand DNA-specific nuclease.

PubMed

Liu, Yibin; Song, Chen; Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Makrigiorgos, G Mike

2017-04-07

Aberrant methylation changes, often present in a minor allelic fraction in clinical samples such as plasma-circulating DNA (cfDNA), are potentially powerful prognostic and predictive biomarkers in human disease including cancer. We report on a novel, highly-multiplexed approach to facilitate analysis of clinically useful methylation changes in minor DNA populations. Methylation Specific Nuclease-assisted Minor-allele Enrichment (MS-NaME) employs a double-strand-specific DNA nuclease (DSN) to remove excess DNA with normal methylation patterns. The technique utilizes oligonucleotide-probes that direct DSN activity to multiple targets in bisulfite-treated DNA, simultaneously. Oligonucleotide probes targeting unmethylated sequences generate local double stranded regions resulting to digestion of unmethylated targets, and leaving methylated targets intact; and vice versa. Subsequent amplification of the targeted regions results in enrichment of the targeted methylated or unmethylated minority-epigenetic-alleles. We validate MS-NaME by demonstrating enrichment of RARb2, ATM, MGMT and GSTP1 promoters in multiplexed MS-NaME reactions (177-plex) using dilutions of methylated/unmethylated DNA and in DNA from clinical lung cancer samples and matched normal tissue. MS-NaME is a highly scalable single-step approach performed at the genomic DNA level in solution that combines with most downstream detection technologies including Sanger sequencing, methylation-sensitive-high-resolution melting (MS-HRM) and methylation-specific-Taqman-based-digital-PCR (digital Methylight) to boost detection of low-level aberrant methylation-changes. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library.

PubMed

Traxlmayr, Michael W; Kiefer, Jonathan D; Srinivas, Raja R; Lobner, Elisabeth; Tisdale, Alison W; Mehta, Naveen K; Yang, Nicole J; Tidor, Bruce; Wittrup, K Dane

2016-10-21

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (T m of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A new method for the construction of a mutant library with a predictable occurrence rate using Poisson distribution.

PubMed

Seong, Ki Moon; Park, Hweon; Kim, Seong Jung; Ha, Hyo Nam; Lee, Jae Yung; Kim, Joon

2007-06-01

A yeast transcriptional activator, Gcn4p, induces the expression of genes that are involved in amino acid and purine biosynthetic pathways under amino acid starvation. Gcn4p has an acidic activation domain in the central region and a bZIP domain in the C-terminus that is divided into the DNA-binding motif and dimerization leucine zipper motif. In order to identify amino acids in the DNA-binding motif of Gcn4p which are involved in transcriptional activation, we constructed mutant libraries in the DNA-binding motif through an innovative application of random mutagenesis. Mutant library made by oligonucleotides which were mutated randomly using the Poisson distribution showed that the actual mutation frequency was in good agreement with expected values. This method could save the time and effort to create a mutant library with a predictable mutation frequency. Based on the studies using the mutant libraries constructed by the new method, the specific residues of the DNA-binding domain in Gcn4p appear to be involved in the transcriptional activities on a conserved binding site.

Libraries of Synthetic TALE-Activated Promoters: Methods and Applications.

PubMed

Schreiber, T; Tissier, A

2016-01-01

The discovery of proteins with programmable DNA-binding specificities triggered a whole array of applications in synthetic biology, including genome editing, regulation of transcription, and epigenetic modifications. Among those, transcription activator-like effectors (TALEs) due to their natural function as transcription regulators, are especially well-suited for the development of orthogonal systems for the control of gene expression. We describe here the construction and testing of libraries of synthetic TALE-activated promoters which are under the control of a single TALE with a given DNA-binding specificity. These libraries consist of a fixed DNA-binding element for the TALE, a TATA box, and variable sequences of 19 bases upstream and 43 bases downstream of the DNA-binding element. These libraries were cloned using a Golden Gate cloning strategy making them usable as standard parts in a modular cloning system. The broad range of promoter activities detected and the versatility of these promoter libraries make them valuable tools for applications in the fine-tuning of expression in metabolic engineering projects or in the design and implementation of regulatory circuits. © 2016 Elsevier Inc. All rights reserved.

Apg-2 has a chaperone-like activity similar to Hsp110 and is overexpressed in hepatocellular carcinomas.

PubMed

Gotoh, Kazuhisa; Nonoguchi, Kohsuke; Higashitsuji, Hiroaki; Kaneko, Yoshiyuki; Sakurai, Toshiharu; Sumitomo, Yasuhiko; Itoh, Katsuhiko; Subjeck, John R; Fujita, Jun

2004-02-27

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. We constructed subtracted cDNA libraries enriched with genes overexpressed in HCCs. Among the 17 genes identified were molecular chaperones, Hsp110, Hsp90B, and Hsp70-1. Expression of the Hsp110 family members was further analyzed, and increased transcript levels of Hsp110 and Apg-2, but not Apg-1, were found in 12 and 14, respectively, of 18 HCCs. Immunohistochemical analysis demonstrated the overexpression of the proteins in tumor cells. Apg-2 had chaperone ability similar to Hsp110 in a thermal denaturation assay using luciferase, and showed anti-apoptotic activity. These results suggest that the Hsp110 family members play important roles in hepatocarcinogenesis through their chaperoning activities.

In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies.

PubMed

Lipi, Farhana; Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N

2016-12-01

Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries.

In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies

PubMed Central

Chen, Suxiang; Chakravarthy, Madhuri; Rakesh, Shilpa; Veedu, Rakesh N.

2016-01-01

ABSTRACT Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries. PMID:27715478

Metadata Harvesting in Regional Digital Libraries in the PIONIER Network

ERIC Educational Resources Information Center

Mazurek, Cezary; Stroinski, Maciej; Werla, Marcin; Weglarz, Jan

2006-01-01

Purpose: The paper aims to present the concept of the functionality of metadata harvesting for regional digital libraries, based on the OAI-PMH protocol. This functionality is a part of regional digital libraries platform created in Poland. The platform was required to reach one of main objectives of the Polish PIONIER Programme--to enrich the…

Comparison of EBV DNA viral load in whole blood, plasma, B-cells and B-cell culture supernatant.

PubMed

Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard

2014-05-01

Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B-cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios. © 2013 Wiley Periodicals, Inc.

Enrichment of individual KIR2DL4 sequences from genomic DNA using long-template PCR and allele-specific hybridization to magnetic bead-bound oligonucleotide probes.

PubMed

Roberts, C H; Turino, C; Madrigal, J A; Marsh, S G E

2007-06-01

DNA enrichment by allele-specific hybridization (DEASH) was used as a means to isolate individual alleles of the killer cell immunoglobulin-like receptor (KIR2DL4) gene from heterozygous genomic DNA. Using long-template polymerase chain reaction (LT-PCR), the complete KIR2DL4 gene was amplified from a cell line that had previously been characterized for its KIR gene content by PCR using sequence-specific primers (PCR-SSP). The whole gene amplicons were sequenced and we identified two heterozygous positions in accordance with the predictions of the PCR-SSP. The amplicons were then hybridized to allele-specific, biotinylated oligonucleotide probes and through binding to streptavidin-coated beads, the targeted alleles were enriched. A second PCR amplified only the exonic regions of the enriched allele, and these were then sequenced in full. We show DEASH to be capable of enriching single alleles from a heterozygous PCR product, and through sequencing the enriched DNA, we are able to produce complete coding sequences of the KIR2DL4 alleles in accordance with the typing predicted by PCR-SSP.

BOKP: A DNA Barcode Reference Library for Monitoring Herbal Drugs in the Korean Pharmacopeia

PubMed Central

Liu, Jinxin; Shi, Linchun; Song, Jingyuan; Sun, Wei; Han, Jianping; Liu, Xia; Hou, Dianyun; Yao, Hui; Li, Mingyue; Chen, Shilin

2017-01-01

Herbal drug authentication is an important task in traditional medicine; however, it is challenged by the limitations of traditional authentication methods and the lack of trained experts. DNA barcoding is conspicuous in almost all areas of the biological sciences and has already been added to the British pharmacopeia and Chinese pharmacopeia for routine herbal drug authentication. However, DNA barcoding for the Korean pharmacopeia still requires significant improvements. Here, we present a DNA barcode reference library for herbal drugs in the Korean pharmacopeia and developed a species identification engine named KP-IDE to facilitate the adoption of this DNA reference library for the herbal drug authentication. Using taxonomy records, specimen records, sequence records, and reference records, KP-IDE can identify an unknown specimen. Currently, there are 6,777 taxonomy records, 1,054 specimen records, 30,744 sequence records (ITS2 and psbA-trnH) and 285 reference records. Moreover, 27 herbal drug materials were collected from the Seoul Yangnyeongsi herbal medicine market to give an example for real herbal drugs authentications. Our study demonstrates the prospects of the DNA barcode reference library for the Korean pharmacopeia and provides future directions for the use of DNA barcoding for authenticating herbal drugs listed in other modern pharmacopeias. PMID:29326593

A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA.

PubMed

Ludgate, Jackie L; Wright, James; Stockwell, Peter A; Morison, Ian M; Eccles, Michael R; Chatterjee, Aniruddha

2017-08-31

Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis. Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing. The main features and advantages of this protocol are: An optimized method for extracting good quality DNA from FFPE tissues. An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue. Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing. We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.

Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada.

PubMed

Kuzmina, Maria L; Braukmann, Thomas W A; Fazekas, Aron J; Graham, Sean W; Dewaard, Stephanie L; Rodrigues, Anuar; Bennett, Bruce A; Dickinson, Timothy A; Saarela, Jeffery M; Catling, Paul M; Newmaster, Steven G; Percy, Diana M; Fenneman, Erin; Lauron-Moreau, Aurélien; Ford, Bruce; Gillespie, Lynn; Subramanyam, Ragupathy; Whitton, Jeannette; Jennings, Linda; Metsger, Deborah; Warne, Connor P; Brown, Allison; Sears, Elizabeth; Dewaard, Jeremy R; Zakharov, Evgeny V; Hebert, Paul D N

2017-12-01

Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa.

Using herbarium-derived DNAs to assemble a large-scale DNA barcode library for the vascular plants of Canada1

PubMed Central

Kuzmina, Maria L.; Braukmann, Thomas W. A.; Fazekas, Aron J.; Graham, Sean W.; Dewaard, Stephanie L.; Rodrigues, Anuar; Bennett, Bruce A.; Dickinson, Timothy A.; Saarela, Jeffery M.; Catling, Paul M.; Newmaster, Steven G.; Percy, Diana M.; Fenneman, Erin; Lauron-Moreau, Aurélien; Ford, Bruce; Gillespie, Lynn; Subramanyam, Ragupathy; Whitton, Jeannette; Jennings, Linda; Metsger, Deborah; Warne, Connor P.; Brown, Allison; Sears, Elizabeth; Dewaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

2017-01-01

Premise of the study: Constructing complete, accurate plant DNA barcode reference libraries can be logistically challenging for large-scale floras. Here we demonstrate the promise and challenges of using herbarium collections for building a DNA barcode reference library for the vascular plant flora of Canada. Methods: Our study examined 20,816 specimens representing 5076 of 5190 vascular plant species in Canada (98%). For 98% of the specimens, at least one of the DNA barcode regions was recovered from the plastid loci rbcL and matK and from the nuclear ITS2 region. We used beta regression to quantify the effects of age, type of preservation, and taxonomic affiliation (family) on DNA sequence recovery. Results: Specimen age and method of preservation had significant effects on sequence recovery for all markers, but influenced some families more (e.g., Boraginaceae) than others (e.g., Asteraceae). Discussion: Our DNA barcode library represents an unparalleled resource for metagenomic and ecological genetic research working on temperate and arctic biomes. An observed decline in sequence recovery with specimen age may be associated with poor primer matches, intragenomic variation (for ITS2), or inhibitory secondary compounds in some taxa. PMID:29299394

Construction of cDNA library and preliminary analysis of expressed sequence tags from Siberian tiger

PubMed Central

Liu, Chang-Qing; Lu, Tao-Feng; Feng, Bao-Gang; Liu, Dan; Guan, Wei-Jun; Ma, Yue-Hui

2010-01-01

In this study we successfully constructed a full-length cDNA library from Siberian tiger, Panthera tigris altaica, the most well-known wild Animal. Total RNA was extracted from cultured Siberian tiger fibroblasts in vitro. The titers of primary and amplified libraries were 1.30×106 pfu/ml and 1.62×109 pfu/ml respectively. The proportion of recombinants from unamplified library was 90.5% and average length of exogenous inserts was 1.13 kb. A total of 282 individual ESTs with sizes ranging from 328 to 1,142bps were then analyzed the BLASTX score revealed that 53.9% of the sequences were classified as strong match, 38.6% as nominal and 7.4% as weak match. 28.0% of them were found to be related to enzyme/catalytic protein, 20.9% ESTs to metabolism, 13.1% ESTs to transport, 12.1% ESTs to signal transducer/cell communication, 9.9% ESTs to structure protein, 3.9% ESTs to immunity protein/defense metabolism, 3.2% ESTs to cell cycle, and 8.9 ESTs classified as novel genes. These results demonstrated that the reliability and representativeness of the cDNA library attained to the requirements of a standard cDNA library. This library provided a useful platform for the functional genomic research of Siberian tigers. PMID:20941376

Simple, multiplexed, PCR-based barcoding of DNA enables sensitive mutation detection in liquid biopsies using sequencing.

PubMed

Ståhlberg, Anders; Krzyzanowski, Paul M; Jackson, Jennifer B; Egyud, Matthew; Stein, Lincoln; Godfrey, Tony E

2016-06-20

Detection of cell-free DNA in liquid biopsies offers great potential for use in non-invasive prenatal testing and as a cancer biomarker. Fetal and tumor DNA fractions however can be extremely low in these samples and ultra-sensitive methods are required for their detection. Here, we report an extremely simple and fast method for introduction of barcodes into DNA libraries made from 5 ng of DNA. Barcoded adapter primers are designed with an oligonucleotide hairpin structure to protect the molecular barcodes during the first rounds of polymerase chain reaction (PCR) and prevent them from participating in mis-priming events. Our approach enables high-level multiplexing and next-generation sequencing library construction with flexible library content. We show that uniform libraries of 1-, 5-, 13- and 31-plex can be generated. Utilizing the barcodes to generate consensus reads for each original DNA molecule reduces background sequencing noise and allows detection of variant alleles below 0.1% frequency in clonal cell line DNA and in cell-free plasma DNA. Thus, our approach bridges the gap between the highly sensitive but specific capabilities of digital PCR, which only allows a limited number of variants to be analyzed, with the broad target capability of next-generation sequencing which traditionally lacks the sensitivity to detect rare variants. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Polymorphic DNA microsatellite markers for forensic individual identification and parentage analyses of seven threatened species of parrots (family Psittacidae)

PubMed Central

Jan, Catherine

2016-01-01

The parrot family represents one of the bird group with the largest number of endangered species, as a result of habitat destruction and illegal trade. This illicit traffic involves the smuggling of eggs and animals, and the laundering through captive breeding facilities of wild-caught animals. Despite the huge potential of wildlife DNA forensics to determine with conclusive evidence illegal trade, current usage of DNA profiling approaches in parrots has been limited by the lack of suitable molecular markers specifically developed for the focal species and by low cross-species polymorphism. In this study, we isolated DNA microsatellite markers in seven parrot species threatened with extinction (Amazona brasiliensis, A. oratrix, A. pretrei, A. rhodocorytha, Anodorhynchus leari, Ara rubrogenys and Primolius couloni). From an enriched genomic library followed by 454 pyrosequencing, we characterized a total of 106 polymorphic microsatellite markers (mostly tetranucleotides) in the seven species and tested them across an average number of 19 individuals per species. The mean number of alleles per species and across loci varied from 6.4 to 8.3, with the mean observed heterozygosities ranging from 0.65 to 0.84. Identity and parentage exclusion probabilities were highly discriminatory. The high variability displayed by these microsatellite loci demonstrates their potential utility to perform individual genotyping and parentage analyses, in order to develop a DNA testing framework to determine illegal traffic in these threatened species. PMID:27688959

Polymorphic DNA microsatellite markers for forensic individual identification and parentage analyses of seven threatened species of parrots (family Psittacidae).

PubMed

Jan, Catherine; Fumagalli, Luca

2016-01-01

The parrot family represents one of the bird group with the largest number of endangered species, as a result of habitat destruction and illegal trade. This illicit traffic involves the smuggling of eggs and animals, and the laundering through captive breeding facilities of wild-caught animals. Despite the huge potential of wildlife DNA forensics to determine with conclusive evidence illegal trade, current usage of DNA profiling approaches in parrots has been limited by the lack of suitable molecular markers specifically developed for the focal species and by low cross-species polymorphism. In this study, we isolated DNA microsatellite markers in seven parrot species threatened with extinction (Amazona brasiliensis, A. oratrix, A. pretrei, A. rhodocorytha, Anodorhynchus leari, Ara rubrogenys and Primolius couloni). From an enriched genomic library followed by 454 pyrosequencing, we characterized a total of 106 polymorphic microsatellite markers (mostly tetranucleotides) in the seven species and tested them across an average number of 19 individuals per species. The mean number of alleles per species and across loci varied from 6.4 to 8.3, with the mean observed heterozygosities ranging from 0.65 to 0.84. Identity and parentage exclusion probabilities were highly discriminatory. The high variability displayed by these microsatellite loci demonstrates their potential utility to perform individual genotyping and parentage analyses, in order to develop a DNA testing framework to determine illegal traffic in these threatened species.

Analyses of chicken immunoglobulin light chain cDNA clones indicate a few germline V lambda genes and allotypes of the C lambda locus.

PubMed

Parvari, R; Ziv, E; Lentner, F; Tel-Or, S; Burstein, Y; Schechter, I

1987-01-01

cDNA libraries of chicken spleen and Harder gland (a gland enriched with immunocytes) constructed in pBR322 were screened by differential hybridization and by mRNA hybrid-selected translation. Eleven L-chain cDNA clones were identified from which VL probes were prepared and each was annealed with kidney DNA restriction digests. All VL probes revealed the same set of bands, corresponding to about 15 germline VL genes of one subgroup. The nucleotide sequences of six VL clones showed greater than or equal to 85% homology, and the predicted amino acid sequences were identical or nearly identical to the major N-terminal sequence of L-chains in chicken serum. These findings, and the fact that the VL clones were randomly selected from normal lymphoid tissues, strongly indicate that the bulk of chicken L-chains is encoded by a few germline VL genes, probably much less than 15 since many of the VL genes are known to be pseudogenes. Therefore, it is likely that somatic mechanisms operating prior to specific triggering by antigen play a major role in the generation of antibody diversity in chicken. Analysis of the constant region locus (sequencing of CL gene and cDNAs) demonstrate a single CL isotype and suggest the presence of CL allotypes.

Lessons from Library Power: Enriching Teaching and Learning. Final Report of the Evaluation of the National Library Power Initiative, an Initiative of the DeWitt Wallace-Reader's Digest Fund.

ERIC Educational Resources Information Center

Zweizig, Douglas L.; Hopkins, Dianne McAfee

This book presents the results of an evaluation of Library Power, an initiative of the DeWitt Wallace-Reader's Digest Fund that provided support for school library development in 19 communities. Following an introductory chapter, the chapters are organized around key questions of the evaluation. Chapters 2 through 4 address the implementation of…

Identification of immune protective genes of Eimeria maxima through cDNA expression library screening.

PubMed

Yang, XinChao; Li, MengHui; Liu, JianHua; Ji, YiHong; Li, XiangRui; Xu, LiXin; Yan, RuoFeng; Song, XiaoKai

2017-02-16

Eimeria maxima is one of the most prevalent Eimeria species causing avian coccidiosis, and results in huge economic loss to the global poultry industry. Current control strategies, such as anti-coccidial medication and live vaccines have been limited because of their drawbacks. The third generation anticoccidial vaccines including the recombinant vaccines as well as DNA vaccines have been suggested as a promising alternative strategy. To date, only a few protective antigens of E. maxima have been reported. Hence, there is an urgent need to identify novel protective antigens of E. maxima for the development of neotype anticoccidial vaccines. With the aim of identifying novel protective genes of E. maxima, a cDNA expression library of E. maxima sporozoites was constructed using Gateway technology. Subsequently, the cDNA expression library was divided into 15 sub-libraries for cDNA expression library immunization (cDELI) using parasite challenged model in chickens. Protective sub-libraries were selected for the next round of screening until individual protective clones were obtained, which were further sequenced and analyzed. Adopting the Gateway technology, a high-quality entry library was constructed, containing 9.2 × 10 6 clones with an average inserted fragments length of 1.63 kb. The expression library capacity was 2.32 × 10 7 colony-forming units (cfu) with an average inserted fragments length of 1.64 Kb. The expression library was screened using parasite challenged model in chickens. The screening yielded 6 immune protective genes including four novel protective genes of EmJS-1, EmRP, EmHP-1 and EmHP-2, and two known protective genes of EmSAG and EmCKRS. EmJS-1 is the selR domain-containing protein of E. maxima whose function is unknown. EmHP-1 and EmHP-2 are the hypothetical proteins of E. maxima. EmRP and EmSAG are rhomboid-like protein and surface antigen glycoproteins of E. maxima respectively, and involved in invasion of the parasite. Our results provide a cDNA expression library for further screening of T cell stimulating or inhibiting antigens of E. maxima. Moreover, our results provide six candidate protective antigens for developing new vaccines against E. maxima.

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

PubMed

MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

2015-09-14

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

Accelerated construction of a regional DNA-barcode reference library: Caddisflies (Trichoptera) in the Great Smoky Mountains National Park

USGS Publications Warehouse

Zhou, X.; Robinson, J.L.; Geraci, C.J.; Parker, C.R.; Flint, O.S.; Etnier, D.A.; Ruiter, D.; DeWalt, R.E.; Jacobus, L.M.; Hebert, P.D.N.

2011-01-01

Deoxyribonucleic acid (DNA) barcoding is an effective tool for species identification and lifestage association in a wide range of animal taxa. We developed a strategy for rapid construction of a regional DNA-barcode reference library and used the caddisflies (Trichoptera) of the Great Smoky Mountains National Park (GSMNP) as a model. Nearly 1000 cytochrome c oxidase subunit I (COI) sequences, representing 209 caddisfly species previously recorded from GSMNP, were obtained from the global Trichoptera Barcode of Life campaign. Most of these sequences were collected from outside the GSMNP area. Another 645 COI sequences, representing 80 species, were obtained from specimens collected in a 3-d bioblitz (short-term, intense sampling program) in GSMNP. The joint collections provided barcode coverage for 212 species, 91% of the GSMNP fauna. Inclusion of samples from other localities greatly expedited construction of the regional DNA-barcode reference library. This strategy increased intraspecific divergence and decreased average distances to nearest neighboring species, but the DNA-barcode library was able to differentiate 93% of the GSMNP Trichoptera species examined. Global barcoding projects will aid construction of regional DNA-barcode libraries, but local surveys make crucial contributions to progress by contributing rare or endemic species and full-length barcodes generated from high-quality DNA. DNA taxonomy is not a goal of our present work, but the investigation of COI divergence patterns in caddisflies is providing new insights into broader biodiversity patterns in this group and has directed attention to various issues, ranging from the need to re-evaluate species taxonomy with integrated morphological and molecular evidence to the necessity of an appropriate interpretation of barcode analyses and its implications in understanding species diversity (in contrast to a simple claim for barcoding failure).

Illuminating choices for library prep: a comparison of library preparation methods for whole genome sequencing of Cryptococcus neoformans using Illumina HiSeq.

PubMed

Rhodes, Johanna; Beale, Mathew A; Fisher, Matthew C

2014-01-01

The industry of next-generation sequencing is constantly evolving, with novel library preparation methods and new sequencing machines being released by the major sequencing technology companies annually. The Illumina TruSeq v2 library preparation method was the most widely used kit and the market leader; however, it has now been discontinued, and in 2013 was replaced by the TruSeq Nano and TruSeq PCR-free methods, leaving a gap in knowledge regarding which is the most appropriate library preparation method to use. Here, we used isolates from the pathogenic fungi Cryptococcus neoformans var. grubii and sequenced them using the existing TruSeq DNA v2 kit (Illumina), along with two new kits: the TruSeq Nano DNA kit (Illumina) and the NEBNext Ultra DNA kit (New England Biolabs) to provide a comparison. Compared to the original TruSeq DNA v2 kit, both newer kits gave equivalent or better sequencing data, with increased coverage. When comparing the two newer kits, we found little difference in cost and workflow, with the NEBNext Ultra both slightly cheaper and faster than the TruSeq Nano. However, the quality of data generated using the TruSeq Nano DNA kit was superior due to higher coverage at regions of low GC content, and more SNPs identified. Researchers should therefore evaluate their resources and the type of application (and hence data quality) being considered when ultimately deciding on which library prep method to use.

Encoded Library Synthesis Using Chemical Ligation and the Discovery of sEH Inhibitors from a 334-Million Member Library

PubMed Central

Litovchick, Alexander; Dumelin, Christoph E.; Habeshian, Sevan; Gikunju, Diana; Guié, Marie-Aude; Centrella, Paolo; Zhang, Ying; Sigel, Eric A.; Cuozzo, John W.; Keefe, Anthony D.; Clark, Matthew A.

2015-01-01

A chemical ligation method for construction of DNA-encoded small-molecule libraries has been developed. Taking advantage of the ability of the Klenow fragment of DNA polymerase to accept templates with triazole linkages in place of phosphodiesters, we have designed a strategy for chemically ligating oligonucleotide tags using cycloaddition chemistry. We have utilized this strategy in the construction and selection of a small molecule library, and successfully identified inhibitors of the enzyme soluble epoxide hydrolase. PMID:26061191

Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill.

PubMed

Mulet, Magdalena; David, Zoyla; Nogales, Balbina; Bosch, Rafael; Lalucat, Jorge; García-Valdés, Elena

2011-02-01

The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifying Pseudomonas strains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index for Pseudomonas species was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the species P. stutzeri, P. putida, P. anguilliseptica, and P. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novel Pseudomonas species. One isolate was considered representative of a novel P. stutzeri genomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by the rpoD PCR method was a useful tool for evaluating Pseudomonas communities and also for microdiversity studies of Pseudomonas populations.

Microfluidic droplet enrichment for targeted sequencing

PubMed Central

Eastburn, Dennis J.; Huang, Yong; Pellegrino, Maurizio; Sciambi, Adam; Ptáček, Louis J.; Abate, Adam R.

2015-01-01

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications. PMID:25873629

Plans and progress for building a Great Lakes fauna DNA barcode reference library

EPA Science Inventory

DNA reference libraries provide researchers with an important tool for assessing regional biodiversity by allowing unknown genetic sequences to be assigned identities, while also providing a means for taxonomists to validate identifications. Expanding the representation of Great...

Comparative 454 pyrosequencing of transcripts from two olive genotypes during fruit development

PubMed Central

Alagna, Fiammetta; D'Agostino, Nunzio; Torchia, Laura; Servili, Maurizio; Rao, Rosa; Pietrella, Marco; Giuliano, Giovanni; Chiusano, Maria Luisa; Baldoni, Luciana; Perrotta, Gaetano

2009-01-01

Background Despite its primary economic importance, genomic information on olive tree is still lacking. 454 pyrosequencing was used to enrich the very few sequence data currently available for the Olea europaea species and to identify genes involved in expression of fruit quality traits. Results Fruits of Coratina, a widely cultivated variety characterized by a very high phenolic content, and Tendellone, an oleuropein-lacking natural variant, were used as starting material for monitoring the transcriptome. Four different cDNA libraries were sequenced, respectively at the beginning and at the end of drupe development. A total of 261,485 reads were obtained, for an output of about 58 Mb. Raw sequence data were processed using a four step pipeline procedure and data were stored in a relational database with a web interface. Conclusion Massively parallel sequencing of different fruit cDNA collections has provided large scale information about the structure and putative function of gene transcripts accumulated during fruit development. Comparative transcript profiling allowed the identification of differentially expressed genes with potential relevance in regulating the fruit metabolism and phenolic content during ripening. PMID:19709400

A novel extracellular matrix protein from tomato associated with lignified secondary cell walls.

PubMed Central

Domingo, C; Gómez, M D; Cañas, L; Hernández-Yago, J; Conejero, V; Vera, P

1994-01-01

A cDNA clone representing a novel cell wall protein was isolated from a tomato cDNA library. The deduced amino acid sequence shows that the encoded protein is very small (88 amino acids), contains an N-terminal hydrophobic signal peptide, and is enriched in lysine and tyrosine. We have designated this protein TLRP for tyrosine- and lysine-rich protein. RNA gel blot hybridization identified TLRP transcripts constitutively present in roots, stems, and leaves from tomato plants. The encoded protein seems to be highly insolubilized in the cell wall, and we present evidence that this protein is specifically localized in the modified secondary cell walls of the xylem and in cells of the sclerenchyma. In addition, the protein is localized in the protective periderm layer of the growing root. The highly localized deposition in cells destined to give support and protection to the plant indicates that this cell wall protein alone and/or in collaboration with other cell wall structural proteins may have a specialized structural function by mechanically strengthening the walls. PMID:7919979

Identification of Methanoculleus spp. as active methanogens during anoxic incubations of swine manure storage tank samples.

PubMed

Barret, Maialen; Gagnon, Nathalie; Kalmokoff, Martin L; Topp, Edward; Verastegui, Yris; Brooks, Stephen P J; Matias, Fernando; Neufeld, Josh D; Talbot, Guylaine

2013-01-01

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-(13)C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of (13)C into DNA was detectable at in situ acetate concentrations (~7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the (13)C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis.

Identification of Methanoculleus spp. as Active Methanogens during Anoxic Incubations of Swine Manure Storage Tank Samples

PubMed Central

Barret, Maialen; Gagnon, Nathalie; Kalmokoff, Martin L.; Topp, Edward; Verastegui, Yris; Brooks, Stephen P. J.; Matias, Fernando; Neufeld, Josh D.

2013-01-01

Methane emissions represent a major environmental concern associated with manure management in the livestock industry. A more thorough understanding of how microbial communities function in manure storage tanks is a prerequisite for mitigating methane emissions. Identifying the microorganisms that are metabolically active is an important first step. Methanogenic archaea are major contributors to methanogenesis in stored swine manure, and we investigated active methanogenic populations by DNA stable isotope probing (DNA-SIP). Following a preincubation of manure samples under anoxic conditions to induce substrate starvation, [U-13C]acetate was added as a labeled substrate. Fingerprint analysis of density-fractionated DNA, using length-heterogeneity analysis of PCR-amplified mcrA genes (encoding the alpha subunit of methyl coenzyme M reductase), showed that the incorporation of 13C into DNA was detectable at in situ acetate concentrations (∼7 g/liter). Fingerprints of DNA retrieved from heavy fractions of the 13C treatment were primarily enriched in a 483-bp amplicon and, to a lesser extent, in a 481-bp amplicon. Analyses based on clone libraries of the mcrA and 16S rRNA genes revealed that both of these heavy DNA amplicons corresponded to Methanoculleus spp. Our results demonstrate that uncultivated methanogenic archaea related to Methanoculleus spp. were major contributors to acetate-C assimilation during the anoxic incubation of swine manure storage tank samples. Carbon assimilation and dissimilation rate estimations suggested that Methanoculleus spp. were also major contributors to methane emissions and that the hydrogenotrophic pathway predominated during methanogenesis. PMID:23104405

Isolation of a DNA Probe for Lactobacillus curvatus

PubMed Central

Petrick, Hendrik A. R.; Ambrosio, Riccardo E.; Holzapfel, Wilhelm H.

1988-01-01

A genomic library of Lactobacillus curvatus DSM 20019 was constructed in bacteriophage λ gt11. A 1.2-kilobase DNA probe specific for L. curvatus was isolated from this library. When this probe was hybridized to DNA from Lactobacillus isolates from different sources classified by conventional techniques, differing degrees of hybridization were obtained. This could imply that these isolates may have been incorrectly classified. Images PMID:16347554

Citations as Data: Harvesting the Scholarly Record of Your University to Enrich Institutional Knowledge and Support Research

ERIC Educational Resources Information Center

Sterman, Leila Belle; Clark, Jason A.

2017-01-01

Many research libraries are looking for new ways to demonstrate value for their parent institutions. Metrics, assessment, and promotion of research continue to grow in importance, but they have not always fallen into the scope of services for the research library. Montana State University (MSU) Library recognized a need and interest to quantify…

A Century of Change: The Evolution of School Library Resources, 1915-2015

ERIC Educational Resources Information Center

Lamb, Annette

2015-01-01

School libraries have been in existence since at least the eighth century. However, it wasn't until the twentieth century that the school library was seen primarily as "a source of enrichment for the curriculum, and a means of developing reading and study habits in the pupils" (Clyde 1981, 263). While the formats available and tools for…

Enriching screening libraries with bioactive fragment space.

PubMed

Zhang, Na; Zhao, Hongtao

2016-08-01

By deconvoluting 238,073 bioactive molecules in the ChEMBL library into extended Murcko ring systems, we identified a set of 2245 ring systems present in at least 10 molecules. These ring systems belong to 2221 clusters by ECFP4 fingerprints with a minimum intracluster similarity of 0.8. Their overlap with ring systems in commercial libraries was further quantified. Our findings suggest that success of a small fragment library is driven by the convergence of effective coverage of bioactive ring systems (e.g., 10% coverage by 1000 fragments vs. 40% by 2million HTS compounds), high enrichment of bioactive ring systems, and low molecular complexity enhancing the probability of a match with the protein targets. Reconciling with the previous studies, bioactive ring systems are underrepresented in screening libraries. As such, we propose a library of virtual fragments with key functionalities via fragmentation of bioactive molecules. Its utility is exemplified by a prospective application on protein kinase CK2, resulting in the discovery of a series of novel inhibitors with the most potent compound having an IC50 of 0.5μM and a ligand efficiency of 0.41kcal/mol per heavy atom. Copyright © 2016 Elsevier Ltd. All rights reserved.

Single cell transcriptomics of hypothalamic warm sensitive neurons that control core body temperature and fever response Signaling asymmetry and an extension of chemical neuroanatomy.

PubMed

Eberwine, James; Bartfai, Tamas

2011-03-01

We report on an 'unbiased' molecular characterization of individual, adult neurons, active in a central, anterior hypothalamic neuronal circuit, by establishing cDNA libraries from each individual, electrophysiologically identified warm sensitive neuron (WSN). The cDNA libraries were analyzed by Affymetrix microarray. The presence and frequency of cDNAs were confirmed and enhanced with Illumina sequencing of each single cell cDNA library. cDNAs encoding the GABA biosynthetic enzyme Gad1 and of adrenomedullin, galanin, prodynorphin, somatostatin, and tachykinin were found in the WSNs. The functional cellular and in vivo studies on dozens of the more than 500 neurotransmitters, hormone receptors and ion channels, whose cDNA was identified and sequence confirmed, suggest little or no discrepancy between the transcriptional and functional data in WSNs; whenever agonists were available for a receptor whose cDNA was identified, a functional response was found. Sequencing single neuron libraries permitted identification of rarely expressed receptors like the insulin receptor, adiponectin receptor 2 and of receptor heterodimers; information that is lost when pooling cells leads to dilution of signals and mixing signals. Despite the common electrophysiological phenotype and uniform Gad1 expression, WSN transcriptomes show heterogeneity, suggesting strong epigenetic influence on the transcriptome. Our study suggests that it is well-worth interrogating the cDNA libraries of single neurons by sequencing and chipping. Copyright © 2010 Elsevier Inc. All rights reserved.

An efficient and sensitive method for preparing cDNA libraries from scarce biological samples

PubMed Central

Sterling, Catherine H.; Veksler-Lublinsky, Isana; Ambros, Victor

2015-01-01

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate that the method provides a significant improvement in sensitivity compared to previous cloning methods while maintaining reproducible identification of diverse small RNA species. This method should have widespread applications in a variety of contexts, including biomarker discovery from scarce samples of human tissue or body fluids. PMID:25056322

[Construction of fetal mesenchymal stem cell cDNA subtractive library].

PubMed

Yang, Li; Wang, Dong-Mei; Li, Liang; Bai, Ci-Xian; Cao, Hua; Li, Ting-Yu; Pei, Xue-Tao

2002-04-01

To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp. SSH is a convenient and effective method for screening differentially expressed genes. The constructed cDNA subtractive library of fetal MSC cDNA lays solid foundation for screening and cloning new and specific function related genes of fetal MSC.

Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

PubMed Central

Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

2016-01-01

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

The Library Work Order Processing System: A New Approach to Motivate Employees and to Increase Production in the Technical Service Department of Mercer County Community College Library. Applied Educational Research and Evaluation.

ERIC Educational Resources Information Center

Sim, Yong Sup

After reviewing the current movement toward job enrichment, a system was designed for the technical services department of the Mercer County Community College Library. The Library Work Order Processing System, as tried between January and March, 1974, was designed to permit each worker more variety of jobs. The technical services department was…

Identification of genes differentially expressed in association with acquired cisplatin resistance

PubMed Central

Johnsson, A; Zeelenberg, I; Min, Y; Hilinski, J; Berry, C; Howell, S B; Los, G

2000-01-01

The goal of this study was to identify genes whose mRNA levels are differentially expressed in human cells with acquired cisplatin (cDDP) resistance. Using the parental UMSCC10b head and neck carcinoma cell line and the 5.9-fold cDDP-resistant subline, UMSCC10b/Pt-S15, two suppressive subtraction hybridization (SSH) cDNA libraries were prepared. One library represented mRNAs whose levels were increased in the cDDP resistant variant (the UP library), the other one represented mRNAs whose levels were decreased in the resistant cells (the DOWN library). Arrays constructed with inserts recovered from these libraries were hybridized with SSH products to identify truly differentially expressed elements. A total of 51 cDNA fragments present in the UP library and 16 in the DOWN library met the criteria established for differential expression. The sequences of 87% of these cDNA fragments were identified in Genbank. Among the mRNAs in the UP library that were frequently isolated and that showed high levels of differential expression were cytochrome oxidase I, ribosomal protein 28S, elongation factor 1α, α-enolase, stathmin, and HSP70. The approach taken in this study permitted identification of many genes never before linked to the cDDP-resistant phenotype. © 2000 Cancer Research Campaign PMID:10993653

Comparison of Whole-Cell SELEX Methods for the Identification of Staphylococcus Aureus-Specific DNA Aptamers

PubMed Central

Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

2015-01-01

Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria. PMID:25884791

Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus-specific DNA aptamers.

PubMed

Moon, Jihea; Kim, Giyoung; Park, Saet Byeol; Lim, Jongguk; Mo, Changyeun

2015-04-15

Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria.

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

PubMed

Goldman, D; Sapru, M K; Stewart, S; Plotkin, J; Libermann, T A; Wasylyk, B; Guan, K

1998-10-15

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Highly multiplexed targeted DNA sequencing from single nuclei.

PubMed

Leung, Marco L; Wang, Yong; Kim, Charissa; Gao, Ruli; Jiang, Jerry; Sei, Emi; Navin, Nicholas E

2016-02-01

Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

Selection of stable scFv antibodies by phage display.

PubMed

Brockmann, Eeva-Christine

2012-01-01

ScFv fragments are popular recombinant antibody formats but often suffer from limited stability. Phage display is a powerful tool in antibody engineering and applicable also for stability selection. ScFv variants with improved stability can be selected from large randomly mutated phage displayed libraries with a specific antigen after the unstable variants have been inactivated by heat or GdmCl. Irreversible scFv denaturation, which is a prerequisite for efficient selection, is achieved by combining denaturation with reduction of the intradomain disulfide bonds. Repeated selection cycles of increasing stringency result in enrichment of stabilized scFv fragments. Procedures for constructing a randomly mutated scFv library by error-prone PCR and phage display selection for enrichment of stable scFv antibodies from the library are described here.

Construction and characterization of normalized cDNA libraries by 454 pyrosequencing and estimation of DNA methylation levels in three distantly related termite species.

PubMed

Hayashi, Yoshinobu; Shigenobu, Shuji; Watanabe, Dai; Toga, Kouhei; Saiki, Ryota; Shimada, Keisuke; Bourguignon, Thomas; Lo, Nathan; Hojo, Masaru; Maekawa, Kiyoto; Miura, Toru

2013-01-01

In termites, division of labor among castes, categories of individuals that perform specialized tasks, increases colony-level productivity and is the key to their ecological success. Although molecular studies on caste polymorphism have been performed in termites, we are far from a comprehensive understanding of the molecular basis of this phenomenon. To facilitate future molecular studies, we aimed to construct expressed sequence tag (EST) libraries covering wide ranges of gene repertoires in three representative termite species, Hodotermopsis sjostedti, Reticulitermes speratus and Nasutitermes takasagoensis. We generated normalized cDNA libraries from whole bodies, except for guts containing microbes, of almost all castes, sexes and developmental stages and sequenced them with the 454 GS FLX titanium system. We obtained >1.2 million quality-filtered reads yielding >400 million bases for each of the three species. Isotigs, which are analogous to individual transcripts, and singletons were produced by assembling the reads and annotated using public databases. Genes related to juvenile hormone, which plays crucial roles in caste differentiation of termites, were identified from the EST libraries by BLAST search. To explore the potential for DNA methylation, which plays an important role in caste differentiation of honeybees, tBLASTn searches for DNA methyltransferases (dnmt1, dnmt2 and dnmt3) and methyl-CpG binding domain (mbd) were performed against the EST libraries. All four of these genes were found in the H. sjostedti library, while all except dnmt3 were found in R. speratus and N. takasagoensis. The ratio of the observed to the expected CpG content (CpG O/E), which is a proxy for DNA methylation level, was calculated for the coding sequences predicted from the isotigs and singletons. In all of the three species, the majority of coding sequences showed depletion of CpG O/E (less than 1), and the distributions of CpG O/E were bimodal, suggesting the presence of DNA methylation.

Construction and Characterization of Normalized cDNA Libraries by 454 Pyrosequencing and Estimation of DNA Methylation Levels in Three Distantly Related Termite Species

PubMed Central

Hayashi, Yoshinobu; Shigenobu, Shuji; Watanabe, Dai; Toga, Kouhei; Saiki, Ryota; Shimada, Keisuke; Bourguignon, Thomas; Lo, Nathan; Hojo, Masaru; Maekawa, Kiyoto; Miura, Toru

2013-01-01

In termites, division of labor among castes, categories of individuals that perform specialized tasks, increases colony-level productivity and is the key to their ecological success. Although molecular studies on caste polymorphism have been performed in termites, we are far from a comprehensive understanding of the molecular basis of this phenomenon. To facilitate future molecular studies, we aimed to construct expressed sequence tag (EST) libraries covering wide ranges of gene repertoires in three representative termite species, Hodotermopsis sjostedti , Reticulitermessperatus and Nasutitermestakasagoensis . We generated normalized cDNA libraries from whole bodies, except for guts containing microbes, of almost all castes, sexes and developmental stages and sequenced them with the 454 GS FLX titanium system. We obtained >1.2 million quality-filtered reads yielding >400 million bases for each of the three species. Isotigs, which are analogous to individual transcripts, and singletons were produced by assembling the reads and annotated using public databases. Genes related to juvenile hormone, which plays crucial roles in caste differentiation of termites, were identified from the EST libraries by BLAST search. To explore the potential for DNA methylation, which plays an important role in caste differentiation of honeybees, tBLASTn searches for DNA methyltransferases (dnmt1, dnmt2 and dnmt3) and methyl-CpG binding domain (mbd) were performed against the EST libraries. All four of these genes were found in the H . sjostedti library, while all except dnmt3 were found in R . speratus and N . takasagoensis . The ratio of the observed to the expected CpG content (CpG O/E), which is a proxy for DNA methylation level, was calculated for the coding sequences predicted from the isotigs and singletons. In all of the three species, the majority of coding sequences showed depletion of CpG O/E (less than 1), and the distributions of CpG O/E were bimodal, suggesting the presence of DNA methylation. PMID:24098800

Community analysis of hydrogen-producing extreme thermophilic anaerobic microflora enriched from cow manure with five substrates.

PubMed

Yokoyama, Hiroshi; Moriya, Naoko; Ohmori, Hideyuki; Waki, Miyoko; Ogino, Akifumi; Tanaka, Yasuo

2007-11-01

The present study analyzed the community structures of anaerobic microflora producing hydrogen under extreme thermophilic conditions by two culture-independent methods: denaturing gradient gel electrophoresis (DGGE) and clone library analyses. Extreme thermophilic microflora (ETM) was enriched from cow manure by repeated batch cultures at 75 degrees C, using a substrate of xylose, glucose, lactose, cellobiose, or soluble starch, and produced hydrogen at yields of 0.56, 2.65, 2.17, 2.68, and 1.73 mol/mol-monosaccharide degraded, respectively. The results from the DGGE and clone library analyses were consistent and demonstrated that the community structures of ETM enriched with the four hexose-based substrates (glucose, lactose, cellobiose, and soluble starch) consisted of a single species, closely related to a hydrogen-producing extreme thermophile, Caldoanaerobacter subterraneus, with diversity at subspecies levels. The ETM enriched with xylose was more diverse than those enriched with the other substrates, and contained the bacterium related to C. subterraneus and an unclassified bacterium, distantly related to a xylan-degrading and hydrogen-producing extreme thermophile, Caloramator fervidus.

Chemical Biology Probes from Advanced DNA-encoded Libraries.

PubMed

Salamon, Hazem; Klika Škopić, Mateja; Jung, Kathrin; Bugain, Olivia; Brunschweiger, Andreas

2016-02-19

The identification of bioactive compounds is a crucial step toward development of probes for chemical biology studies. Screening of DNA-encoded small molecule libraries (DELs) has emerged as a validated technology to interrogate vast chemical space. DELs consist of chimeric molecules composed of a low-molecular weight compound that is conjugated to a DNA identifier tag. They are screened as pooled libraries using selection to identify "hits." Screening of DELs has identified numerous bioactive compounds. Some of these molecules were instrumental in gaining a deeper understanding of biological systems. One of the main challenges in the field is the development of synthesis methodology for DELs.

Developmental validation of a Nextera XT mitogenome Illumina MiSeq sequencing method for high-quality samples.

PubMed

Peck, Michelle A; Sturk-Andreaggi, Kimberly; Thomas, Jacqueline T; Oliver, Robert S; Barritt-Ross, Suzanne; Marshall, Charla

2018-05-01

Generating mitochondrial genome (mitogenome) data from reference samples in a rapid and efficient manner is critical to harnessing the greater power of discrimination of the entire mitochondrial DNA (mtDNA) marker. The method of long-range target enrichment, Nextera XT library preparation, and Illumina sequencing on the MiSeq is a well-established technique for generating mitogenome data from high-quality samples. To this end, a validation was conducted for this mitogenome method processing up to 24 samples simultaneously along with analysis in the CLC Genomics Workbench and utilizing the AQME (AFDIL-QIAGEN mtDNA Expert) tool to generate forensic profiles. This validation followed the Federal Bureau of Investigation's Quality Assurance Standards (QAS) for forensic DNA testing laboratories and the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines. The evaluation of control DNA, non-probative samples, blank controls, mixtures, and nonhuman samples demonstrated the validity of this method. Specifically, the sensitivity was established at ≥25 pg of nuclear DNA input for accurate mitogenome profile generation. Unreproducible low-level variants were observed in samples with low amplicon yields. Further, variant quality was shown to be a useful metric for identifying sequencing error and crosstalk. Success of this method was demonstrated with a variety of reference sample substrates and extract types. These studies further demonstrate the advantages of using NGS techniques by highlighting the quantitative nature of heteroplasmy detection. The results presented herein from more than 175 samples processed in ten sequencing runs, show this mitogenome sequencing method and analysis strategy to be valid for the generation of reference data. Copyright © 2018 Elsevier B.V. All rights reserved.

Physical mapping of complex genomes

DOEpatents

Evans, Glen A.

1993-01-01

Method for simultaneous identification of overlapping cosmid clones among multiple cosmid clones and the use of the method for mapping complex genomes are provided. A library of cosmid clones that contains the DNA to be mapped is constructed and arranged in a manner such that individual clones can be identified and replicas of the arranged clones prepared. In preferred embodiments, the clones are arranged in a two dimensional matrix. In such embodiments, the cosmid clones in a row are pooled, mixed probes complementary to the ends of the DNA inserts int he pooled clones are synthesized, hybridized to a first replica of the library. Hybridizing clones, which include the pooled row, are identified. A second portion of clones is prepared by pooling cosmid clones that correspond to a column in the matrix. The second pool thereby includes one clone from the first portion pooled clones. This common clone is located on the replica at the intersection of the column and row. Mixed probes complementary to the ends of the DNA inserts in the second pooled portion of clones are prepared and hybridized to a second replica of the library. The hybridization pattern on the first and second replicas of the library are compared and cross-hybridizing clones, other than the clones in the pooled column and row, that hybridize to identical clones in the first and second replicas are identified. These clones necessarily include DNA inserts that overlap with the DNA insert int he common clone located at the intersection of the pooled row and pooled column. The DNA in the entire library may be mapped by pooling the clones in each of the rows and columns of the matrix, preparing mixed end-specific probes and hybridizing the probes from each row or column to a replica of the library. Since all clones in the library are located at the intersection of a column and a row, the overlapping clones for all clones in the library may be identified and a physical map constructed. In other preferred embodiments, the cosmid clones are arranged in a three dimensional matrix, pooled and compared in threes according to intersecting planes of the three dimensional matrix. Arrangements corresponding to geometries of higher dimensions may also be prepared and used to simultaneously identify overlapping clones in highly complex libraries with relatively few hybridization reactions.

Principles and application of antibody libraries for infectious diseases.

PubMed

Lim, Bee Nar; Tye, Gee Jun; Choong, Yee Siew; Ong, Eugene Boon Beng; Ismail, Asma; Lim, Theam Soon

2014-12-01

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

ORF phage display to identify cellular proteins with different functions.

PubMed

Li, Wei

2012-09-01

Open reading frame (ORF) phage display is a new branch of phage display aimed at improving its efficiency to identify cellular proteins with specific binding or functional activities. Despite the success of phage display with antibody libraries and random peptide libraries, phage display with cDNA libraries of cellular proteins identifies a high percentage of non-ORF clones encoding unnatural short peptides with minimal biological implications. This is mainly because of the uncontrollable reading frames of cellular proteins in conventional cDNA libraries. ORF phage display solves this problem by eliminating non-ORF clones to generate ORF cDNA libraries. Here I summarize the procedures of ORF phage display, discuss the factors influencing its efficiency, present examples of its versatile applications, and highlight evidence of its capability of identifying biologically relevant cellular proteins. ORF phage display coupled with different selection strategies is capable of delineating diverse functions of cellular proteins with unique advantages. Copyright © 2012 Elsevier Inc. All rights reserved.

Algorithms for optimizing cross-overs in DNA shuffling.

PubMed

He, Lu; Friedman, Alan M; Bailey-Kellogg, Chris

2012-03-21

DNA shuffling generates combinatorial libraries of chimeric genes by stochastically recombining parent genes. The resulting libraries are subjected to large-scale genetic selection or screening to identify those chimeras with favorable properties (e.g., enhanced stability or enzymatic activity). While DNA shuffling has been applied quite successfully, it is limited by its homology-dependent, stochastic nature. Consequently, it is used only with parents of sufficient overall sequence identity, and provides no control over the resulting chimeric library. This paper presents efficient methods to extend the scope of DNA shuffling to handle significantly more diverse parents and to generate more predictable, optimized libraries. Our CODNS (cross-over optimization for DNA shuffling) approach employs polynomial-time dynamic programming algorithms to select codons for the parental amino acids, allowing for zero or a fixed number of conservative substitutions. We first present efficient algorithms to optimize the local sequence identity or the nearest-neighbor approximation of the change in free energy upon annealing, objectives that were previously optimized by computationally-expensive integer programming methods. We then present efficient algorithms for more powerful objectives that seek to localize and enhance the frequency of recombination by producing "runs" of common nucleotides either overall or according to the sequence diversity of the resulting chimeras. We demonstrate the effectiveness of CODNS in choosing codons and allocating substitutions to promote recombination between parents targeted in earlier studies: two GAR transformylases (41% amino acid sequence identity), two very distantly related DNA polymerases, Pol X and β (15%), and beta-lactamases of varying identity (26-47%). Our methods provide the protein engineer with a new approach to DNA shuffling that supports substantially more diverse parents, is more deterministic, and generates more predictable and more diverse chimeric libraries.

Novel and highly informative Capsicum SSR markers and their cross-species transferability.

PubMed

Buso, G S C; Reis, A M M; Amaral, Z P S; Ferreira, M E

2016-09-23

This study was undertaken primarily to develop new simple sequence repeat (SSR) markers for Capsicum. As part of this project aimed at broadening the use of molecular tools in Capsicum breeding, two genomic libraries enriched for AG/TC repeat sequences were constructed for Capsicum annuum. A total of 475 DNA clones were sequenced from both libraries and 144 SSR markers were tested on cultivated and wild species of Capsicum. Forty-five SSR markers were randomly selected to genotype a panel of 48 accessions of the Capsicum germplasm bank. The number of alleles per locus ranged from 2 to 11, with an average of 6 alleles. The polymorphism information content was on average 0.60, ranging from 0.20 to 0.83. The cross-species transferability to seven cultivated and wild Capsicum species was tested with a set of 91 SSR markers. We found that a high proportion of the loci produced amplicons in all species tested. C. frutescens had the highest number of transferable markers, whereas the wild species had the lowest. Our results indicate that the new markers can be readily used in genetic analyses of Capsicum.

Informational Aspects of Isotopic Diversity in Biology and Medicine

NASA Astrophysics Data System (ADS)

Berezin, Alexander A.

2004-10-01

Use of stable and radioactive isotopes in biology and medicine is intensive, yet informational aspects of isotopes as such are largely neglected (A.A.Berezin, J.Theor.Biol.,1992). Classical distinguishability (``labelability'') of isotopes allows for pattern generation dynamics. Quantum mechanically advantages of isotopicity (diversity of stable isotopes) arise from (almost perfect) degeneracy of various isotopic configurations; this in turn allows for isotopic sweeps (hoppings) by resonance neutron tunneling (Eccles mechanism). Isotopic variations of de Broglie wavelength affect quantum tunneling, diffusivity, magnetic interactions (e.g. by Lorentz force), etc. Ergodicity principle (all isoenergetic states are eventually accessed) implies possibility of fast scanning of library of morphogenetic patterns (cf metaphors of universal ``Platonic'' Library of Patterns: e.g. J.L.Borges, R.Sheldrake) with subsequent Darwinian reinforcement (e.g. by targeted mutations) of evolutionary advantageous patterns and structures. Isotopic shifts in organisms, from viruses and protozoa to mammalians, (e.g. DNA with enriched or depleted C-13) are tools to elucidate possible informational (e.g. Shannon entropy) role of isotopicity in genetic (e.g. evolutionary and morphological), dynamical (e.g. physiological and neurological) as well as medical (e.g. carcinogenesis, aging) aspects of biology and medicine.

Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method.

PubMed

Dalmasso, Marion; Bolocan, Andrei Sorin; Hernandez, Marta; Kapetanakou, Anastasia E; Kuchta, Tomáš; Manios, Stavros G; Melero, Beatriz; Minarovičová, Jana; Muhterem, Meryem; Nicolau, Anca Ioana; Rovira, Jordi; Skandamis, Panagiotis N; Stessl, Beatrix; Wagner, Martin; Jordan, Kieran; Rodríguez-Lázaro, David

2014-03-01

Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second enrichment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1. Copyright © 2014 Elsevier B.V. All rights reserved.

Using Institute of Museum and Library Services Grants to Support Out-of-School Time Programs. Funding Note

ERIC Educational Resources Information Center

Griffin, Shawn Stelow

2010-01-01

Out-of-school time programs give many youth the chance to engage in interesting and enriching opportunities in the arts. One source of funding for art and cultural activities in out-of-school time programs is The Institute of Museum and Library Services (IMLS). This federal agency is charged with creating strong libraries and museums that connect…

Errors in CGAP xProfiler and cDNA DGED: the importance of library parsing and gene selection algorithms.

PubMed

Milnthorpe, Andrew T; Soloviev, Mikhail

2011-04-15

The Cancer Genome Anatomy Project (CGAP) xProfiler and cDNA Digital Gene Expression Displayer (DGED) have been made available to the scientific community over a decade ago and since then were used widely to find genes which are differentially expressed between cancer and normal tissues. The tissue types are usually chosen according to the ontology hierarchy developed by NCBI. The xProfiler uses an internally available flat file database to determine the presence or absence of genes in the chosen libraries, while cDNA DGED uses the publicly available UniGene Expression and Gene relational databases to count the sequences found for each gene in the presented libraries. We discovered that the CGAP approach often includes libraries from dependent or irrelevant tissues (one third of libraries were incorrect on average, with some tissue searches no correct libraries being selected at all). We also discovered that the CGAP approach reported genes from outside the selected libraries and may omit genes found within the libraries. Other errors include the incorrect estimation of the significance values and inaccurate settings for the library size cut-off values. We advocated a revised approach to finding libraries associated with tissues. In doing so, libraries from dependent or irrelevant tissues do not get included in the final library pool. We also revised the method for determining the presence or absence of a gene by searching the UniGene relational database, revised calculation of statistical significance and sorted the library cut-off filter. Our results justify re-evaluation of all previously reported results where NCBI CGAP expression data and tools were used.

Errors in CGAP xProfiler and cDNA DGED: the importance of library parsing and gene selection algorithms

PubMed Central

2011-01-01

Background The Cancer Genome Anatomy Project (CGAP) xProfiler and cDNA Digital Gene Expression Displayer (DGED) have been made available to the scientific community over a decade ago and since then were used widely to find genes which are differentially expressed between cancer and normal tissues. The tissue types are usually chosen according to the ontology hierarchy developed by NCBI. The xProfiler uses an internally available flat file database to determine the presence or absence of genes in the chosen libraries, while cDNA DGED uses the publicly available UniGene Expression and Gene relational databases to count the sequences found for each gene in the presented libraries. Results We discovered that the CGAP approach often includes libraries from dependent or irrelevant tissues (one third of libraries were incorrect on average, with some tissue searches no correct libraries being selected at all). We also discovered that the CGAP approach reported genes from outside the selected libraries and may omit genes found within the libraries. Other errors include the incorrect estimation of the significance values and inaccurate settings for the library size cut-off values. We advocated a revised approach to finding libraries associated with tissues. In doing so, libraries from dependent or irrelevant tissues do not get included in the final library pool. We also revised the method for determining the presence or absence of a gene by searching the UniGene relational database, revised calculation of statistical significance and sorted the library cut-off filter. Conclusion Our results justify re-evaluation of all previously reported results where NCBI CGAP expression data and tools were used. PMID:21496233

Preconcentration of DNA using magnetic ionic liquids that are compatible with real-time PCR for rapid nucleic acid quantification.

PubMed

Emaus, Miranda N; Clark, Kevin D; Hinners, Paige; Anderson, Jared L

2018-04-28

Nucleic acid extraction and purification represents a major bottleneck in DNA analysis. Traditional methods for DNA purification often require reagents that may inhibit quantitative polymerase chain reaction (qPCR) if not sufficiently removed from the sample. Approaches that employ magnetic beads may exhibit lower extraction efficiencies due to sedimentation and aggregation. In this study, four hydrophobic magnetic ionic liquids (MILs) were investigated as DNA extraction solvents with the goal of improving DNA enrichment factors and compatibility with downstream bioanalytical techniques. By designing custom qPCR buffers, we directly incorporated DNA-enriched MILs including trihexyl(tetradecyl)phosphonium tris(hexafluoroacetylaceto)nickelate(II) ([P 6,6,6,14 + ][Ni(hfacac) 3 - ]), [P 6,6,6,14 + ] tris(hexafluoroacetylaceto)colbaltate(II) ([Co(hfacac) 3 - ]), [P 6,6,6,14 + ] tris(hexafluoroacetylaceto)manganate(II) ([Mn(hfacac) 3 - ]), or [P 6,6,6,14 + ] tetrakis(hexafluoroacetylaceto)dysprosate(III) ([Dy(hfacac) 4 - ]) into reaction systems, thereby circumventing the need for time-consuming DNA recovery steps. Incorporating MILs into the reaction buffer did not significantly impact the amplification efficiency of the reaction (91.1%). High enrichment factors were achieved using the [P 6,6,6,14 + ][Ni(hfacac) 3 - ] MIL for the extraction of single-stranded and double-stranded DNA with extraction times as short as 2 min. When compared to a commercial magnetic bead-based platform, the [P 6,6,6,14 + ][Ni(hfacac) 3 - ] MIL was capable of producing higher enrichment factors for single-stranded DNA and similar enrichment factors for double-stranded DNA. The MIL-based method was applied for the extraction and direct qPCR amplification of mutation prone-KRAS oncogene fragment in plasma samples. Graphical abstract Magnetic ionic liquid solvents are shown to preconcentrate sufficient KRAS DNA template from an aqueous solution in as short as 2 min without using chaotropic salts or toxic organic solvents. By using custom-designed qPCR buffers, DNA can be directly amplified and quantified from four MILs examined in this study.

Generation and Analysis of a Large-Scale Expressed Sequence Tag Database from a Full-Length Enriched cDNA Library of Developing Leaves of Gossypium hirsutum L

PubMed Central

Pang, Chaoyou; Fan, Shuli; Song, Meizhen; Yu, Shuxun

2013-01-01

Background Cotton (Gossypium hirsutum L.) is one of the world’s most economically-important crops. However, its entire genome has not been sequenced, and limited resources are available in GenBank for understanding the molecular mechanisms underlying leaf development and senescence. Methodology/Principal Findings In this study, 9,874 high-quality ESTs were generated from a normalized, full-length cDNA library derived from pooled RNA isolated from throughout leaf development during the plant blooming stage. After clustering and assembly of these ESTs, 5,191 unique sequences, representative 1,652 contigs and 3,539 singletons, were obtained. The average unique sequence length was 682 bp. Annotation of these unique sequences revealed that 84.4% showed significant homology to sequences in the NCBI non-redundant protein database, and 57.3% had significant hits to known proteins in the Swiss-Prot database. Comparative analysis indicated that our library added 2,400 ESTs and 991 unique sequences to those known for cotton. The unigenes were functionally characterized by gene ontology annotation. We identified 1,339 and 200 unigenes as potential leaf senescence-related genes and transcription factors, respectively. Moreover, nine genes related to leaf senescence and eleven MYB transcription factors were randomly selected for quantitative real-time PCR (qRT-PCR), which revealed that these genes were regulated differentially during senescence. The qRT-PCR for three GhYLSs revealed that these genes express express preferentially in senescent leaves. Conclusions/Significance These EST resources will provide valuable sequence information for gene expression profiling analyses and functional genomics studies to elucidate their roles, as well as for studying the mechanisms of leaf development and senescence in cotton and discovering candidate genes related to important agronomic traits of cotton. These data will also facilitate future whole-genome sequence assembly and annotation in G. hirsutum and comparative genomics among Gossypium species. PMID:24146870

Impact of an indigenous microbial enhanced oil recovery field trial on microbial community structure in a high pour-point oil reservoir.

PubMed

Zhang, Fan; She, Yue-Hui; Li, Hua-Min; Zhang, Xiao-Tao; Shu, Fu-Chang; Wang, Zheng-Liang; Yu, Long-Jiang; Hou, Du-Jie

2012-08-01

Based on preliminary investigation of microbial populations in a high pour-point oil reservoir, an indigenous microbial enhanced oil recovery (MEOR) field trial was carried out. The purpose of the study is to reveal the impact of the indigenous MEOR process on microbial community structure in the oil reservoir using 16Sr DNA clone library technique. The detailed monitoring results showed significant response of microbial communities during the field trial and large discrepancies of stimulated microorganisms in the laboratory and in the natural oil reservoir. More specifically, after nutrients injection, the original dominant populations of Petrobacter and Alishewanella in the production wells almost disappeared. The expected desirable population of Pseudomonas aeruginosa, determined by enrichment experiments in laboratory, was stimulated successfully in two wells of the five monitored wells. Unexpectedly, another potential population of Pseudomonas pseudoalcaligenes which were not detected in the enrichment culture in laboratory was stimulated in the other three monitored production wells. In this study, monitoring of microbial community displayed a comprehensive alteration of microbial populations during the field trial to remedy the deficiency of culture-dependent monitoring methods. The results would help to develop and apply more MEOR processes.

Cinnamides as selective small-molecule inhibitors of a cellular model of breast cancer stem cells.

PubMed

Germain, Andrew R; Carmody, Leigh C; Nag, Partha P; Morgan, Barbara; Verplank, Lynn; Fernandez, Cristina; Donckele, Etienne; Feng, Yuxiong; Perez, Jose R; Dandapani, Sivaraman; Palmer, Michelle; Lander, Eric S; Gupta, Piyush B; Schreiber, Stuart L; Munoz, Benito

2013-03-15

A high-throughput screen (HTS) was conducted against stably propagated cancer stem cell (CSC)-enriched populations using a library of 300,718 compounds from the National Institutes of Health (NIH) Molecular Libraries Small Molecule Repository (MLSMR). A cinnamide analog displayed greater than 20-fold selective inhibition of the breast CSC-like cell line (HMLE_sh_Ecad) over the isogenic control cell line (HMLE_sh_eGFP). Herein, we report structure-activity relationships of this class of cinnamides for selective lethality towards CSC-enriched populations. Copyright © 2013. Published by Elsevier Ltd.

Improving cell mixture deconvolution by identifying optimal DNA methylation libraries (IDOL).

PubMed

Koestler, Devin C; Jones, Meaghan J; Usset, Joseph; Christensen, Brock C; Butler, Rondi A; Kobor, Michael S; Wiencke, John K; Kelsey, Karl T

2016-03-08

Confounding due to cellular heterogeneity represents one of the foremost challenges currently facing Epigenome-Wide Association Studies (EWAS). Statistical methods leveraging the tissue-specificity of DNA methylation for deconvoluting the cellular mixture of heterogenous biospecimens offer a promising solution, however the performance of such methods depends entirely on the library of methylation markers being used for deconvolution. Here, we introduce a novel algorithm for Identifying Optimal Libraries (IDOL) that dynamically scans a candidate set of cell-specific methylation markers to find libraries that optimize the accuracy of cell fraction estimates obtained from cell mixture deconvolution. Application of IDOL to training set consisting of samples with both whole-blood DNA methylation data (Illumina HumanMethylation450 BeadArray (HM450)) and flow cytometry measurements of cell composition revealed an optimized library comprised of 300 CpG sites. When compared existing libraries, the library identified by IDOL demonstrated significantly better overall discrimination of the entire immune cell landscape (p = 0.038), and resulted in improved discrimination of 14 out of the 15 pairs of leukocyte subtypes. Estimates of cell composition across the samples in the training set using the IDOL library were highly correlated with their respective flow cytometry measurements, with all cell-specific R (2)>0.99 and root mean square errors (RMSEs) ranging from [0.97 % to 1.33 %] across leukocyte subtypes. Independent validation of the optimized IDOL library using two additional HM450 data sets showed similarly strong prediction performance, with all cell-specific R (2)>0.90 and R M S E<4.00 %. In simulation studies, adjustments for cell composition using the IDOL library resulted in uniformly lower false positive rates compared to competing libraries, while also demonstrating an improved capacity to explain epigenome-wide variation in DNA methylation within two large publicly available HM450 data sets. Despite consisting of half as many CpGs compared to existing libraries for whole blood mixture deconvolution, the optimized IDOL library identified herein resulted in outstanding prediction performance across all considered data sets and demonstrated potential to improve the operating characteristics of EWAS involving adjustments for cell distribution. In addition to providing the EWAS community with an optimized library for whole blood mixture deconvolution, our work establishes a systematic and generalizable framework for the assembly of libraries that improve the accuracy of cell mixture deconvolution.

Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation.

PubMed

Mauger, Florence; Kernaleguen, Magali; Lallemand, Céline; Kristensen, Vessela N; Deleuze, Jean-François; Tost, Jörg

2018-05-01

The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.

PubMed

Shi, Liang; Khandurina, Julia; Ronai, Zsolt; Li, Bi-Yu; Kwan, Wai King; Wang, Xun; Guttman, András

2003-01-01

A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment-pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by BsaJ I and EcoR I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high-resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer-controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96-well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments (i.e., single-base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR-amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.

Detection of regional DNA methylation using DNA-graphene affinity interactions.

PubMed

Haque, Md Hakimul; Gopalan, Vinod; Yadav, Sharda; Islam, Md Nazmul; Eftekhari, Ehsan; Li, Qin; Carrascosa, Laura G; Nguyen, Nam-Trung; Lam, Alfred K; Shiddiky, Muhammad J A

2017-01-15

We report a new method for the detection of regional DNA methylation using base-dependent affinity interaction (i.e., adsorption) of DNA with graphene. Due to the strongest adsorption affinity of guanine bases towards graphene, bisulfite-treated guanine-enriched methylated DNA leads to a larger amount of the adsorbed DNA on the graphene-modified electrodes in comparison to the adenine-enriched unmethylated DNA. The level of the methylation is quantified by monitoring the differential pulse voltammetric current as a function of the adsorbed DNA. The assay is sensitive to distinguish methylated and unmethylated DNA sequences at single CpG resolution by differentiating changes in DNA methylation as low as 5%. Furthermore, this method has been used to detect methylation levels in a collection of DNA samples taken from oesophageal cancer tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

Identifying insects with incomplete DNA barcode libraries, African fruit flies (Diptera: Tephritidae) as a test case.

PubMed

Virgilio, Massimiliano; Jordaens, Kurt; Breman, Floris C; Backeljau, Thierry; De Meyer, Marc

2012-01-01

We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods.

Identifying Insects with Incomplete DNA Barcode Libraries, African Fruit Flies (Diptera: Tephritidae) as a Test Case

PubMed Central

Virgilio, Massimiliano; Jordaens, Kurt; Breman, Floris C.; Backeljau, Thierry; De Meyer, Marc

2012-01-01

We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods. PMID:22359600

Anaerobic Redox Cycling of Iron by Freshwater Sediment Microorganisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weber, Karrie A.; Urrutia, Matilde M.; Churchill, Perry F.

2006-01-01

The potential for microbially-mediated anaerobic redox cycling of iron (Fe) was examined in a first-generation enrichment culture of freshwater wetland sediment microorganisms. MPN enumerations revealed the presence of significant populations of Fe(III)-reducing (ca. 108 cells mL-1) and Fe(II)-oxidizing, nitrate-reducing organisms (ca. 105 cells mL-1) in the sediment used to inoculate the enrichment cultures. Nitrate reduction commenced immediately following inoculation of acetate-containing (ca. 1 mM) medium with a small quantity (1% vol/vol) of wetland sediment, and resulted in the transient accumulation of NO2- and production of a mixture of end-products including NH4+. Fe(III) oxide (high surface area goethite) reduction took placemore » - after NO3- was depleted and continued until all the acetate was utilized. Addition of NO3 after Fe(III) reduction ceased resulted in the immediate oxidation of Fe(II) coupled to reduction of + NO3-to NH4 . No significant NO2- accumulation was observed during nitrate-dependent Fe(II) oxidation. No Fe(II) oxidation occurred in pasteurized controls. Microbial community structure in the enrichment was monitored by DGGE analysis of PCR amplified 16s rDNA and RT-PCR amplified 16S rRNA, as well as by construction of 16S rDNA clone libraries for four different time points during the experiment. Strong similarities in dominant members of the microbial community were observed in the Fe(III) reduction and nitrate-dependent Fe(II) oxidation phases of the experiment, specifically the common presence of organisms closely related (= 95% sequence similarity) to the genera Geobacter and Dechloromonas. These results indicate that the wetland sediments contained organisms such as Geobacter sp. which are capable of both + dissimilatory Fe(III) reduction and oxidation of Fe(II) with reduction of NO3-reduction to NH4 . Our findings suggest that microbially-catalyzed nitrate-dependent Fe(II) oxidation has the potential to contribute to a dynamic anaerobic Fe redox cycle in freshwater sediments.« less

Targeting a Complex Transcriptome: The Construction of the Mouse Full-Length cDNA Encyclopedia

PubMed Central

Carninci, Piero; Waki, Kazunori; Shiraki, Toshiyuki; Konno, Hideaki; Shibata, Kazuhiro; Itoh, Masayoshi; Aizawa, Katsunori; Arakawa, Takahiro; Ishii, Yoshiyuki; Sasaki, Daisuke; Bono, Hidemasa; Kondo, Shinji; Sugahara, Yuichi; Saito, Rintaro; Osato, Naoki; Fukuda, Shiro; Sato, Kenjiro; Watahiki, Akira; Hirozane-Kishikawa, Tomoko; Nakamura, Mari; Shibata, Yuko; Yasunishi, Ayako; Kikuchi, Noriko; Yoshiki, Atsushi; Kusakabe, Moriaki; Gustincich, Stefano; Beisel, Kirk; Pavan, William; Aidinis, Vassilis; Nakagawara, Akira; Held, William A.; Iwata, Hiroo; Kono, Tomohiro; Nakauchi, Hiromitsu; Lyons, Paul; Wells, Christine; Hume, David A.; Fagiolini, Michela; Hensch, Takao K.; Brinkmeier, Michelle; Camper, Sally; Hirota, Junji; Mombaerts, Peter; Muramatsu, Masami; Okazaki, Yasushi; Kawai, Jun; Hayashizaki, Yoshihide

2003-01-01

We report the construction of the mouse full-length cDNA encyclopedia,the most extensive view of a complex transcriptome,on the basis of preparing and sequencing 246 libraries. Before cloning,cDNAs were enriched in full-length by Cap-Trapper,and in most cases,aggressively subtracted/normalized. We have produced 1,442,236 successful 3′-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5′ end reads,which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU),which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC),which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large numbers of clusters (and TUs) of this project,which also include non-protein-coding RNAs,and the lower gene number estimation of genome annotations. Altogether,5′-end clusters identify regions that are potential promoters for 8637 known genes and 5′-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete. PMID:12819125

Cloning and Identification of Recombinant Argonaute-Bound Small RNAs Using Next-Generation Sequencing.

PubMed

Gangras, Pooja; Dayeh, Daniel M; Mabin, Justin W; Nakanishi, Kotaro; Singh, Guramrit

2018-01-01

Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both. Therefore, a single method that yields quantitative, bias-free NGS libraries to identify small and long RNAs from low levels of input will be of wide interest. Here, we introduce such a procedure that is based on several modifications of two published protocols and allows robust, sensitive, and reproducible cloning and sequencing of small amounts of RNAs of variable lengths. The method was applied to the identification of small RNAs bound to a purified eukaryotic AGO. Following ligation of a DNA adapter to RNA 3'-end, the key feature of this method is to use the adapter for priming reverse transcription (RT) wherein biotinylated deoxyribonucleotides specifically incorporated into the extended complementary DNA. Such RT products are enriched on streptavidin beads, circularized while immobilized on beads and directly used for PCR amplification. We provide a stepwise guide to generate RNA-Seq libraries, their purification, quantification, validation, and preparation for next-generation sequencing. We also provide basic steps in post-NGS data analyses using Galaxy, an open-source, web-based platform.

Ribosome display: next-generation display technologies for production of antibodies in vitro.

PubMed

He, Mingyue; Khan, Farid

2005-06-01

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

Genes differentially expressed in Theobroma cacao associated with resistance to witches' broom disease caused by Crinipellis perniciosa.

PubMed

Leal, Gildemberg Amorim; Albuquerque, Paulo S B; Figueira, Antonio

2007-05-01

SUMMARY The basidiomycete Crinipellis perniciosa is the causal agent of witches' broom disease of Theobroma cacao (cocoa). Hypertrophic growth of infected buds ('brooms') is the most dramatic symptom, but the main economic losses derive from pod infection. To identify cocoa genes differentially expressed during the early stages of infection, two cDNA libraries were constructed using the suppression subtractive hybridization (SSH) approach. Subtraction hybridization was conducted between cDNAs from infected shoot-tips of the susceptible genotype 'ICS 39' and the resistant 'CAB 214', in both directions. A total of 187 unique sequences were obtained, with 83 from the library enriched for the susceptible 'ICS 39' sequences, and 104 for the resistant 'CAB 214'. By homology search and ontology analyses, the identified sequences were mainly putatively categorized as belonging to 'signal transduction', 'response to biotic and abiotic stress', 'metabolism', 'RNA and DNA metabolism', 'protein metabolism' and 'cellular maintenance' classes. Quantitative reverse transcription amplification (RT-qPCR) of 23 transcripts identified as differentially expressed between genotypes revealed distinct kinetics of gene up-regulation at the asymptomatic stage of the disease. Expression induction in the susceptible 'ICS 39' in response to C. perniciosa was delayed and limited, while in 'CAB 214' there was a quicker and more intense reaction, with two peaks of gene induction at 48 and 120 h after inoculation, corresponding to morphological and biochemical changes previously described during colonization. Similar differences in gene induction were validated for another resistant genotype ('CAB 208') in an independent experiment. Validation of these genes corroborated similar hypothetical mechanisms of resistance described in other pathosystems.

Pros and cons of methylation-based enrichment methods for ancient DNA.

PubMed

Seguin-Orlando, Andaine; Gamba, Cristina; Der Sarkissian, Clio; Ermini, Luca; Louvel, Guillaume; Boulygina, Eugenia; Sokolov, Alexey; Nedoluzhko, Artem; Lorenzen, Eline D; Lopez, Patricio; McDonald, H Gregory; Scott, Eric; Tikhonov, Alexei; Stafford, Thomas W; Alfarhan, Ahmed H; Alquraishi, Saleh A; Al-Rasheid, Khaled A S; Shapiro, Beth; Willerslev, Eske; Prokhortchouk, Egor; Orlando, Ludovic

2015-07-02

The recent discovery that DNA methylation survives in fossil material provides an opportunity for novel molecular approaches in palaeogenomics. Here, we apply to ancient DNA extracts the probe-independent Methylated Binding Domains (MBD)-based enrichment method, which targets DNA molecules containing methylated CpGs. Using remains of a Palaeo-Eskimo Saqqaq individual, woolly mammoths, polar bears and two equine species, we confirm that DNA methylation survives in a variety of tissues, environmental contexts and over a large temporal range (4,000 to over 45,000 years before present). MBD enrichment, however, appears principally biased towards the recovery of CpG-rich and long DNA templates and is limited by the fast post-mortem cytosine deamination rates of methylated epialleles. This method, thus, appears only appropriate for the analysis of ancient methylomes from very well preserved samples, where both DNA fragmentation and deamination have been limited. This work represents an essential step toward the characterization of ancient methylation signatures, which will help understanding the role of epigenetic changes in past environmental and cultural transitions.

Pros and cons of methylation-based enrichment methods for ancient DNA

PubMed Central

Seguin-Orlando, Andaine; Gamba, Cristina; Sarkissian, Clio Der; Ermini, Luca; Louvel, Guillaume; Boulygina, Eugenia; Sokolov, Alexey; Nedoluzhko, Artem; Lorenzen, Eline D.; Lopez, Patricio; McDonald, H. Gregory; Scott, Eric; Tikhonov, Alexei; Stafford,, Thomas W.; Alfarhan, Ahmed H.; Alquraishi, Saleh A.; Al-Rasheid, Khaled A. S.; Shapiro, Beth; Willerslev, Eske; Prokhortchouk, Egor; Orlando, Ludovic

2015-01-01

The recent discovery that DNA methylation survives in fossil material provides an opportunity for novel molecular approaches in palaeogenomics. Here, we apply to ancient DNA extracts the probe-independent Methylated Binding Domains (MBD)-based enrichment method, which targets DNA molecules containing methylated CpGs. Using remains of a Palaeo-Eskimo Saqqaq individual, woolly mammoths, polar bears and two equine species, we confirm that DNA methylation survives in a variety of tissues, environmental contexts and over a large temporal range (4,000 to over 45,000 years before present). MBD enrichment, however, appears principally biased towards the recovery of CpG-rich and long DNA templates and is limited by the fast post-mortem cytosine deamination rates of methylated epialleles. This method, thus, appears only appropriate for the analysis of ancient methylomes from very well preserved samples, where both DNA fragmentation and deamination have been limited. This work represents an essential step toward the characterization of ancient methylation signatures, which will help understanding the role of epigenetic changes in past environmental and cultural transitions. PMID:26134828

Primer-Free Aptamer Selection Using A Random DNA Library

PubMed Central

Pan, Weihua; Xin, Ping; Patrick, Susan; Dean, Stacey; Keating, Christine; Clawson, Gary

2010-01-01

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues. Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10. We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format. PMID:20689511

OCLC in Asia Pacific.

ERIC Educational Resources Information Center

Chang, Min-min

1998-01-01

Discusses the Online Computer Library Center (OCLC) and the changing Asia Pacific library scene under the broad headings of the three phases of technology innovation. Highlights include WorldCat and the OCLC shared cataloging system; resource sharing and interlibrary loan; enriching OCLC online catalog with Asian collections; and future outlooks.…

Autoantibodies associated with RNA are more enriched than anti-dsDNA antibodies in circulating immune complexes in SLE.

PubMed

Ahlin, E; Mathsson, L; Eloranta, M-L; Jonsdottir, T; Gunnarsson, I; Rönnblom, L; Rönnelid, J

2012-05-01

To what extent different autoantibodies accumulate in systemic lupus erythematosus (SLE) immune complexes (ICs), and whether such accumulation is associated with disease activity has been investigated. ICs were isolated from SLE sera by both polyethylene glycol (PEG) precipitation and C1q-binding. Autoantibody specificities were determined using a lineblot assay quantified by densitometry. To compare the relative levels of autoantibodies, levels were normalized to the total levels of IgG measured by ELISA in sera and parallel ICs. Samples were investigated both in a cross-sectional design as well as in a paired design with samples obtained during both active and inactive SLE. All investigated autoantibody specificities except anti-dsDNA were enriched in circulating ICs as compared with parallel sera. The group of antibodies against RNA-associated antigens (anti-RNP/Sm, anti-Sm, anti-SSA/Ro60, anti-SSA/Ro52, anti-SSB/La) all exhibited higher median enrichment than the DNA-associated (anti-dsDNA, anti-histones, anti-nucleosomes) or cytoplasmic (anti-ribosomal P) antigens. In particular autoantibodies against RNP/Sm and SSA/Ro52 had the highest degree of enrichment in SLE PEG precipitates. These findings were corroborated by analysis of autoantibody content in C1q-bound ICs. There was no difference in degree of IC accumulation of the investigated autoantibodies during active and inactive SLE. Our findings demonstrate a difference in enrichment between autoantibodies against RNA- and DNA-associated autoantigens in isolated SLE IC, suggesting that the RNA-associated autoantibodies are more prone to form circulating ICs in SLE, in contrast to antibodies against DNA-associated autoantigens such as dsDNA. These finding have implications in understanding mechanisms of differential autoantibody accumulation in target organs in SLE.

Epigenetics insights into chronic pain: DNA hypomethylation in fibromyalgia-a controlled pilot-study.

PubMed

Ciampi de Andrade, Daniel; Maschietto, Mariana; Galhardoni, Ricardo; Gouveia, Gisele; Chile, Thais; Victorino Krepischi, Ana C; Dale, Camila S; Brunoni, André R; Parravano, Daniella C; Cueva Moscoso, Ana S; Raicher, Irina; Kaziyama, Helena H S; Teixeira, Manoel J; Brentani, Helena P

2017-08-01

To evaluate changes in DNA methylation profiles in patients with fibromyalgia (FM) compared to matched healthy controls (HCs). All individuals underwent full clinical and neurophysiological assessment by cortical excitability (CE) parameters measured by transcranial magnetic stimulation. DNA from the peripheral blood of patients with FM (n = 24) and HC (n = 24) were assessed using the Illumina-HumanMethylation450 BeadChips. We identified 1610 differentially methylated positions (DMPs) in patients with FM displaying a nonrandom distribution in regions of the genome. Sixty-nine percent of DMP in FM were hypomethylated compared to HC. Differentially methylated positions were enriched in 5 genomic regions (1p34; 6p21; 10q26; 17q25; 19q13). The functional characterization of 960 genes related to DMPs revealed an enrichment for MAPK signaling pathway (n = 18 genes), regulation of actin cytoskeleton (n = 15 genes), and focal adhesion (n = 13 genes). A gene-gene interaction network enrichment analysis revealed the participation of DNA repair pathways, mitochondria-related processes, and synaptic signaling. Even though DNA was extracted from peripheral blood, this set of genes was enriched for disorders such as schizophrenia, mood disorders, bulimia, hyperphagia, and obesity. Remarkably, the hierarchical clusterization based on the methylation levels of the 1610 DMPs showed an association with neurophysiological measurements of CE in FM and HC. Fibromyalgia has a hypomethylation DNA pattern, which is enriched in genes implicated in stress response and DNA repair/free radical clearance. These changes occurred parallel to changes in CE parameters. New epigenetic insights into the pathophysiology of FM may provide the basis for the development of biomarkers of this disorder.

Library construction for next-generation sequencing: Overviews and challenges

PubMed Central

Head, Steven R.; Komori, H. Kiyomi; LaMere, Sarah A.; Whisenant, Thomas; Van Nieuwerburgh, Filip; Salomon, Daniel R.; Ordoukhanian, Phillip

2014-01-01

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed. PMID:24502796

Sequencing historical specimens: successful preparation of small specimens with low amounts of degraded DNA.

PubMed

Sproul, John S; Maddison, David R

2017-11-01

Despite advances that allow DNA sequencing of old museum specimens, sequencing small-bodied, historical specimens can be challenging and unreliable as many contain only small amounts of fragmented DNA. Dependable methods to sequence such specimens are especially critical if the specimens are unique. We attempt to sequence small-bodied (3-6 mm) historical specimens (including nomenclatural types) of beetles that have been housed, dried, in museums for 58-159 years, and for which few or no suitable replacement specimens exist. To better understand ideal approaches of sample preparation and produce preparation guidelines, we compared different library preparation protocols using low amounts of input DNA (1-10 ng). We also explored low-cost optimizations designed to improve library preparation efficiency and sequencing success of historical specimens with minimal DNA, such as enzymatic repair of DNA. We report successful sample preparation and sequencing for all historical specimens despite our low-input DNA approach. We provide a list of guidelines related to DNA repair, bead handling, reducing adapter dimers and library amplification. We present these guidelines to facilitate more economical use of valuable DNA and enable more consistent results in projects that aim to sequence challenging, irreplaceable historical specimens. © 2017 John Wiley & Sons Ltd.

Large-Scale Concatenation cDNA Sequencing

PubMed Central

Yu, Wei; Andersson, Björn; Worley, Kim C.; Muzny, Donna M.; Ding, Yan; Liu, Wen; Ricafrente, Jennifer Y.; Wentland, Meredith A.; Lennon, Greg; Gibbs, Richard A.

1997-01-01

A total of 100 kb of DNA derived from 69 individual human brain cDNA clones of 0.7–2.0 kb were sequenced by concatenated cDNA sequencing (CCS), whereby multiple individual DNA fragments are sequenced simultaneously in a single shotgun library. The method yielded accurate sequences and a similar efficiency compared with other shotgun libraries constructed from single DNA fragments (>20 kb). Computer analyses were carried out on 65 cDNA clone sequences and their corresponding end sequences to examine both nucleic acid and amino acid sequence similarities in the databases. Thirty-seven clones revealed no DNA database matches, 12 clones generated exact matches (≥98% identity), and 16 clones generated nonexact matches (57%–97% identity) to either known human or other species genes. Of those 28 matched clones, 8 had corresponding end sequences that failed to identify similarities. In a protein similarity search, 27 clone sequences displayed significant matches, whereas only 20 of the end sequences had matches to known protein sequences. Our data indicate that full-length cDNA insert sequences provide significantly more nucleic acid and protein sequence similarity matches than expressed sequence tags (ESTs) for database searching. [All 65 cDNA clone sequences described in this paper have been submitted to the GenBank data library under accession nos. U79240–U79304.] PMID:9110174

Primer Extension Mutagenesis Powered by Selective Rolling Circle Amplification

PubMed Central

Huovinen, Tuomas; Brockmann, Eeva-Christine; Akter, Sultana; Perez-Gamarra, Susan; Ylä-Pelto, Jani; Liu, Yuan; Lamminmäki, Urpo

2012-01-01

Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material. PMID:22355397

Enriching the Catalog

ERIC Educational Resources Information Center

Tennant, Roy

2004-01-01

After decades of costly and time-consuming effort, nearly all libraries have completed the retrospective conversion of their card catalogs to electronic form. However, bibliographic systems still are really not much more than card catalogs on wheels. Enriched content that Amazon.com takes for granted--such as digitized tables of contents, cover…

SALP, a new single-stranded DNA library preparation method especially useful for the high-throughput characterization of chromatin openness states.

PubMed

Wu, Jian; Dai, Wei; Wu, Lin; Wang, Jinke

2018-02-13

Next-generation sequencing (NGS) is fundamental to the current biological and biomedical research. Construction of sequencing library is a key step of NGS. Therefore, various library construction methods have been explored. However, the current methods are still limited by some shortcomings. This study developed a new NGS library construction method, Single strand Adaptor Library Preparation (SALP), by using a novel single strand adaptor (SSA). SSA is a double-stranded oligonucleotide with a 3' overhang of 3 random nucleotides, which can be efficiently ligated to the 3' end of single strand DNA by T4 DNA ligase. SALP can be started with any denatured DNA fragments such as those sheared by Tn5 tagmentation, enzyme digestion and sonication. When started with Tn5-tagmented chromatin, SALP can overcome a key limitation of ATAC-seq and become a high-throughput NGS library construction method, SALP-seq, which can be used to comparatively characterize the chromatin openness state of multiple cells unbiasly. In this way, this study successfully characterized the comparative chromatin openness states of four different cell lines, including GM12878, HepG2, HeLa and 293T, with SALP-seq. Similarly, this study also successfully characterized the chromatin openness states of HepG2 cells with SALP-seq by using 10 5 to 500 cells. This study developed a new NGS library construction method, SALP, by using a novel kind of single strand adaptor (SSA), which should has wide applications in the future due to its unique performance.

Prospective identification of parasitic sequences in phage display screens

PubMed Central

Matochko, Wadim L.; Cory Li, S.; Tang, Sindy K.Y.; Derda, Ratmir

2014-01-01

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones. PMID:24217917

Development of polymorphic genic-SSR markers by cDNA library sequencing in boxwood, Buxus spp. (Buxaceae)

USDA-ARS?s Scientific Manuscript database

Genic microsatellites or simple sequence repeat (genic-SSR) markers were developed in boxwood (Buxus taxa) for genetic diversity analysis, identification of taxa, and to facilitate breeding. cDNA libraries were developed from mRNA extracted from leaves of Buxus sempervirens ‘Vardar Valley’ and seque...

An innovative strategy to clone positive modifier genes of defects caused by mtDNA mutations: MRPS18C as suppressor gene of m.3946G>A mutation in MT-ND1 gene.

PubMed

Rodríguez-García, María Elena; Cotrina-Vinagre, Francisco Javier; Carnicero-Rodríguez, Patricia; Martínez-Azorín, Francisco

2017-07-01

We have developed a new functional complementation approach to clone modifier genes which overexpression is able to suppress the biochemical defects caused by mtDNA mutations (suppressor genes). This strategy consists in transferring human genes into respiratory chain-deficient fibroblasts, followed by a metabolic selection in a highly selective medium. We used a normalized expression cDNA library in an episomal vector (pREP4) to transfect the fibroblasts, and a medium with glutamine and devoid of any carbohydrate source to select metabolically. Growing the patient's fibroblasts in this selective medium, the deficient cells rapidly disappear unless they are rescued by the cDNA of a suppressor gene. The use of an episomal vector allows us to carry out several rounds of transfection/selection (cyclical phenotypic rescue) to enrich the rescue with true clones of suppressor genes. Using fibroblasts from a patient with epileptic encephalopathy with the m.3946G>A (p.E214K) mutation in the MT-ND1 gene, several candidate genes were identified and one of them was characterized functionally. Thus, overexpression of MRPS18C gene (that encode for bS18m protein) suppressed the molecular defects produced by this mtDNA mutation, recovering the complex I activity and reducing the ROS produced by this complex to normal levels. We suggest that modulation of bS18m expression may be an effective therapeutic strategy for the patients with this mutation.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

PubMed

Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

2009-09-01

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

[The study of complex-formation of DNA with the antimicrobial drug decamethoxine].

PubMed

Sorokin, V A; Blagoĭ, Iu P; Valeev, V A; Gladchenko, G O; Sukhodub, L F; Volianskiĭ, Iu L

1990-01-01

The interaction of effective antibacterial drug decametoxyn with natural DNA was studied by UV-spectroscopy. Decametoxyn shows a specificity to nucleotides: it decreases the cooperativity of melting and the thermal stability of DNA parts enriched by AT pairs. The characteristics of the helix-coil transition on the DNA parts enriched by GC-pairs are invariable. Interaction with AT-pairs results in their partial or complete melting at room temperature, followed by intermolecule aggregation. Interacting with phosphates decametoxyn manifests itself not as a dication but as two single-charged ions.

Creation of reference DNA barcode library and authentication of medicinal plant raw drugs used in Ayurvedic medicine.

PubMed

Vassou, Sophie Lorraine; Nithaniyal, Stalin; Raju, Balaji; Parani, Madasamy

2016-07-18

Ayurveda is a system of traditional medicine that originated in ancient India, and it is still in practice. Medicinal plants are the backbone of Ayurveda, which heavily relies on the plant-derived therapeutics. While Ayurveda is becoming more popular in several countries throughout the World, lack of authenticated medicinal plant raw drugs is a growing concern. Our aim was to DNA barcode the medicinal plants that are listed in the Ayurvedic Pharmacopoeia of India (API) to create a reference DNA barcode library, and to use the same to authenticate the raw drugs that are sold in markets. We have DNA barcoded 347 medicinal plants using rbcL marker, and curated rbcL DNA barcodes for 27 medicinal plants from public databases. These sequences were used to create Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL). This library was used to authenticate 100 medicinal plant raw drugs, which were in the form of powders (82) and seeds (18). Ayurvedic Pharmacopoeia of India - Reference DNA Barcode Library (API-RDBL) was created with high quality and authentic rbcL barcodes for 374 out of the 395 medicinal plants that are included in the API. The rbcL DNA barcode differentiated 319 species (85 %) with the pairwise divergence ranging between 0.2 and 29.9 %. PCR amplification and DNA sequencing success rate of rbcL marker was 100 % even for the poorly preserved medicinal plant raw drugs that were collected from local markets. DNA barcoding revealed that only 79 % raw drugs were authentic, and the remaining 21 % samples were adulterated. Further, adulteration was found to be much higher with powders (ca. 25 %) when compared to seeds (ca. 5 %). The present study demonstrated the utility of DNA barcoding in authenticating medicinal plant raw drugs, and found that approximately one fifth of the market samples were adulterated. Powdered raw drugs, which are very difficult to be identified by taxonomists as well as common people, seem to be the easy target for adulteration. Developing a quality control protocol for medicinal plant raw drugs by incorporating DNA barcoding as a component is essential to ensure safety to the consumers.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

PubMed Central

Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

2016-01-01

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

The Hemiptera (Insecta) of Canada: Constructing a Reference Library of DNA Barcodes

PubMed Central

Gwiazdowski, Rodger A.; Foottit, Robert G.; Maw, H. Eric L.; Hebert, Paul D. N.

2015-01-01

DNA barcode reference libraries linked to voucher specimens create new opportunities for high-throughput identification and taxonomic re-evaluations. This study provides a DNA barcode library for about 45% of the recognized species of Canadian Hemiptera, and the publically available R workflow used for its generation. The current library is based on the analysis of 20,851 specimens including 1849 species belonging to 628 genera and 64 families. These individuals were assigned to 1867 Barcode Index Numbers (BINs), sequence clusters that often coincide with species recognized through prior taxonomy. Museum collections were a key source for identified specimens, but we also employed high-throughput collection methods that generated large numbers of unidentified specimens. Many of these specimens represented novel BINs that were subsequently identified by taxonomists, adding barcode coverage for additional species. Our analyses based on both approaches includes 94 species not listed in the most recent Canadian checklist, representing a potential 3% increase in the fauna. We discuss the development of our workflow in the context of prior DNA barcode library construction projects, emphasizing the importance of delineating a set of reference specimens to aid investigations in cases of nomenclatural and DNA barcode discordance. The identification for each specimen in the reference set can be annotated on the Barcode of Life Data System (BOLD), allowing experts to highlight questionable identifications; annotations can be added by any registered user of BOLD, and instructions for this are provided. PMID:25923328

Improving the Conservation of Mediterranean Chondrichthyans: The ELASMOMED DNA Barcode Reference Library

PubMed Central

Arculeo, Marco; Bonello, Juan J.; Bonnici, Leanne; Cannas, Rita; Carbonara, Pierluigi; Cau, Alessandro; Charilaou, Charis; El Ouamari, Najib; Fiorentino, Fabio; Follesa, Maria Cristina; Garofalo, Germana; Golani, Daniel; Guarniero, Ilaria; Hanner, Robert; Hemida, Farid; Kada, Omar; Lo Brutto, Sabrina; Mancusi, Cecilia; Morey, Gabriel; Schembri, Patrick J.; Serena, Fabrizio; Sion, Letizia; Stagioni, Marco; Tursi, Angelo; Vrgoc, Nedo; Steinke, Dirk; Tinti, Fausto

2017-01-01

Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera), including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions. PMID:28107413

Methods for transforming and expression screening of filamentous fungal cells with a DNA library

DOEpatents

Teter, Sarah; Lamsa, Michael; Cherry, Joel; Ward, Connie

2015-06-02

The present invention relates to methods for expression screening of filamentous fungal transformants, comprising: (a) isolating single colony transformants of a DNA library introduced into E. coli; (b) preparing DNA from each of the single colony E. coli transformants; (c) introducing a sample of each of the DNA preparations of step (b) into separate suspensions of protoplasts of a filamentous fungus to obtain transformants thereof, wherein each transformant contains one or more copies of an individual polynucleotide from the DNA library; (d) growing the individual filamentous fungal transformants of step (c) on selective growth medium, thereby permitting growth of the filamentous fungal transformants, while suppressing growth of untransformed filamentous fungi; and (e) measuring activity or a property of each polypeptide encoded by the individual polynucleotides. The present invention also relates to isolated polynucleotides encoding polypeptides of interest obtained by such methods, to nucleic acid constructs, expression vectors, and recombinant host cells comprising the isolated polynucleotides, and to methods of producing the polypeptides encoded by the isolated polynucleotides.

Deep Learning through Concept-Based Inquiry

ERIC Educational Resources Information Center

Donham, Jean

2010-01-01

Learning in the library should present opportunities to enrich student learning activities to address concerns of interest and cognitive complexity, but these must be tasks that call for in-depth analysis--not merely gathering facts. Library learning experiences need to demand enough of students to keep them interested and also need to be…

Examining the Association between the "Imagination Library" Early Childhood Literacy Program and Kindergarten Readiness

ERIC Educational Resources Information Center

Samiei, Shahin; Bush, Andrew J.; Sell, Marie; Imig, Doug

2016-01-01

This study evaluated participation in the "Imagination Library" early childhood literacy enrichment program and children's pre-literacy and pre-numeracy skills at kindergarten entry in an urban school district. Previous studies have demonstrated that program participation is associated with greater early childhood reading practices.…

Teaching Analytics: A Clustering and Triangulation Study of Digital Library User Data

ERIC Educational Resources Information Center

Xu, Beijie; Recker, Mimi

2012-01-01

Teachers and students increasingly enjoy unprecedented access to abundant web resources and digital libraries to enhance and enrich their classroom experiences. However, due to the distributed nature of such systems, conventional educational research methods, such as surveys and observations, provide only limited snapshots. In addition,…

Now's the Time: Online Library Orientations

ERIC Educational Resources Information Center

Farrell, Sandy L.; Driver, Carol; Weathers, Anita

2011-01-01

Increasingly at West Kentucky Community and Technical College (WKCTC), English 101, English 102, and Enrichment 091 are taught online, allowing students more work availability and time with family, while decreasing commute time and expense. In order to meet the library orientation needs of these online students and provide other options for…

No Longer the "Poor Man's University"

ERIC Educational Resources Information Center

Hackett, Abi; Novitzky, Jan

2008-01-01

Museums, libraries and archives can offer fantastic opportunities for adult learning in their own right. Many know that what is on offer can complement and enrich the opportunities offered through the more formal adult learning sector. The schools sector has long-standing links with museums and libraries. In adult education there are less…

Characterization of the translation products of the major mRNA species from rabbit lactating mammary glands and construction of bacterial recombinants containing casein and alpha-lactalbumin complementary DNA.

PubMed Central

Suard, Y M; Tosi, M; Kraehenbuhl, J P

1982-01-01

Total cytoplasmic polyadenylated RNA from lactating rabbit mammary glands was analysed on methylmercury hydroxide-agarose gels. The size of the most abundant mRNA species ranged between 0.5 and 5.0 kb (kilobases), with major bands at 0.55, 0.84, 0.92, 1.18 and 2.4 kb and discrete minor bands of 1.5, 1.7, 3.0 and 3.9 kb. Translation in vitro of total mRNA with [3H]leucine or [35S]methionine as precursor yielded four major bands with apparent Mr values of 16 000, 25 000, 26 000 and 29 000. The four protein bands were identified by immunoprecipitation by using specific antisera as alpha-lactalbumin and x-, kappa- and alpha-caseins, respectively. Labelling with (35S]cysteine followed by immunoprecipitation with anti-transferrin or anti-alpha-lactalbumin sera allowed the identification of two whey proteins. Translated transferrin was resolved as an 80 000-dalton band and alpha-lactalbumin appeared as a 16 000-dalton protein. A library of recombinant plasmids containing cDNA (complementary DNA) sequences representing cytoplasmic polyadenylated RNA was used to isolate clones for the major rabbit caseins and alpha-lactalbumin. A preliminary characterization of these cDNA clones was achieved by colony hybridization with enriched RNA fractions as probes. Positive clones were identified by use of hybrid-promoted translation in vitro and immunoprecipitation of the translation products. The corresponding mRNA species were further identified by hybridizing RNA blots with radioactively labelled cDNA clones. We present the restriction map of alpha-casein and kappa-casein cDNA clones. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6123313

Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

PubMed

Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

2018-01-01

DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

Inhibition of recombinase polymerase amplification by background DNA: a lateral flow-based method for enriching target DNA.

PubMed

Rohrman, Brittany; Richards-Kortum, Rebecca

2015-02-03

Recombinase polymerase amplification (RPA) may be used to detect a variety of pathogens, often after minimal sample preparation. However, previous work has shown that whole blood inhibits RPA. In this paper, we show that the concentrations of background DNA found in whole blood prevent the amplification of target DNA by RPA. First, using an HIV-1 RPA assay with known concentrations of nonspecific background DNA, we show that RPA tolerates more background DNA when higher HIV-1 target concentrations are present. Then, using three additional assays, we demonstrate that the maximum amount of background DNA that may be tolerated in RPA reactions depends on the DNA sequences used in the assay. We also show that changing the RPA reaction conditions, such as incubation time and primer concentration, has little effect on the ability of RPA to function when high concentrations of background DNA are present. Finally, we develop and characterize a lateral flow-based method for enriching the target DNA concentration relative to the background DNA concentration. This sample processing method enables RPA of 10(4) copies of HIV-1 DNA in a background of 0-14 μg of background DNA. Without lateral flow sample enrichment, the maximum amount of background DNA tolerated is 2 μg when 10(6) copies of HIV-1 DNA are present. This method requires no heating or other external equipment, may be integrated with upstream DNA extraction and purification processes, is compatible with the components of lysed blood, and has the potential to detect HIV-1 DNA in infant whole blood with high proviral loads.

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

An improved protocol for DNA extraction from alkaline soil and sediment samples for constructing metagenomic libraries.

PubMed

Verma, Digvijay; Satyanarayana, T

2011-09-01

An improved single-step protocol has been developed for extracting pure community humic substance-free DNA from alkaline soils and sediments. The method is based on direct cell lysis in the presence of powdered activated charcoal and polyvinylpolypyrrolidone followed by precipitation with polyethyleneglycol and isopropanol. The strategy allows simultaneous isolation and purification of DNA while minimizing the loss of DNA with respect to other available protocols for metagenomic DNA extraction. Moreover, the purity levels are significant, which are difficult to attain with any of the methods reported in the literature for DNA extraction from soils. The DNA thus extracted was free from humic substances and, therefore, could be processed for restriction digestion, PCR amplification as well as for the construction of metagenomic libraries.

Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

PubMed

Maggi, Elaine C; Gravina, Silvia; Cheng, Haiying; Piperdi, Bilal; Yuan, Ziqiang; Dong, Xiao; Libutti, Steven K; Vijg, Jan; Montagna, Cristina

2018-01-01

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells

PubMed Central

Fernández, Agustín F.; Bayón, Gustavo F.; Urdinguio, Rocío G.; Toraño, Estela G.; García, María G.; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M.; Riancho, José A.; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A.

2015-01-01

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type–independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. PMID:25271306

AXM mutagenesis: an efficient means for the production of libraries for directed evolution of proteins.

PubMed

Holland, Erika G; Buhr, Diane L; Acca, Felicity E; Alderman, Dawn; Bovat, Kristin; Busygina, Valeria; Kay, Brian K; Weiner, Michael P; Kiss, Margaret M

2013-08-30

Affinity maturation is an important part of the recombinant antibody development process. There are several well-established approaches for generating libraries of mutated antibody genes for affinity maturation, but these approaches are generally too laborious or expensive to allow high-throughput, parallel processing of multiple antibodies. Here, we describe a scalable approach that enables the generation of libraries with greater than 10(8) clones from a single Escherichia coli transformation. In our method, a mutated DNA fragment is produced using PCR conditions that promote nucleotide misincorporation into newly synthesized DNA. In the PCR reaction, one of the primers contains at least three phosphorothioate linkages at its 5' end, and treatment of the PCR product with a 5' to 3' exonuclease is used to preferentially remove the strand synthesized with the non-modified primer, resulting in a single-stranded DNA fragment. This fragment then serves as a megaprimer to prime DNA synthesis on a uracilated, circular, single-stranded template in a Kunkel-like mutagenesis reaction that biases nucleotide base-changes between the megaprimer and uracilated DNA sequence in favor of the in vitro synthesized megaprimer. This method eliminates the inefficient subcloning steps that are normally required for the construction of affinity maturation libraries from randomly mutagenized antibody genes. Copyright © 2013. Published by Elsevier B.V.

Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples.

PubMed

Borsu, Laetitia; Intrieri, Julie; Thampi, Linta; Yu, Helena; Riely, Gregory; Nafa, Khedoudja; Chandramohan, Raghu; Ladanyi, Marc; Arcila, Maria E

2016-11-01

Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Genomic clones for human cholinesterase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kott, M.; Venta, P.J.; Larsen, J.

1987-05-01

A human genomic library was prepared from peripheral white blood cells from a single donor by inserting an MboI partial digest into BamHI poly-linker sites of EMBL3. This library was screened using an oligolabeled human cholinesterase cDNA probe over 700 bp long. The latter probe was obtained from a human basal ganglia cDNA library. Of approximately 2 million clones screened with high stringency conditions several positive clones were identified; two have been plaque purified. One of these clones has been partially mapped using restriction enzymes known to cut within the coded region of the cDNA for human serum cholinesterase. Hybridizationmore » of the fragments and their sizes are as expected if the genomic clone is cholinesterase. Sequencing of the DNA fragments in M13 is in progress to verify the identify of the clone and the location of introns.« less

Rapid and Easy Protocol for Quantification of Next-Generation Sequencing Libraries.

PubMed

Hawkins, Steve F C; Guest, Paul C

2018-01-01

The emergence of next-generation sequencing (NGS) over the last 10 years has increased the efficiency of DNA sequencing in terms of speed, ease, and price. However, the exact quantification of a NGS library is crucial in order to obtain good data on sequencing platforms developed by the current market leader Illumina. Different approaches for DNA quantification are available currently and the most commonly used are based on analysis of the physical properties of the DNA through spectrophotometric or fluorometric methods. Although these methods are technically simple, they do not allow exact quantification as can be achieved using a real-time quantitative PCR (qPCR) approach. A qPCR protocol for DNA quantification with applications in NGS library preparation studies is presented here. This can be applied in various fields of study such as medical disorders resulting from nutritional programming disturbances.

A DNA Barcode Library for Korean Chironomidae (Insecta: Diptera) and Indexes for Defining Barcode Gap

PubMed Central

Kim, Sungmin; Song, Kyo-Hong; Ree, Han-Il; Kim, Won

2012-01-01

Non-biting midges (Diptera: Chironomidae) are a diverse population that commonly causes respiratory allergies in humans. Chironomid larvae can be used to indicate freshwater pollution, but accurate identification on the basis of morphological characteristics is difficult. In this study, we constructed a mitochondrial cytochrome c oxidase subunit I (COI)-based DNA barcode library for Korean chironomids. This library consists of 211 specimens from 49 species, including adults and unidentified larvae. The interspecies and intraspecies COI sequence variations were analyzed. Sophisticated indexes were developed in order to properly evaluate indistinct barcode gaps that are created by insufficient sampling on both the interspecies and intraspecies levels and by variable mutation rates across taxa. In a variety of insect datasets, these indexes were useful for re-evaluating large barcode datasets and for defining COI barcode gaps. The COI-based DNA barcode library will provide a rapid and reliable tool for the molecular identification of Korean chironomid species. Furthermore, this reverse-taxonomic approach will be improved by the continuous addition of other speceis’ sequences to the library. PMID:22138764

Comparison Of A Neutron Kinetics Parameter For A Polyethylene Moderated Highly Enriched Uranium System

DOE Office of Scientific and Technical Information (OSTI.GOV)

McKenzie, IV, George Espy; Goda, Joetta Marie; Grove, Travis Justin

This paper examines the comparison of MCNP® code’s capability to calculate kinetics parameters effectively for a thermal system containing highly enriched uranium (HEU). The Rossi-α parameter was chosen for this examination because it is relatively easy to measure as well as easy to calculate using MCNP®’s kopts card. The Rossi-α also incorporates many other parameters of interest in nuclear kinetics most of which are more difficult to precisely measure. The comparison looks at two different nuclear data libraries for comparison to the experimental data. These libraries are ENDF/BVI (.66c) and ENDF/BVII (.80c).

Repair of DNA damage caused by cytosine deamination in mitochondrial DNA of forensic case samples.

PubMed

Gorden, Erin M; Sturk-Andreaggi, Kimberly; Marshall, Charla

2018-05-01

DNA sequence damage from cytosine deamination is well documented in degraded samples, such as those from ancient and forensic contexts. This study examined the effect of a DNA repair treatment on mitochondrial DNA (mtDNA) from aged and degraded skeletal samples. DNA extracts from 21 non-probative, degraded skeletal samples (aged 50-70 years) were utilized for the analysis. A portion of each sample extract was subjected to DNA repair using a commercial repair kit, the New England BioLabs' NEBNext FFPE DNA Repair Kit (Ipswich, MA). MtDNA was enriched using PCR and targeted capture in a side-by-side experiment of untreated and repaired DNA. Sequencing was performed using both traditional (Sanger-type; STS) and next-generation sequencing (NGS) methods Although cytosine deamination was evident in the mtDNA sequence data, the observed level of damaged bases varied by sequencing method as well as by enrichment type. The STS PCR amplicon data did not show evidence of cytosine deamination that could be distinguished from background signal in either the untreated or repaired sample set. However, the same PCR amplicons showed 850 C → T/G → A substitutions consistent with cytosine deamination with variant frequencies (VFs) of up to 25% when sequenced using NGS methods The occurrence of base misincorporation due to cytosine deamination was reduced by 98% (to 10) in the NGS amplicon data after repair. The NGS capture data indicated low levels (1-2%) of cytosine deamination in mtDNA fragments that was effectively mitigated by DNA repair. The observed difference in the level of cytosine deamination between the PCR and capture enrichment methods can be attributed to the greater propensity for stochastic effects from the PCR enrichment technique employed (e.g., low template input, increased PCR cycles). Altogether these results indicate that DNA repair may be required when sequencing PCR-amplified DNA from degraded forensic case samples with NGS methods. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

EPA Science Inventory

We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development - Poster

EPA Science Inventory

We examined the bacterial composition of chlorinated drinking water using 16S rRNA gene clone libraries derived from RNA and DNA extracted from twelve water samples collected in three different months (June, August, and September of 2007). Phylogenetic analysis of 1234 and 1117 ...

Construction and EST sequencing of full-length, drought stress cDNA libraries for common beans (Phaseolus vulgaris L.)

PubMed Central

2011-01-01

Background Common bean is an important legume crop with only a moderate number of short expressed sequence tags (ESTs) made with traditional methods. The goal of this research was to use full-length cDNA technology to develop ESTs that would overlap with the beginning of open reading frames and therefore be useful for gene annotation of genomic sequences. The library was also constructed to represent genes expressed under drought, low soil phosphorus and high soil aluminum toxicity. We also undertook comparisons of the full-length cDNA library to two previous non-full clone EST sets for common bean. Results Two full-length cDNA libraries were constructed: one for the drought tolerant Mesoamerican genotype BAT477 and the other one for the acid-soil tolerant Andean genotype G19833 which has been selected for genome sequencing. Plants were grown in three soil types using deep rooting cylinders subjected to drought and non-drought stress and tissues were collected from both roots and above ground parts. A total of 20,000 clones were selected robotically, half from each library. Then, nearly 10,000 clones from the G19833 library were sequenced with an average read length of 850 nucleotides. A total of 4,219 unigenes were identified consisting of 2,981 contigs and 1,238 singletons. These were functionally annotated with gene ontology terms and placed into KEGG pathways. Compared to other EST sequencing efforts in common bean, about half of the sequences were novel or represented the 5' ends of known genes. Conclusions The present full-length cDNA libraries add to the technological toolbox available for common bean and our sequencing of these clones substantially increases the number of unique EST sequences available for the common bean genome. All of this should be useful for both functional gene annotation, analysis of splice site variants and intron/exon boundary determination by comparison to soybean genes or with common bean whole-genome sequences. In addition the library has a large number of transcription factors and will be interesting for discovery and validation of drought or abiotic stress related genes in common bean. PMID:22118559

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

NASA Technical Reports Server (NTRS)

Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

2003-01-01

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.

Phylogenetic Analysis of Anaerobic Psychrophilic Enrichment Cultures Obtained from a Greenland Glacier Ice Core

PubMed Central

Sheridan, Peter P.; Miteva, Vanya I.; Brenchley, Jean E.

2003-01-01

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at −9°C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 × 107 cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at −2°C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years. PMID:12676695

An evaluation of two-channel ChIP-on-chip and DNA methylation microarray normalization strategies

PubMed Central

2012-01-01

Background The combination of chromatin immunoprecipitation with two-channel microarray technology enables genome-wide mapping of binding sites of DNA-interacting proteins (ChIP-on-chip) or sites with methylated CpG di-nucleotides (DNA methylation microarray). These powerful tools are the gateway to understanding gene transcription regulation. Since the goals of such studies, the sample preparation procedures, the microarray content and study design are all different from transcriptomics microarrays, the data pre-processing strategies traditionally applied to transcriptomics microarrays may not be appropriate. Particularly, the main challenge of the normalization of "regulation microarrays" is (i) to make the data of individual microarrays quantitatively comparable and (ii) to keep the signals of the enriched probes, representing DNA sequences from the precipitate, as distinguishable as possible from the signals of the un-enriched probes, representing DNA sequences largely absent from the precipitate. Results We compare several widely used normalization approaches (VSN, LOWESS, quantile, T-quantile, Tukey's biweight scaling, Peng's method) applied to a selection of regulation microarray datasets, ranging from DNA methylation to transcription factor binding and histone modification studies. Through comparison of the data distributions of control probes and gene promoter probes before and after normalization, and assessment of the power to identify known enriched genomic regions after normalization, we demonstrate that there are clear differences in performance between normalization procedures. Conclusion T-quantile normalization applied separately on the channels and Tukey's biweight scaling outperform other methods in terms of the conservation of enriched and un-enriched signal separation, as well as in identification of genomic regions known to be enriched. T-quantile normalization is preferable as it additionally improves comparability between microarrays. In contrast, popular normalization approaches like quantile, LOWESS, Peng's method and VSN normalization alter the data distributions of regulation microarrays to such an extent that using these approaches will impact the reliability of the downstream analysis substantially. PMID:22276688

Development of High Throughput Process for Constructing 454 Titanium and Illumina Libraries

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deshpande, Shweta; Hack, Christopher; Tang, Eric

2010-05-28

We have developed two processes with the Biomek FX robot to construct 454 titanium and Illumina libraries in order to meet the increasing library demands. All modifications in the library construction steps were made to enable the adaptation of the entire processes to work with the 96-well plate format. The key modifications include the shearing of DNA with Covaris E210 and the enzymatic reaction cleaning and fragment size selection with SPRI beads and magnetic plate holders. The construction of 96 Titanium libraries takes about 8 hours from sheared DNA to ssDNA recovery. The processing of 96 Illumina libraries takes lessmore » time than that of the Titanium library process. Although both processes still require manual transfer of plates from robot to other work stations such as thermocyclers, these robotic processes represent about 12- to 24-folds increase of library capacity comparing to the manual processes. To enable the sequencing of many libraries in parallel, we have also developed sets of molecular barcodes for both library types. The requirements for the 454 library barcodes include 10 bases, 40-60percent GC, no consecutive same base, and no less than 3 bases difference between barcodes. We have used 96 of the resulted 270 barcodes to construct libraries and pool to test the ability of accurately assigning reads to the right samples. When allowing 1 base error occurred in the 10 base barcodes, we could assign 99.6percent of the total reads and 100percent of them were uniquely assigned. As for the Illumina barcodes, the requirements include 4 bases, balanced GC, and at least 2 bases difference between barcodes. We have begun to assess the ability to assign reads after pooling different number of libraries. We will discuss the progress and the challenges of these scale-up processes.« less

Comparative analysis of bacteria associated with different mosses by 16S rRNA and 16S rDNA sequencing.

PubMed

Tian, Yang; Li, Yan Hong

2017-01-01

To understand the differences of the bacteria associated with different mosses, a phylogenetic study of bacterial communities in three mosses was carried out based on 16S rDNA and 16S rRNA sequencing. The mosses used were Hygroamblystegium noterophilum, Entodon compressus and Grimmia montana, representing hygrophyte, shady plant and xerophyte, respectively. In total, the operational taxonomic units (OTUs), richness and diversity were different regardless of the moss species and the library level. All the examined 1183 clones were assigned to 248 OTUs, 56 genera were assigned in rDNA libraries and 23 genera were determined at the rRNA level. Proteobacteria and Bacteroidetes were considered as the most dominant phyla in all the libraries, whereas abundant Actinobacteria and Acidobacteria were detected in the rDNA library of Entodon compressus and approximately 24.7% clones were assigned to Candidate division TM7 in Grimmia montana at rRNA level. The heatmap showed the bacterial profiles derived from rRNA and rDNA were partly overlapping. However, the principle component analysis of all the profiles derived from rDNA showed sharper differences between the different mosses than that of rRNA-based profiles. This suggests that the metabolically active bacterial compositions in different mosses were more phylogenetically similar and the differences of the bacteria associated with different mosses were mainly detected at the rDNA level. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA sequencing is preferred approach to have a good understanding on the constitution of the microbial communities in mosses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Next-generation sequencing library construction on a surface.

PubMed

Feng, Kuan; Costa, Justin; Edwards, Jeremy S

2018-05-30

Next-generation sequencing (NGS) has revolutionized almost all fields of biology, agriculture and medicine, and is widely utilized to analyse genetic variation. Over the past decade, the NGS pipeline has been steadily improved, and the entire process is currently relatively straightforward. However, NGS instrumentation still requires upfront library preparation, which can be a laborious process, requiring significant hands-on time. Herein, we present a simple but robust approach to streamline library preparation by utilizing surface bound transposases to construct DNA libraries directly on a flowcell surface. The surface bound transposases directly fragment genomic DNA while simultaneously attaching the library molecules to the flowcell. We sequenced and analysed a Drosophila genome library generated by this surface tagmentation approach, and we showed that our surface bound library quality was comparable to the quality of the library from a commercial kit. In addition to the time and cost savings, our approach does not require PCR amplification of the library, which eliminates potential problems associated with PCR duplicates. We described the first study to construct libraries directly on a flowcell. We believe our technique could be incorporated into the existing Illumina sequencing pipeline to simplify the workflow, reduce costs, and improve data quality.

Selection dynamic of Escherichia coli host in M13 combinatorial peptide phage display libraries.

PubMed

Zanconato, Stefano; Minervini, Giovanni; Poli, Irene; De Lucrezia, Davide

2011-01-01

Phage display relies on an iterative cycle of selection and amplification of random combinatorial libraries to enrich the initial population of those peptides that satisfy a priori chosen criteria. The effectiveness of any phage display protocol depends directly on library amino acid sequence diversity and the strength of the selection procedure. In this study we monitored the dynamics of the selective pressure exerted by the host organism on a random peptide library in the absence of any additional selection pressure. The results indicate that sequence censorship exerted by Escherichia coli dramatically reduces library diversity and can significantly impair phage display effectiveness.

Evaluation of anonymous and expressed sequence tag derived polymorphic microsatellite markers in the tobacco budworm Heliothis virescens (Lepidoptera: noctuidae)

USDA-ARS?s Scientific Manuscript database

Polymorphic genetic markers were identified and characterized using a partial genomic library of Heliothis virescens enriched for simple sequence repeats (SSR) and nucleotide sequences of expressed sequence tags (EST). Nucleotide sequences of 192 clones from the partial genomic library yielded 147 u...

Towards isolation of the gene for X-linked retinitis pigmentosa (RP3)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dry, K.L.; Aldred, M.A.; Hardwick, L.J.

1994-09-01

Until recently the region of interest containing the gene for X-linked retinitis pigmentosa (RP3) was thought to lie between CYBB (Xp21.1) and the proximal end of the deletion in patient BB (JBBprox). This region was thought to span 100-150 kb. Here we present new mapping data to show that the distance between the 5{prime} (most proximal) end of CYBB and JBBprox is only 50 kb. Recently Roux et al. (1994) have described the isolation of a gene within this region but this showed no disease-associated changes. Further evidence from mapping the deletion in patient NF (who suffered from McLead`s syndromemore » and CGD but not RP) and from linkage analysis of our RP3 families with a new dinucleotide repeat suggests that the gene must extend proximally from JBBprox. In order to extend the region of search we have constructed a YAC contig spanning 800 kb to OTC. We are continuing our search for the RP3 gene using a variety of strategies including exon trapping and cDNA enrichment as well as direct screening of cDNA libraries with subclones from this region.« less

Sequences characterization of microsatellite DNA sequences in Pacific abalone ( Haliotis discus hannai)

NASA Astrophysics Data System (ADS)

Li, Qi; Akihiro, Kijima

2007-01-01

The microsatellite-enriched library was constructed using magnetic bead hybridization selection method, and the microsatellite DNA sequences were analyzed in Pacific abalone Haliotis discus hannai. Three hundred and fifty white colonies were screened using PCR-based technique, and 84 clones were identified to potentially contain microsatellite repeat motif. The 84 clones were sequenced, and 42 microsatellites and 4 minisatellites with a minimum of five repeats were found (13.1% of white colonies screened). Besides the motif of CA contained in the oligoprobe, we also found other 16 types of microsatellite repeats including a dinucleotide repeat, two tetranucleotide repeats, twelve pentanucleotide repeats and a hexanucleotide repeat. According to Weber (1990), the microsatellite sequences obtained could be categorized structurally into perfect repeats (73.3%), imperfect repeats (13.3%), and compound repeats (13.4%). Among the microsatellite repeats, relatively short arrays (<20 repeats) were most abundant, accounting for 75.0%. The largest length of microsatellites was 48 repeats, and the average number of repeats was 13.4. The data on the composition and length distribution of microsatellites obtained in the present study can be useful for choosing the repeat motifs for microsatellite isolation in other abalone species.

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy.

PubMed

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-12-10

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.

Microbial life in Champagne Pool, a geothermal spring in Waiotapu, New Zealand.

PubMed

Hetzer, Adrian; Morgan, Hugh W; McDonald, Ian R; Daughney, Christopher J

2007-07-01

Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy

PubMed Central

Chen, Man; Yu, Yuanyuan; Jiang, Feng; Zhou, Junwei; Li, Yongshu; Liang, Chao; Dang, Lei; Lu, Aiping; Zhang, Ge

2016-01-01

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed. PMID:27973403

A genetic linkage map of the long arm of human chromosome 22.

PubMed

Rouleau, G A; Haines, J L; Bazanowski, A; Colella-Crowley, A; Trofatter, J A; Wexler, N S; Conneally, P M; Gusella, J F

1989-01-01

We have used a recombinant phage library enriched for chromosome 22 sequences to isolate and characterize eight anonymous DNA probes detecting restriction fragment length polymorphisms on this autosome. These were used in conjunction with eight previously reported loci, including the genes BCR, IGLV, and PDGFB, four anonymous DNA markers, and the P1 blood group antigen, to construct a linkage map for chromosome 22. The linkage group is surprisingly large, spanning 97 cM on the long arm of the chromosome. There are no large gaps in the map; the largest intermarker interval is 14 cM. Unlike several other chromosomes, little overall difference was observed for sex-specific recombination rates on chromosome 22. The availability of a genetic map will facilitate investigation of chromosome 22 rearrangements in such disorders as cat eye syndrome and DiGeorge syndrome, deletions in acoustic neuroma and meningioma, and translocations in Ewing sarcoma. This defined set of linked markers will also permit testing chromosome 22 for the presence of particular disease genes by family studies and should immediately support more precise mapping and identification of flanking markers for NF2, the defective gene causing bilateral acoustic neurofibromatosis.

Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues

PubMed Central

Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Terry, Richard; Turczyk, Brian M.; Yang, Joyce L.; Lee, Ho Suk; Aach, John; Zhang, Kun; Church, George M.

2014-01-01

RNA sequencing measures the quantitative change in gene expression over the whole transcriptome, but it lacks spatial context. On the other hand, in situ hybridization provides the location of gene expression, but only for a small number of genes. Here we detail a protocol for genome-wide profiling of gene expression in situ in fixed cells and tissues, in which RNA is converted into cross-linked cDNA amplicons and sequenced manually on a confocal microscope. Unlike traditional RNA-seq our method enriches for context-specific transcripts over house-keeping and/or structural RNA, and it preserves the tissue architecture for RNA localization studies. Our protocol is written for researchers experienced in cell microscopy with minimal computing skills. Library construction and sequencing can be completed within 14 d, with image analysis requiring an additional 2 d. PMID:25675209

Determination of buoyant density and sensitivity to chloroform and freon for the etiological agent of infectious salmonid anaemia

USGS Publications Warehouse

Christie, K.E.; Hjeltnes, B.; Uglenes , I.; Winton, J.R.

1993-01-01

Plasma was collected from Atlantic salmon Salrno salar with acute infectious salmon anaemia (ISA) and used to challenge Atlantic salmon parr by intraperitoneal injection. Treatment of plasma with the lipid solvent, chloroform, showed that the etiological agent of ISA contained essential lipids, probably as a viral envelope. Some infectivity remained following treatment with freon. Injection challenges using fractions from equilibrium density gradient centrifugation of plasma from fish with acute ISA revealed a band of infectivity in the range 1.184 to 1.262 g cm-3. The band was believed to conta~n both complete ISA-virus particles and infectious particles lacking a complete envelope, nucleocapsid or genome. Density gradient centrifugation of infectious plasma for enrichment of the putative ISA virus appeared to offer a suitable method for obtaining virus-specific nucleic acid for use in the construction of cDNA libraries. 

Single molecule targeted sequencing for cancer gene mutation detection.

PubMed

Gao, Yan; Deng, Liwei; Yan, Qin; Gao, Yongqian; Wu, Zengding; Cai, Jinsen; Ji, Daorui; Li, Gailing; Wu, Ping; Jin, Huan; Zhao, Luyang; Liu, Song; Ge, Liangjin; Deem, Michael W; He, Jiankui

2016-05-19

With the rapid decline in cost of sequencing, it is now affordable to examine multiple genes in a single disease-targeted clinical test using next generation sequencing. Current targeted sequencing methods require a separate step of targeted capture enrichment during sample preparation before sequencing. Although there are fast sample preparation methods available in market, the library preparation process is still relatively complicated for physicians to use routinely. Here, we introduced an amplification-free Single Molecule Targeted Sequencing (SMTS) technology, which combined targeted capture and sequencing in one step. We demonstrated that this technology can detect low-frequency mutations using artificially synthesized DNA sample. SMTS has several potential advantages, including simple sample preparation thus no biases and errors are introduced by PCR reaction. SMTS has the potential to be an easy and quick sequencing technology for clinical diagnosis such as cancer gene mutation detection, infectious disease detection, inherited condition screening and noninvasive prenatal diagnosis.

Novel Technology for Enrichment of Biomolecules from Cell-Free Body Fluids and Subsequent DNA Sizing.

PubMed

Patel, Vipulkumar; Celec, Peter; Grunt, Magdalena; Schwarzenbach, Heidi; Jenneckens, Ingo; Hillebrand, Timo

2016-01-01

Circulating cell-free DNA (ccfDNA) is a promising diagnostic tool and its size fractionation is of interest. However, kits for isolation of ccfDNA available on the market are designed for small volumes hence processing large sample volumes is laborious. We have tested a new method that enables enrichment of ccfDNA from large volumes of plasma and subsequently allows size-fractionation of isolated ccfDNA into two fractions with individually established cut-off levels of ccfDNA length. This method allows isolation of low-abundant DNA as well as separation of long and short DNA molecules. This procedure may be important e.g., in prenatal diagnostics and cancer research that have been already confirmed by our primary experiments. Here, we report the results of selective separation of 200- and 500-bp long synthetic DNA fragments spiked in plasma samples. Furthermore, we size-fractionated ccfDNA from the plasma of pregnant women and verified the prevalence of fetal ccfDNA in all fractions.

Enriched Title-Based Keyword Index Generation Using dBase II.

ERIC Educational Resources Information Center

Rajendran, P. P.

1986-01-01

Describes the use of a database management system (DBMS)--dBaseII--to create an enriched title-based keyword index for a collection of news items at the Renewable Energy Resources Information Center of the Asian Institute of Technology. The use of DBMSs in libraries in developing countries is emphasized. (Author/LRW)

A comparative study of ChIP-seq sequencing library preparation methods.

PubMed

Sundaram, Arvind Y M; Hughes, Timothy; Biondi, Shea; Bolduc, Nathalie; Bowman, Sarah K; Camilli, Andrew; Chew, Yap C; Couture, Catherine; Farmer, Andrew; Jerome, John P; Lazinski, David W; McUsic, Andrew; Peng, Xu; Shazand, Kamran; Xu, Feng; Lyle, Robert; Gilfillan, Gregor D

2016-10-21

ChIP-seq is the primary technique used to investigate genome-wide protein-DNA interactions. As part of this procedure, immunoprecipitated DNA must undergo "library preparation" to enable subsequent high-throughput sequencing. To facilitate the analysis of biopsy samples and rare cell populations, there has been a recent proliferation of methods allowing sequencing library preparation from low-input DNA amounts. However, little information exists on the relative merits, performance, comparability and biases inherent to these procedures. Notably, recently developed single-cell ChIP procedures employing microfluidics must also employ library preparation reagents to allow downstream sequencing. In this study, seven methods designed for low-input DNA/ChIP-seq sample preparation (Accel-NGS® 2S, Bowman-method, HTML-PCR, SeqPlex™, DNA SMART™, TELP and ThruPLEX®) were performed on five replicates of 1 ng and 0.1 ng input H3K4me3 ChIP material, and compared to a "gold standard" reference PCR-free dataset. The performance of each method was examined for the prevalence of unmappable reads, amplification-derived duplicate reads, reproducibility, and for the sensitivity and specificity of peak calling. We identified consistent high performance in a subset of the tested reagents, which should aid researchers in choosing the most appropriate reagents for their studies. Furthermore, we expect this work to drive future advances by identifying and encouraging use of the most promising methods and reagents. The results may also aid judgements on how comparable are existing datasets that have been prepared with different sample library preparation reagents.

Microbial communities involved in methane production from hydrocarbons in oil sands tailings.

PubMed

Siddique, Tariq; Penner, Tara; Klassen, Jonathan; Nesbø, Camilla; Foght, Julia M

2012-09-04

Microbial metabolism of residual hydrocarbons, primarily short-chain n-alkanes and certain monoaromatic hydrocarbons, in oil sands tailings ponds produces large volumes of CH(4) in situ. We characterized the microbial communities involved in methanogenic biodegradation of whole naphtha (a bitumen extraction solvent) and its short-chain n-alkane (C(6)-C(10)) and BTEX (benzene, toluene, ethylbenzene, and xylenes) components using primary enrichment cultures derived from oil sands tailings. Clone libraries of bacterial 16S rRNA genes amplified from these enrichments showed increased proportions of two orders of Bacteria: Clostridiales and Syntrophobacterales, with Desulfotomaculum and Syntrophus/Smithella as the closest named relatives, respectively. In parallel archaeal clone libraries, sequences affiliated with cultivated acetoclastic methanogens (Methanosaetaceae) were enriched in cultures amended with n-alkanes, whereas hydrogenotrophic methanogens (Methanomicrobiales) were enriched with BTEX. Naphtha-amended cultures harbored a blend of these two archaeal communities. The results imply syntrophic oxidation of hydrocarbons in oil sands tailings, with the activities of different carbon flow pathways to CH(4) being influenced by the primary hydrocarbon substrate. These results have implications for predicting greenhouse gas emissions from oil sands tailings repositories.

Hybrid selection for sequencing pathogen genomes from clinical samples

PubMed Central

2011-01-01

We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla. PMID:21835008

Current and future resources for functional metagenomics.

PubMed

Lam, Kathy N; Cheng, Jiujun; Engel, Katja; Neufeld, Josh D; Charles, Trevor C

2015-01-01

Functional metagenomics is a powerful experimental approach for studying gene function, starting from the extracted DNA of mixed microbial populations. A functional approach relies on the construction and screening of metagenomic libraries-physical libraries that contain DNA cloned from environmental metagenomes. The information obtained from functional metagenomics can help in future annotations of gene function and serve as a complement to sequence-based metagenomics. In this Perspective, we begin by summarizing the technical challenges of constructing metagenomic libraries and emphasize their value as resources. We then discuss libraries constructed using the popular cloning vector, pCC1FOS, and highlight the strengths and shortcomings of this system, alongside possible strategies to maximize existing pCC1FOS-based libraries by screening in diverse hosts. Finally, we discuss the known bias of libraries constructed from human gut and marine water samples, present results that suggest bias may also occur for soil libraries, and consider factors that bias metagenomic libraries in general. We anticipate that discussion of current resources and limitations will advance tools and technologies for functional metagenomics research.

BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction.

PubMed

Townsley, Brad T; Covington, Michael F; Ichihashi, Yasunori; Zumstein, Kristina; Sinha, Neelima R

2015-01-01

Next Generation Sequencing (NGS) is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq) has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing the terminal breathing of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq) reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE) libraries and can easily extend to full transcript coverage shotgun (SHO) type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

A simple and novel method for RNA-seq library preparation of single cell cDNA analysis by hyperactive Tn5 transposase.

PubMed

Brouilette, Scott; Kuersten, Scott; Mein, Charles; Bozek, Monika; Terry, Anna; Dias, Kerith-Rae; Bhaw-Rosun, Leena; Shintani, Yasunori; Coppen, Steven; Ikebe, Chiho; Sawhney, Vinit; Campbell, Niall; Kaneko, Masahiro; Tano, Nobuko; Ishida, Hidekazu; Suzuki, Ken; Yashiro, Kenta

2012-10-01

Deep sequencing of single cell-derived cDNAs offers novel insights into oncogenesis and embryogenesis. However, traditional library preparation for RNA-seq analysis requires multiple steps with consequent sample loss and stochastic variation at each step significantly affecting output. Thus, a simpler and better protocol is desirable. The recently developed hyperactive Tn5-mediated library preparation, which brings high quality libraries, is likely one of the solutions. Here, we tested the applicability of hyperactive Tn5-mediated library preparation to deep sequencing of single cell cDNA, optimized the protocol, and compared it with the conventional method based on sonication. This new technique does not require any expensive or special equipment, which secures wider availability. A library was constructed from only 100 ng of cDNA, which enables the saving of precious specimens. Only a few steps of robust enzymatic reaction resulted in saved time, enabling more specimens to be prepared at once, and with a more reproducible size distribution among the different specimens. The obtained RNA-seq results were comparable to the conventional method. Thus, this Tn5-mediated preparation is applicable for anyone who aims to carry out deep sequencing for single cell cDNAs. Copyright © 2012 Wiley Periodicals, Inc.

Sequence evaluation of four specific cDNA libraries for developmental genomics of sunflower.

PubMed

Tamborindeguy, C; Ben, C; Liboz, T; Gentzbittel, L

2004-04-01

Four different cDNA libraries were constructed from sunflower protoplasts growing under embryogenic and non-embryogenic conditions: one standard library from each condition and two subtractive libraries in opposite sense. A total of 22,876 cDNA clones were obtained and 4800 ESTs were sequenced, giving rise to 2479 high quality ESTs representing an unigene set of 1502 sequences. This set was compared with ESTs represented in public databases using the programs BLASTN and BLASTX, and its members were classified according to putative function using the catalog in the Kyoto Encyclopedia of Genes and Genomes (KEGG). Some 33% of sequences failed to align with existing plant ESTs and therefore represent putative novel genes. The libraries show a low level of redundancy and, on average, 50% of the present ESTs have not been previously reported for sunflower. Several potentially interesting genes were identified, based on their homology with genes involved in animal zygotic division or plant embryogenesis. We also identified two ESTs that show significantly different levels of expression under embryogenic and non-embryogenic conditions. The libraries described here represent an original and valuable resource for the discovery of yet unknown genes putatively involved in dicot embryogenesis and improving our knowledge of the mechanisms involved in polarity acquisition by plant embryos.

Miniaturization Technologies for Efficient Single-Cell Library Preparation for Next-Generation Sequencing.

PubMed

Mora-Castilla, Sergio; To, Cuong; Vaezeslami, Soheila; Morey, Robert; Srinivasan, Srimeenakshi; Dumdie, Jennifer N; Cook-Andersen, Heidi; Jenkins, Joby; Laurent, Louise C

2016-08-01

As the cost of next-generation sequencing has decreased, library preparation costs have become a more significant proportion of the total cost, especially for high-throughput applications such as single-cell RNA profiling. Here, we have applied novel technologies to scale down reaction volumes for library preparation. Our system consisted of in vitro differentiated human embryonic stem cells representing two stages of pancreatic differentiation, for which we prepared multiple biological and technical replicates. We used the Fluidigm (San Francisco, CA) C1 single-cell Autoprep System for single-cell complementary DNA (cDNA) generation and an enzyme-based tagmentation system (Nextera XT; Illumina, San Diego, CA) with a nanoliter liquid handler (mosquito HTS; TTP Labtech, Royston, UK) for library preparation, reducing the reaction volume down to 2 µL and using as little as 20 pg of input cDNA. The resulting sequencing data were bioinformatically analyzed and correlated among the different library reaction volumes. Our results showed that decreasing the reaction volume did not interfere with the quality or the reproducibility of the sequencing data, and the transcriptional data from the scaled-down libraries allowed us to distinguish between single cells. Thus, we have developed a process to enable efficient and cost-effective high-throughput single-cell transcriptome sequencing. © 2016 Society for Laboratory Automation and Screening.

Discovering Drugs with DNA-Encoded Library Technology: From Concept to Clinic with an Inhibitor of Soluble Epoxide Hydrolase.

PubMed

Belyanskaya, Svetlana L; Ding, Yun; Callahan, James F; Lazaar, Aili L; Israel, David I

2017-05-04

DNA-encoded chemical library technology was developed with the vision of its becoming a transformational platform for drug discovery. The hope was that a new paradigm for the discovery of low-molecular-weight drugs would be enabled by combining the vast molecular diversity achievable with combinatorial chemistry, the information-encoding attributes of DNA, the power of molecular biology, and a streamlined selection-based discovery process. Here, we describe the discovery and early clinical development of GSK2256294, an inhibitor of soluble epoxide hydrolase (sEH, EPHX2), by using encoded-library technology (ELT). GSK2256294 is an orally bioavailable, potent and selective inhibitor of sEH that has a long half life and produced no serious adverse events in a first-time-in-human clinical study. To our knowledge, GSK2256294 is the first molecule discovered from this technology to enter human clinical testing and represents a realization of the vision that DNA-encoded chemical library technology can efficiently yield molecules with favorable properties that can be readily progressed into high-quality drugs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expanded Genetic Codes in Next Generation Sequencing Enable Decontamination and Mitochondrial Enrichment

PubMed Central

McKernan, Kevin J.; Spangler, Jessica; Zhang, Lei; Tadigotla, Vasisht; McLaughlin, Stephen; Warner, Jason; Zare, Amir; Boles, Richard G.

2014-01-01

We have developed a PCR method, coined Déjà vu PCR, that utilizes six nucleotides in PCR with two methyl specific restriction enzymes that respectively digest these additional nucleotides. Use of this enzyme-and-nucleotide combination enables what we term a “DNA diode”, where DNA can advance in a laboratory in only one direction and cannot feedback into upstream assays. Here we describe aspects of this method that enable consecutive amplification with the introduction of a 5th and 6th base while simultaneously providing methylation dependent mitochondrial DNA enrichment. These additional nucleotides enable a novel DNA decontamination technique that generates ephemeral and easy to decontaminate DNA. PMID:24788618

Improving enrichment of circulating fetal DNA for genetic testing: size fractionation followed by whole gene amplification.

PubMed

Jorgez, Carolina J; Bischoff, Farideh Z

2009-01-01

Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA. Copyright 2009 S. Karger AG, Basel.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells.

PubMed

Fernández, Agustín F; Bayón, Gustavo F; Urdinguio, Rocío G; Toraño, Estela G; García, María G; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M; Riancho, José A; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A; Fraga, Mario F

2015-01-01

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. © 2015 Fernández et al.; Published by Cold Spring Harbor Laboratory Press.

Genomic sequencing of Pleistocene cave bears

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noonan, James P.; Hofreiter, Michael; Smith, Doug

2005-04-01

Despite the information content of genomic DNA, ancient DNA studies to date have largely been limited to amplification of mitochondrial DNA due to technical hurdles such as contamination and degradation of ancient DNAs. In this study, we describe two metagenomic libraries constructed using unamplified DNA extracted from the bones of two 40,000-year-old extinct cave bears. Analysis of {approx}1 Mb of sequence from each library showed that, despite significant microbial contamination, 5.8 percent and 1.1 percent of clones in the libraries contain cave bear inserts, yielding 26,861 bp of cave bear genome sequence. Alignment of this sequence to the dog genome,more » the closest sequenced genome to cave bear in terms of evolutionary distance, revealed roughly the expected ratio of cave bear exons, repeats and conserved noncoding sequences. Only 0.04 percent of all clones sequenced were derived from contamination with modern human DNA. Comparison of cave bear with orthologous sequences from several modern bear species revealed the evolutionary relationship of these lineages. Using the metagenomic approach described here, we have recovered substantial quantities of mammalian genomic sequence more than twice as old as any previously reported, establishing the feasibility of ancient DNA genomic sequencing programs.« less

Informatic selection of a neural crest-melanocyte cDNA set for microarray analysis

PubMed Central

Loftus, S. K.; Chen, Y.; Gooden, G.; Ryan, J. F.; Birznieks, G.; Hilliard, M.; Baxevanis, A. D.; Bittner, M.; Meltzer, P.; Trent, J.; Pavan, W.

1999-01-01

With cDNA microarrays, it is now possible to compare the expression of many genes simultaneously. To maximize the likelihood of finding genes whose expression is altered under the experimental conditions, it would be advantageous to be able to select clones for tissue-appropriate cDNA sets. We have taken advantage of the extensive sequence information in the dbEST expressed sequence tag (EST) database to identify a neural crest-derived melanocyte cDNA set for microarray analysis. Analysis of characterized genes with dbEST identified one library that contained ESTs representing 21 neural crest-expressed genes (library 198). The distribution of the ESTs corresponding to these genes was biased toward being derived from library 198. This is in contrast to the EST distribution profile for a set of control genes, characterized to be more ubiquitously expressed in multiple tissues (P < 1 × 10−9). From library 198, a subset of 852 clustered ESTs were selected that have a library distribution profile similar to that of the 21 neural crest-expressed genes. Microarray analysis demonstrated the majority of the neural crest-selected 852 ESTs (Mel1 array) were differentially expressed in melanoma cell lines compared with a non-neural crest kidney epithelial cell line (P < 1 × 10−8). This was not observed with an array of 1,238 ESTs that was selected without library origin bias (P = 0.204). This study presents an approach for selecting tissue-appropriate cDNAs that can be used to examine the expression profiles of developmental processes and diseases. PMID:10430933

The Drosophila gene collection: Identification of putative full-length cDNAs for 70 percent of D. melanogaster genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stapleton, Mark; Liao, Guochun; Brokstein, Peter

2002-08-12

Collections of full-length nonredundant cDNA clones are critical reagents for functional genomics. The first step toward these resources is the generation and single-pass sequencing of cDNA libraries that contain a high proportion of full-length clones. The first release of the Drosophila Gene Collection Release 1 (DGCr1) was produced from six libraries representing various tissues, developmental stages, and the cultured S2 cell line. Nearly 80,000 random 5prime expressed sequence tags (EST) from these libraries were collapsed into a nonredundant set of 5849 cDNAs, corresponding to {approx}40 percent of the 13,474 predicted genes in Drosophila. To obtain cDNA clones representing the remainingmore » genes, we have generated an additional 157,835 5prime ESTs from two previously existing and three new libraries. One new library is derived from adult testis, a tissue we previously did not exploit for gene discovery; two new cap-trapped normalized libraries are derived from 0-22hr embryos and adult heads. Taking advantage of the annotated D. melanogaster genome sequence, we clustered the ESTs by aligning them to the genome. Clusters that overlap genes not already represented by cDNA clones in the DGCr1 were analyzed further, and putative full-length clones were selected for inclusion in the new DGC. This second release of the DGC (DGCr2) contains 5061 additional clones, extending the collection to 10,910 cDNAs representing >70 percent of the predicted genes in Drosophila.« less

Discovery of Potent and Selective Inhibitors for ADAMTS-4 through DNA-Encoded Library Technology (ELT).

PubMed

Ding, Yun; O'Keefe, Heather; DeLorey, Jennifer L; Israel, David I; Messer, Jeffrey A; Chiu, Cynthia H; Skinner, Steven R; Matico, Rosalie E; Murray-Thompson, Monique F; Li, Fan; Clark, Matthew A; Cuozzo, John W; Arico-Muendel, Christopher; Morgan, Barry A

2015-08-13

The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-encoded triazine library by affinity selection. Structure-activity relationship studies based on the selection information led to the identification of potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC50 of 10 nM against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE, and ADAMTS-13. These inhibitors have no obvious zinc ligand functionality.

Discovery of Potent and Selective Inhibitors for ADAMTS-4 through DNA-Encoded Library Technology (ELT)

PubMed Central

2015-01-01

The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-encoded triazine library by affinity selection. Structure–activity relationship studies based on the selection information led to the identification of potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC50 of 10 nM against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE, and ADAMTS-13. These inhibitors have no obvious zinc ligand functionality. PMID:26288689

Construction of a general human chromosome jumping library, with application to cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Collins, F.S.; Drumm, M.L.; Cole, J.L.

1987-02-27

In many genetic disorders, the responsible gene and its protein product are unknown. The technique known as reverse genetics, in which chromosomal map positions and genetically linked DNA markers are used to identify and clone such genes, is complicated by the fact that the molecular distances from the closest DNA markers to the gene itself are often too large to traverse by standard cloning techniques. To address this situation, a general human chromosome jumping library was constructed that allows the cloning of DNA sequences approximately 100 kilobases away from any starting point in genomic DNA. As an illustration of itsmore » usefulness, this library was searched for a jumping clone, starting at the met oncogene, which is a marker tightly linked to the cystic fibrosis gene that is located on human chromosome 7. Mapping of the new genomic fragment by pulsed field gel electrophoresis confirmed that it resides on chromosome 7 within 240 kilobases downstream of the met gene. The use of chromosome jumping should be applicable to any genetic locus for which a closely linked DNA marker is available.« less

Diversity of ribosomal 16S DNA- and RNA-based bacterial community in an office building drinking water system.

PubMed

Inkinen, J; Jayaprakash, B; Santo Domingo, J W; Keinänen-Toivola, M M; Ryu, H; Pitkänen, T

2016-06-01

Next-generation sequencing of 16S ribosomal RNA genes (rDNA) and ribosomal RNA (rRNA) was used to characterize water and biofilm microbiome collected from a drinking water distribution system of an office building after its first year of operation. The total bacterial community (rDNA) and active bacterial members (rRNA) sequencing databases were generated by Illumina MiSeq PE250 platform. As estimated by Chao1 index, species richness in cold water system was lower (180-260) in biofilms (Sphingomonas spp., Methylobacterium spp., Limnohabitans spp., Rhizobiales order) than in waters (250-580), (also Methylotenera spp.) (P = 0·005, n = 20). Similarly species richness (Chao1) was slightly higher (210-580) in rDNA libraries compared to rRNA libraries (150-400; P = 0·054, n = 24). Active Mycobacterium spp. was found in cross-linked polyethylene (PEX), but not in corresponding copper pipeline biofilm. Nonpathogenic Legionella spp. was found in rDNA libraries but not in rRNA libraries. Microbial communities differed between water and biofilms, between cold and hot water systems, locations in the building and between water rRNA and rDNA libraries, as shown by clear clusters in principal component analysis (PcoA). By using the rRNA method, we found that not all bacterial community members were active (e.g. Legionella spp.), whereas other members showed increased activity in some locations; for example, Pseudomonas spp. in hot water circulations' biofilm and order Rhizobiales and Limnohabitans spp. in stagnated locations' water and biofilm. rRNA-based methods may be better than rDNA-based methods for evaluating human health implications as rRNA methods can be used to describe the active bacterial fraction. This study indicates that copper as a pipeline material might have an adverse impact on the occurrence of Mycobacterium spp. The activity of Legionella spp. maybe questionable when detected solely by using DNA-based methods. © 2016 The Society for Applied Microbiology.

Construction of a complementary DNA library for Parelaphostrongylus tenuis and identification of a potentially sero-diagnostic recombinant antigen.

PubMed

Ogunremi, Oladele; Benjamin, Jane; MacDonald, Lily; Schimpf, Robert

2008-12-01

Newly developed serological tests for diagnosing parelaphostrongylosis in cervids, using the excretory-secretory products (ES) of the infective larvae of Parelaphostrongylus tenuis in enzyme-linked immunosorbent assays (ELISAs), have demonstrable superiority over the traditional method of larval recovery and microscopic identification. To generate a source of ELISA antigen by genetic engineering, we created a complementary DNA (cDNA) expression library by the reverse transcription of mRNA of P. tenuis adult worms, and ligation with the vector lambda-ZAP II. The library was screened using antisera produced in mice by immunization with a somatic antigen preparation of adult worms. Seventeen clones were isolated, sequenced, and checked for similarity to other DNA sequences in GenBank. A previously identified parasite gene encoding an aspartyl protease inhibitor (API) was isolated from the cDNA library, subcloned and expressed using the pET expression vector to produce a glutathione S transferase (GST)-His-S.Tag-P. tenuis API fusion protein (molecular weight = 63 kDa). An enzyme-linked immunosorbent assay utilizing the API fusion protein as the coating antigen was used to serologically diagnose all white-tailed deer (WTD, 10 out of 10) that had been inoculated with 6 - 150 L3 P. tenuis, indicating that the antigen may be a useful serodiagnostic antigen for P. tenuis infection in this cervid species.

In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses ▿ †

PubMed Central

Grimm, Dirk; Lee, Joyce S.; Wang, Lora; Desai, Tushar; Akache, Bassel; Storm, Theresa A.; Kay, Mark A.

2008-01-01

Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications. PMID:18400866

[Application of gene capture technology on mutation screening of RB1 gene in retinoblastoma patients].

PubMed

Meng, Q Y; Huang, L Z; Wang, B; Li, X X; Liang, J H

2017-06-11

Objectives: To analyze RB1 gene mutation in retinoblastoma (RB) patients using gene capture technology. Methods: Experimental research. The clinical data of 17 RB patients were collected at Department of Ophthalmology, Peking University People's Hospital from June 2010 to Jun 2014. Peripheral blood samples of seventeen RB patients and their parents were collected and genomic DNA were extracted. DNA library from RB patients was mixed with designed gene capture probe of RB1 exons and its flanking sequences. The data were analyzed using bioinformatics software. To avoid the false positive, the abnormal sites were verified using the Sanger sequencing method. Results: Totally, there were 17 RB patients, including 12 males and 5 females, from 0.5 to 23 years old, average ages were (3.2±5.2) years old. Both eyes were involved in 6 patients. The other 11 cases were only one eye was attacked. Four RB patients were found to have germline mutations, among whom 2 had bilateral tumors and 2 had unilateral tumors. 2 novel missense mutations were identified, including 15(th) exon c.1408A>T (p. Ile470Phe) and c.1960G>C (p. Val654Leu) at 19(th) exon. No RB1 mutation was identified in any of their parents. We also identified 2 mutations reported previously. One is c.1030C>T termination mutation at 10(th) exon in a bilateral RB patients and his father, who was diagnosed with unilateral RB. The other is c.371-372delTA frame shift mutation at 3(rd) exon. No mutation was found in their parents. Conclusions: Two novel germline RB1 mutations were found using gene capture technology, which enriched RB1 mutations library. (Chin J Ophthalmol, 2017, 53: 455-459) .

High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency.

PubMed

Takahashi, Mayumi; Wu, Xiwei; Ho, Michelle; Chomchan, Pritsana; Rossi, John J; Burnett, John C; Zhou, Jiehua

2016-09-22

The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct "biased sequences" and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the "biased sequences" was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy.

High throughput sequencing analysis of RNA libraries reveals the influences of initial library and PCR methods on SELEX efficiency

PubMed Central

Takahashi, Mayumi; Wu, Xiwei; Ho, Michelle; Chomchan, Pritsana; Rossi, John J.; Burnett, John C.; Zhou, Jiehua

2016-01-01

The systemic evolution of ligands by exponential enrichment (SELEX) technique is a powerful and effective aptamer-selection procedure. However, modifications to the process can dramatically improve selection efficiency and aptamer performance. For example, droplet digital PCR (ddPCR) has been recently incorporated into SELEX selection protocols to putatively reduce the propagation of byproducts and avoid selection bias that result from differences in PCR efficiency of sequences within the random library. However, a detailed, parallel comparison of the efficacy of conventional solution PCR versus the ddPCR modification in the RNA aptamer-selection process is needed to understand effects on overall SELEX performance. In the present study, we took advantage of powerful high throughput sequencing technology and bioinformatics analysis coupled with SELEX (HT-SELEX) to thoroughly investigate the effects of initial library and PCR methods in the RNA aptamer identification. Our analysis revealed that distinct “biased sequences” and nucleotide composition existed in the initial, unselected libraries purchased from two different manufacturers and that the fate of the “biased sequences” was target-dependent during selection. Our comparison of solution PCR- and ddPCR-driven HT-SELEX demonstrated that PCR method affected not only the nucleotide composition of the enriched sequences, but also the overall SELEX efficiency and aptamer efficacy. PMID:27652575

Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression

PubMed Central

Aze, Antoine; Sannino, Vincenzo; Soffientini, Paolo; Bachi, Angela; Costanzo, Vincenzo

2016-01-01

Half of human genome is made of repetitive DNA. However, mechanisms underlying replication of chromosome regions containing repetitive DNA are poorly understood. We reconstituted replication of defined human chromosome segments using Bacterial Artificial Chromosomes (BACs) in Xenopus laevis egg extract. Using this approach we characterized chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. Proteomic analysis of centromeric chromatin revealed replication dependent enrichment of a network of DNA repair factors among which the MSH2-6 complex, which was required for efficient centromeric DNA replication. However, contrary to expectations, the ATR dependent checkpoint monitoring DNA replication fork arrest could not be activated on highly repetitive DNA due to inability of single stranded DNA binding protein RPA to accumulate on chromatin. Electron microscopy of centromeric DNA and supercoil mapping revealed the presence of Topoisomerase I dependent DNA loops embedded in a protein matrix enriched for SMC2-4 proteins. This arrangement suppressed ATR signalling by preventing RPA hyper-loading, facilitating replication of centromeric DNA. These findings have important implications on our understanding of repetitive DNA metabolism and centromere organization under normal and stressful conditions. PMID:27111843

Screening and selection of artificial riboswitches.

PubMed

Harbaugh, Svetlana V; Martin, Jennifer; Weinstein, Jenna; Ingram, Grant; Kelley-Loughnane, Nancy

2018-05-17

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system. Copyright © 2018. Published by Elsevier Inc.

Diversity of anaerobic microbes in spacecraft assembly clean rooms.

PubMed

Probst, Alexander; Vaishampayan, Parag; Osman, Shariff; Moissl-Eichinger, Christine; Andersen, Gary L; Venkateswaran, Kasthuri

2010-05-01

Although the cultivable and noncultivable microbial diversity of spacecraft assembly clean rooms has been previously documented using conventional and state-of-the-art molecular techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain. Therefore, anaerobic bacterial communities of three clean-room facilities were analyzed during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on several media, and DNA was extracted from suitable anaerobic enrichments and examined with conventional 16S rRNA gene clone library, as well as high-density phylogenetic 16S rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses predominantly showed the presence of clostridial and propionibacterial strains. The 16S rRNA gene sequences retrieved from clone libraries revealed distinct microbial populations associated with each clean-room facility, clustered exclusively within gram-positive organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla of bacteria, and provided a deeper insight into the microbial community structure of the clean-room facilities. This study presents an integrated approach for assessing the anaerobic microbial population within clean-room facilities, using both molecular and cultivation-based analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the clean rooms even after the imposition of rigorous maintenance programs and will pose a challenge to planetary protection implementation activities.

Replication, checkpoint suppression and structure of centromeric DNA

PubMed Central

Romeo, Francesco; Costanzo, Vincenzo

2016-01-01

ABSTRACT Human centromeres contain large amounts of repetitive DNA sequences known as α satellite DNA, which can be difficult to replicate and whose functional role is unclear. Recently, we have characterized protein composition, structural organization and checkpoint response to stalled replication forks of centromeric chromatin reconstituted in Xenopus laevis egg extract. We showed that centromeric DNA has high affinity for SMC2-4 subunits of condensins and for CENP-A, it is enriched for DNA repair factors and suppresses the ATR checkpoint to ensure its efficient replication. We also showed that centromeric chromatin forms condensins enriched and topologically constrained DNA loops, which likely contribute to the overall structure of the centromere. These findings have important implications on how chromosomes are organized and genome stability is maintained in mammalian cells. PMID:27893298

Construction and characterization of a bacterial artificial chromosome library for hexaploid wheat line 92R137

USDA-ARS?s Scientific Manuscript database

For map-based cloning of genes conferring important traits in the hexaploid wheat line 92R137, a bacterial artificial chromosome (BAC) library, including two sub libraries, was constructed using the genomic DNA of 92R137 digested with restriction enzymes HindIII and BamHI. The BAC library was compos...

Massive migration from the steppe was a source for Indo-European languages in Europe.

PubMed

Haak, Wolfgang; Lazaridis, Iosif; Patterson, Nick; Rohland, Nadin; Mallick, Swapan; Llamas, Bastien; Brandt, Guido; Nordenfelt, Susanne; Harney, Eadaoin; Stewardson, Kristin; Fu, Qiaomei; Mittnik, Alissa; Bánffy, Eszter; Economou, Christos; Francken, Michael; Friederich, Susanne; Pena, Rafael Garrido; Hallgren, Fredrik; Khartanovich, Valery; Khokhlov, Aleksandr; Kunst, Michael; Kuznetsov, Pavel; Meller, Harald; Mochalov, Oleg; Moiseyev, Vayacheslav; Nicklisch, Nicole; Pichler, Sandra L; Risch, Roberto; Rojo Guerra, Manuel A; Roth, Christina; Szécsényi-Nagy, Anna; Wahl, Joachim; Meyer, Matthias; Krause, Johannes; Brown, Dorcas; Anthony, David; Cooper, Alan; Alt, Kurt Werner; Reich, David

2015-06-11

We generated genome-wide data from 69 Europeans who lived between 8,000-3,000 years ago by enriching ancient DNA libraries for a target set of almost 400,000 polymorphisms. Enrichment of these positions decreases the sequencing required for genome-wide ancient DNA analysis by a median of around 250-fold, allowing us to study an order of magnitude more individuals than previous studies and to obtain new insights about the past. We show that the populations of Western and Far Eastern Europe followed opposite trajectories between 8,000-5,000 years ago. At the beginning of the Neolithic period in Europe, ∼8,000-7,000 years ago, closely related groups of early farmers appeared in Germany, Hungary and Spain, different from indigenous hunter-gatherers, whereas Russia was inhabited by a distinctive population of hunter-gatherers with high affinity to a ∼24,000-year-old Siberian. By ∼6,000-5,000 years ago, farmers throughout much of Europe had more hunter-gatherer ancestry than their predecessors, but in Russia, the Yamnaya steppe herders of this time were descended not only from the preceding eastern European hunter-gatherers, but also from a population of Near Eastern ancestry. Western and Eastern Europe came into contact ∼4,500 years ago, as the Late Neolithic Corded Ware people from Germany traced ∼75% of their ancestry to the Yamnaya, documenting a massive migration into the heartland of Europe from its eastern periphery. This steppe ancestry persisted in all sampled central Europeans until at least ∼3,000 years ago, and is ubiquitous in present-day Europeans. These results provide support for a steppe origin of at least some of the Indo-European languages of Europe.

Massive migration from the steppe was a source for Indo-European languages in Europe

PubMed Central

Haak, Wolfgang; Lazaridis, Iosif; Patterson, Nick; Rohland, Nadin; Mallick, Swapan; Llamas, Bastien; Brandt, Guido; Nordenfelt, Susanne; Harney, Eadaoin; Stewardson, Kristin; Fu, Qiaomei; Mittnik, Alissa; Bánffy, Eszter; Economou, Christos; Francken, Michael; Friederich, Susanne; Pena, Rafael Garrido; Hallgren, Fredrik; Khartanovich, Valery; Khokhlov, Aleksandr; Kunst, Michael; Kuznetsov, Pavel; Meller, Harald; Mochalov, Oleg; Moiseyev, Vayacheslav; Nicklisch, Nicole; Pichler, Sandra L.; Risch, Roberto; Rojo Guerra, Manuel A.; Roth, Christina; Szécsényi-Nagy, Anna; Wahl, Joachim; Meyer, Matthias; Krause, Johannes; Brown, Dorcas; Anthony, David; Cooper, Alan; Alt, Kurt Werner; Reich, David

2016-01-01

We generated genome-wide data from 69 Europeans who lived between 8,000–3,000 years ago by enriching ancient DNA libraries for a target set of almost 400,000 polymorphisms. Enrichment of these positions decreases the sequencing required for genome-wide ancient DNA analysis by a median of around 250-fold, allowing us to study an order of magnitude more individuals than previous studies1–8 and to obtain new insights about the past. We show that the populations of Western and Far Eastern Europe followed opposite trajectories between 8,000–5,000 years ago. At the beginning of the Neolithic period in Europe, 8,000–7,000 years ago, closely related groups of early farmers appeared in Germany, Hungary and Spain, different from indigenous hunter-gatherers, whereas Russia was inhabited by a distinctive population of hunter-gatherers with high affinity to a 24,000-year-old Siberian6. By 6,000–5,000 years ago, farmers throughout much of Europe had more hunter-gatherer ancestry than their predecessors, but in Russia, the Yamnaya steppe herders of this time were descended not only from the preceding eastern European hunter-gatherers, but also from a population of Near Eastern ancestry. Western and Eastern Europe came into contact 4,500 years ago, as the Late Neolithic Corded Ware people from Germany traced 75% of their ancestry to the Yamnaya, documenting a massive migration into the heartland of Europe from its eastern periphery. This steppe ancestry persisted in all sampled central Europeans until at least 3,000 years ago, and is ubiquitous in present-day Europeans. These results provide support for a steppe origin9 of at least some of the Indo-European languages of Europe. PMID:25731166

A biogeochemical and genetic survey of acetylene fermentation by environmental samples and bacterial isolates

USGS Publications Warehouse

Miller, Laurence G.; Baesman, Shaun M.; Kirshtein, Julie; Voytek, Mary A.; Oremland, Ronald S.

2013-01-01

Anoxic samples (sediment and groundwater) from 13 chemically diverse field sites were assayed for their ability to consume acetylene (C2H2). Over incubation periods ranging from ˜ 10 to 80 days, selected samples from 7 of the 13 tested sites displayed significant C2H2 removal. No significant formation of ethylene was noted in these incubations; therefore, C2H2 consumption could be attributed to acetylene hydratase (AH) rather than nitrogenase activity. This putative AH (PAH) activity was observed in only 21% of the total of assayed samples, while amplification of AH genes from extracted DNA using degenerate primers derived from Pelobacter acetylenicus occurred in even fewer (9.8%) samples. Acetylene-fermenting bacteria were isolated as a pure culture from the sediments of a tidal mudflat in San Francisco Bay (SFB93) and as an enrichment culture from freshwater Searsville Lake (SV7). Comparison of 16S rDNA clone libraries revealed that SFB93 was closely related to P. carbolinicus, while SV7 consisted of several unrelated bacteria. AH gene was amplified from SFB93 but not SV7. The inability of the primers to generate amplicons in the SV7 enrichment, as well as from several of the environmental samples that displayed PAH activity, implied that either the primers were too highly constrained in their specificity or that there was a different type of AH gene in these environmental samples than occurs in P. acetylenicus. The significance of this work with regard to the search for life in the outer Solar System, where C2HL2 is abundant, is discussed.

Exploring Microbial Iron Oxidation in Wetland Soils

NASA Astrophysics Data System (ADS)

Wang, J.; Muyzer, G.; Bodelier, P. L. E.; den Oudsten, F.; Laanbroek, H. J.

2009-04-01

Iron is one of the most abundant elements on earth and is essential for life. Because of its importance, iron cycling and its interaction with other chemical and microbial processes has been the focus of many studies. Iron-oxidizing bacteria (FeOB) have been detected in a wide variety of environments. Among those is the rhizosphere of wetland plants roots which release oxygen into the soil creating suboxic conditions required by these organisms. It has been reported that in these rhizosphere microbial iron oxidation proceeds up to four orders of magnitude faster than strictly abiotic oxidation. On the roots of these wetland plants iron plaques are formed by microbial iron oxidation which are involved in the sequestering of heavy metals as well organic pollutants, which of great environmental significance.Despite their important role being catalysts of iron-cycling in wetland environments, little is known about the diversity and distribution of iron-oxidizing bacteria in various environments. This study aimed at developing a PCR-DGGE assay enabling the detection of iron oxidizers in wetland habitats. Gradient tubes were used to enrich iron-oxidizing bacteria. From these enrichments, a clone library was established based on the almost complete 16s rRNA gene using the universal bacterial primers 27f and 1492r. This clone library consisted of mainly α- and β-Proteobacteria, among which two major clusters were closely related to Gallionella spp. Specific probes and primers were developed on the basis of this 16S rRNA gene clone library. The newly designed Gallionella-specific 16S rRNA gene primer set 122f/998r was applied to community DNA obtained from three contrasting wetland environments, and the PCR products were used in denaturing gradient gel electrophoresis (DGGE) analysis. A second 16S rRNA gene clone library was constructed using the PCR products from one of our sampling sites amplified with the newly developed primer set 122f/998r. The cloned 16S rRNA gene sequences all represented novel culturable iron oxidizers most closely related to Gallionella spp. Based on their nucleotide sequences four groups could be identified, which were comparable to the DGGE banding pattern obtained before with the gradient tubes enrichments. The above mentioned nested PCR-DGGE method was used to study the distribution and community composition of Gallionella-like iron-oxidizing bacteria under the influence of plants species, soil depth, as well as season. Soil samples from Appels, Belgium, an intertidal, freshwater marsh known to hold intensive iron cycling, were taken from 5 different vegetation types in April, July and October 2007. Soil cores were sliced at 1-cm intervals and subjected to chemical and molecular analyses. The DGGE patterns showed that the community of iron-oxidizing bacteria differed with vegetation type, and sediment depth. Samples taken in autumn held lower diversity in Gallionella-related iron oxidizers than those sampled in spring and summer.

De novo transcriptome assembly and analysis of differential gene expression following peptidoglycan (PGN) challenge in Antheraea pernyi.

PubMed

Liu, Yu; Xin, Zhao-Zhe; Zhang, Dai-Zhen; Zhu, Xiao-Yu; Wang, Ying; Chen, Li; Tang, Bo-Ping; Zhou, Chun-Lin; Chai, Xin-Yue; Tian, Ji-Wu; Liu, Qiu-Ning

2018-06-01

Antheraea pernyi is not only an important economic insect, it is increasingly employed as a model organism due to a variety of advantages, including ease of rearing and experimental manipulation compared with other Lepidoptera. Peptidoglycan (PGN) is a major component of the bacterial cell wall, and interactions between PGN and A. pernyi cause a series of physiological changes in the insect. In the present study, we constructed cDNA libraries from a A. pernyi PGN-infected group and a control group stimulated with phosphate-buffered saline (PBS). The transcriptome was de novo assembled using the Trinity platform, and 1698 differentially expressed genes (DEGs) were identified, comprising 894 up-regulated and 804 down-regulated genes. To further investigate immune-related DEGs, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed. GO analysis identified major immune-related GO terms and KEGG enrichment indicated gene responses to three pathways related to the insect immune system. Several homologous genes related to the immune response of the A. pernyi fat body post-PGN infection were identified and categorised. Taken together, the results provide insight into the complex molecular mechanisms of the responses to bacterial infection at the transcriptional level. Copyright © 2018 Elsevier B.V. All rights reserved.

A Novel Approach to Assay DNA Methylation in Prostate Cancer

DTIC Science & Technology

2016-10-01

prepared into libraries according to standard protocols using Bioo Scientific’s DNA Sample Kit (cat. no. 514101, Austin , TX , USA). Libraries were...Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited...ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 11. SPONSOR/MONITOR’S

Non-B-Form DNA Is Enriched at Centromeres

PubMed Central

Henikoff, Steven

2018-01-01

Abstract Animal and plant centromeres are embedded in repetitive “satellite” DNA, but are thought to be epigenetically specified. To define genetic characteristics of centromeres, we surveyed satellite DNA from diverse eukaryotes and identified variation in <10-bp dyad symmetries predicted to adopt non-B-form conformations. Organisms lacking centromeric dyad symmetries had binding sites for sequence-specific DNA-binding proteins with DNA-bending activity. For example, human and mouse centromeres are depleted for dyad symmetries, but are enriched for non-B-form DNA and are associated with binding sites for the conserved DNA-binding protein CENP-B, which is required for artificial centromere function but is paradoxically nonessential. We also detected dyad symmetries and predicted non-B-form DNA structures at neocentromeres, which form at ectopic loci. We propose that centromeres form at non-B-form DNA because of dyad symmetries or are strengthened by sequence-specific DNA binding proteins. This may resolve the CENP-B paradox and provide a general basis for centromere specification. PMID:29365169

Analysis of the leaf transcriptome of Musa acuminata during interaction with Mycosphaerella musicola: gene assembly, annotation and marker development

PubMed Central

2013-01-01

Background Although banana (Musa sp.) is an important edible crop, contributing towards poverty alleviation and food security, limited transcriptome datasets are available for use in accelerated molecular-based breeding in this genus. 454 GS-FLX Titanium technology was employed to determine the sequence of gene transcripts in genotypes of Musa acuminata ssp. burmannicoides Calcutta 4 and M. acuminata subgroup Cavendish cv. Grande Naine, contrasting in resistance to the fungal pathogen Mycosphaerella musicola, causal organism of Sigatoka leaf spot disease. To enrich for transcripts under biotic stress responses, full length-enriched cDNA libraries were prepared from whole plant leaf materials, both uninfected and artificially challenged with pathogen conidiospores. Results The study generated 846,762 high quality sequence reads, with an average length of 334 bp and totalling 283 Mbp. De novo assembly generated 36,384 and 35,269 unigene sequences for M. acuminata Calcutta 4 and Cavendish Grande Naine, respectively. A total of 64.4% of the unigenes were annotated through Basic Local Alignment Search Tool (BLAST) similarity analyses against public databases. Assembled sequences were functionally mapped to Gene Ontology (GO) terms, with unigene functions covering a diverse range of molecular functions, biological processes and cellular components. Genes from a number of defense-related pathways were observed in transcripts from each cDNA library. Over 99% of contig unigenes mapped to exon regions in the reference M. acuminata DH Pahang whole genome sequence. A total of 4068 genic-SSR loci were identified in Calcutta 4 and 4095 in Cavendish Grande Naine. A subset of 95 potential defense-related gene-derived simple sequence repeat (SSR) loci were validated for specific amplification and polymorphism across M. acuminata accessions. Fourteen loci were polymorphic, with alleles per polymorphic locus ranging from 3 to 8 and polymorphism information content ranging from 0.34 to 0.82. Conclusions A large set of unigenes were characterized in this study for both M. acuminata Calcutta 4 and Cavendish Grande Naine, increasing the number of public domain Musa ESTs. This transcriptome is an invaluable resource for furthering our understanding of biological processes elicited during biotic stresses in Musa. Gene-based markers will facilitate molecular breeding strategies, forming the basis of genetic linkage mapping and analysis of quantitative trait loci. PMID:23379821

The Influence of Literacy-Enriched Play Settings on Preschoolers' Conceptions of Print.

ERIC Educational Resources Information Center

Neuman, Susan B.; Roskos, Kathy

This study examined the influence of literacy-enriched play centers on preschoolers' conceptions of print. Subjects, 25 boys and 12 girls aged 4 and 5 years from 2 urban preschool classes, were systematically observed before and after 5 basic design changes were made in the classrooms. Four distinct play centers (post office, library, office, and…

A pair of new BAC and BIBAC vectors that facilitate BAC/BIBAC library construction and intact large genomic DNA insert exchange.

PubMed

Shi, Xue; Zeng, Haiyang; Xue, Yadong; Luo, Meizhong

2011-10-11

Large-insert BAC and BIBAC libraries are important tools for structural and functional genomics studies of eukaryotic genomes. To facilitate the construction of BAC and BIBAC libraries and the transfer of complete large BAC inserts into BIBAC vectors, which is desired in positional cloning, we developed a pair of new BAC and BIBAC vectors. The new BAC vector pIndigoBAC536-S and the new BIBAC vector BIBAC-S have the following features: 1) both contain two 18-bp non-palindromic I-SceI sites in an inverted orientation at positions that flank an identical DNA fragment containing the lacZ selection marker and the cloning site. Large DNA inserts can be excised from the vectors as single fragments by cutting with I-SceI, allowing the inserts to be easily sized. More importantly, because the two vectors contain different antibiotic resistance genes for transformant selection and produce the same non-complementary 3' protruding ATAA ends by I-SceI that suppress self- and inter-ligations, the exchange of intact large genomic DNA inserts between the BAC and BIBAC vectors is straightforward; 2) both were constructed as high-copy composite vectors. Reliable linearized and dephosphorylated original low-copy pIndigoBAC536-S and BIBAC-S vectors that are ready for library construction can be prepared from the high-copy composite vectors pHZAUBAC1 and pHZAUBIBAC1, respectively, without the need for additional preparation steps or special reagents, thus simplifying the construction of BAC and BIBAC libraries. BIBAC clones constructed with the new BIBAC-S vector are stable in both E. coli and Agrobacterium. The vectors can be accessed through our website http://GResource.hzau.edu.cn. The two new vectors and their respective high-copy composite vectors can largely facilitate the construction and characterization of BAC and BIBAC libraries. The transfer of complete large genomic DNA inserts from one vector to the other is made straightforward.

Writing DNA with GenoCAD.

PubMed

Czar, Michael J; Cai, Yizhi; Peccoud, Jean

2009-07-01

Chemical synthesis of custom DNA made to order calls for software streamlining the design of synthetic DNA sequences. GenoCAD (www.genocad.org) is a free web-based application to design protein expression vectors, artificial gene networks and other genetic constructs composed of multiple functional blocks called genetic parts. By capturing design strategies in grammatical models of DNA sequences, GenoCAD guides the user through the design process. By successively clicking on icons representing structural features or actual genetic parts, complex constructs composed of dozens of functional blocks can be designed in a matter of minutes. GenoCAD automatically derives the construct sequence from its comprehensive libraries of genetic parts. Upon completion of the design process, users can download the sequence for synthesis or further analysis. Users who elect to create a personal account on the system can customize their workspace by creating their own parts libraries, adding new parts to the libraries, or reusing designs to quickly generate sets of related constructs.

Generation of a total of 6483 expressed sequence tags from 60 day-old bovine whole fetus and fetal placenta.

PubMed

Oishi, M; Gohma, H; Lejukole, H Y; Taniguchi, Y; Yamada, T; Suzuki, K; Shinkai, H; Uenishi, H; Yasue, H; Sasaki, Y

2004-05-01

Expressed sequence tags (ESTs) generated based on characterization of clones isolated randomly from cDNA libraries are used to study gene expression profiles in specific tissues and to provide useful information for characterizing tissue physiology. In this study, two directionally cloned cDNA libraries were constructed from 60 day-old bovine whole fetus and fetal placenta. We have characterized 5357 and 1126 clones, and then identified 3464 and 795 unique sequences for the fetus and placenta cDNA libraries: 1851 and 504 showed homology to already identified genes, and 1613 and 291 showed no significant matches to any of the sequences in DNA databases, respectively. Further, we found 94 unique sequences overlapping in both the fetus and the placenta, leading to a catalog of 4165 genes expressed in 60 day-old fetus and placenta. The catalog is used to examine expression profile of genes in 60 day-old bovine fetus and placenta.

Construction and application of EST library from Setaria italica in response to dehydration stress.

PubMed

Zhang, Jinpeng; Liu, Tingsong; Fu, Junjie; Zhu, Yun; Jia, Jinping; Zheng, Jun; Zhao, Yinhe; Zhang, Ying; Wang, Guoying

2007-07-01

Foxtail millet is a gramineous crop with low water requirement. Despite its high water use efficiency, less attention has been paid to the molecular genetics of foxtail millet. This article reports the construction of subtracted cDNA libraries from foxtail millet seedlings under dehydration stress and the expression profile analysis of 1947 UniESTs from the subtracted cDNA libraries by a cDNA microarray. The results showed that 95 and 57 ESTs were upregulated by dehydration stress, respectively, in roots and shoots of seedlings and that 10 and 27 ESTs were downregulated, respectively, in roots and shoots. The expression profile analysis showed that genes induced in foxtail millet roots were different from those in shoots during dehydration stress and that the early response to dehydration stress in foxtail millet roots was the activation of the glycolysis metabolism. Moreover, protein degradation pathway may also play a pivotal role in drought-tolerant responses of foxtail millet. Finally, Northern blot analysis validated well the cDNA microarray data.

Enrichment of dynamic chromosomal crosslinks drive phase separation of the nucleolus

PubMed Central

Hult, Caitlin; Adalsteinsson, David; Vasquez, Paula A.; Lawrimore, Josh; Bennett, Maggie; York, Alyssa; Cook, Diana; Yeh, Elaine; Forest, Mark Gregory

2017-01-01

Abstract Regions of highly repetitive DNA, such as those found in the nucleolus, show a self-organization that is marked by spatial segregation and frequent self-interaction. The mechanisms that underlie the sequestration of these sub-domains are largely unknown. Using a stochastic, bead-spring representation of chromatin in budding yeast, we find enrichment of protein-mediated, dynamic chromosomal cross-links recapitulates the segregation, morphology and self-interaction of the nucleolus. Rates and enrichment of dynamic crosslinking have profound consequences on domain morphology. Our model demonstrates the nucleolus is phase separated from other chromatin in the nucleus and predicts that multiple rDNA loci will form a single nucleolus independent of their location within the genome. Fluorescent labeling of budding yeast nucleoli with CDC14-GFP revealed that a split rDNA locus indeed forms a single nucleolus. We propose that nuclear sub-domains, such as the nucleolus, result from phase separations within the nucleus, which are driven by the enrichment of protein-mediated, dynamic chromosomal crosslinks. PMID:28977453

Encoded libraries of chemically modified peptides.

PubMed

Heinis, Christian; Winter, Greg

2015-06-01

The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries. Copyright © 2015. Published by Elsevier Ltd.

A new protocol to detect multiple foodborne pathogens with PCR dipstick DNA chromatography after a six-hour enrichment culture in a broad-range food pathogen enrichment broth.

PubMed

Hayashi, Masahiro; Natori, Tatsuya; Kubota-Hayashi, Sayoko; Miyata, Machiko; Ohkusu, Kiyofumi; Kawamoto, Keiko; Kurazono, Hisao; Makino, Souichi; Ezaki, Takayuki

2013-01-01

A quick foodborne pathogen screening method after six-hour enrichment culture with a broad-range food pathogen enrichment broth is described. Pathogenic factors of Salmonella enterica, Shigella spp., enteroinvasive Escherichia coli, and enterohemorrhagic E. coli are amplified with a cocktail primer and rapid polymerase chain reaction (PCR), which finishes amplification in 30 min. The PCR amplicon was differentiated with a dipstick DNA chromatography assay in 5-10 min. Starting from a four- to six-hour enrichment culture, this assay was finished within 45 min. Detection sensitivity of this protocol was less than 2.5 CFU/25 g for S. enterica and 3.3 CFU/25 g for enterohemorrhagic E. coli in spiked ground meat experiments.

Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

PubMed

Osato, Naoki

2018-01-19

Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional enrichments were related to the cellular functions. The normalized number of functional enrichments of human putative transcriptional target genes changed according to the criteria of enhancer-promoter assignments and correlated with the median expression level of the target genes. These analyses and characters of human putative transcriptional target genes would be useful to examine the criteria of enhancer-promoter assignments and to predict the novel mechanisms and factors such as DNA binding proteins and DNA sequences of enhancer-promoter interactions.

Primary culture of cat intestinal epithelial cells in vitro and the cDNA library construction.

PubMed

Zhao, Gui Hua; Liu, Ye; Cheng, Yun Tang; Zhao, Qing Song; Qiu, Xiao; Xu, Chao; Xiao, Ting; Zhu, Song; Liu, Gong Zhen; Yin, Kun

2018-06-26

Felids are the only definitive hosts of Toxoplasma gondii. To lay a foundation for screening the T. gondii-felids interaction factors, we have developed a reproducible primary culture method for cat intestinal epithelial cells (IECs). The primary IECs were isolated from a new born cat's small intestine jejunum region without food ingress, and respectively in vitro cultured by tissue cultivation and combined digestion method with collagenase XI and dispase I, then purified by trypsinization. After identification, the ds cDNA of cat IECs was synthesized for constructing pGADT7 homogenization three-frame plasmid, and transformed into the yeast Y187 for generating the cDNA library. Our results indicated that cultivation of primary cat IECs relays on combined digestion to form polarized and confluent monolayers within 3 days with typical features of normal epithelial cells. The purified cells cultured by digestion method were identified to be nature intestinal epithelial cells using immunohistochemical analysis and were able to maintain viability for at least 15 passages. The homogenizable ds cDNA, which is synthesized from the total RNA extracted from our cultured IECs, distributed among 0.5-2.0 kb, and generated satisfying three-frame cDNA library with the capacity of 1.2 × 106 and the titer of 5.2 × 107 pfu/mL. Our results established an optimal method for the culturing and passage of cat IECs model in vitro, and laid a cDNA library foundation for the subsequent interaction factors screening by yeast two-hybrid.

Establishing a community-wide DNA barcode library as a new tool for arctic research.

PubMed

Wirta, H; Várkonyi, G; Rasmussen, C; Kaartinen, R; Schmidt, N M; Hebert, P D N; Barták, M; Blagoev, G; Disney, H; Ertl, S; Gjelstrup, P; Gwiazdowicz, D J; Huldén, L; Ilmonen, J; Jakovlev, J; Jaschhof, M; Kahanpää, J; Kankaanpää, T; Krogh, P H; Labbee, R; Lettner, C; Michelsen, V; Nielsen, S A; Nielsen, T R; Paasivirta, L; Pedersen, S; Pohjoismäki, J; Salmela, J; Vilkamaa, P; Väre, H; von Tschirnhaus, M; Roslin, T

2016-05-01

DNA sequences offer powerful tools for describing the members and interactions of natural communities. In this study, we establish the to-date most comprehensive library of DNA barcodes for a terrestrial site, including all known macroscopic animals and vascular plants of an intensively studied area of the High Arctic, the Zackenberg Valley in Northeast Greenland. To demonstrate its utility, we apply the library to identify nearly 20 000 arthropod individuals from two Malaise traps, each operated for two summers. Drawing on this material, we estimate the coverage of previous morphology-based species inventories, derive a snapshot of faunal turnover in space and time and describe the abundance and phenology of species in the rapidly changing arctic environment. Overall, 403 terrestrial animal and 160 vascular plant species were recorded by morphology-based techniques. DNA barcodes (CO1) offered high resolution in discriminating among the local animal taxa, with 92% of morphologically distinguishable taxa assigned to unique Barcode Index Numbers (BINs) and 93% to monophyletic clusters. For vascular plants, resolution was lower, with 54% of species forming monophyletic clusters based on barcode regions rbcLa and ITS2. Malaise catches revealed 122 BINs not detected by previous sampling and DNA barcoding. The insect community was dominated by a few highly abundant taxa. Even closely related taxa differed in phenology, emphasizing the need for species-level resolution when describing ongoing shifts in arctic communities and ecosystems. The DNA barcode library now established for Zackenberg offers new scope for such explorations, and for the detailed dissection of interspecific interactions throughout the community. © 2015 John Wiley & Sons Ltd.