Using eDNA to estimate distribution of fish species in a complex river system (presentation)

EPA Science Inventory

Environmental DNA (eDNA) analysis of biological material shed by aquatic organisms is a noninvasive genetic tool that can improve efficiency and reduce costs associated with species detection in aquatic systems. eDNA methods are widely used to assess presence/absence of a target ...

FastID: Extremely Fast Forensic DNA Comparisons

DTIC Science & Technology

2017-05-19

FastID: Extremely Fast Forensic DNA Comparisons Darrell O. Ricke, PhD Bioengineering Systems & Technologies Massachusetts Institute of...Technology Lincoln Laboratory Lexington, MA USA Darrell.Ricke@ll.mit.edu Abstract—Rapid analysis of DNA forensic samples can have a critical impact on...time sensitive investigations. Analysis of forensic DNA samples by massively parallel sequencing is creating the next gold standard for DNA

Role of indirect readout mechanism in TATA box binding protein-DNA interaction.

PubMed

Mondal, Manas; Choudhury, Devapriya; Chakrabarti, Jaydeb; Bhattacharyya, Dhananjay

2015-03-01

Gene expression generally initiates from recognition of TATA-box binding protein (TBP) to the minor groove of DNA of TATA box sequence where the DNA structure is significantly different from B-DNA. We have carried out molecular dynamics simulation studies of TBP-DNA system to understand how the DNA structure alters for efficient binding. We observed rigid nature of the protein while the DNA of TATA box sequence has an inherent flexibility in terms of bending and minor groove widening. The bending analysis of the free DNA and the TBP bound DNA systems indicate presence of some similar structures. Principal coordinate ordination analysis also indicates some structural features of the protein bound and free DNA are similar. Thus we suggest that the DNA of TATA box sequence regularly oscillates between several alternate structures and the one suitable for TBP binding is induced further by the protein for proper complex formation.

BioVLAB-mCpG-SNP-EXPRESS: A system for multi-level and multi-perspective analysis and exploration of DNA methylation, sequence variation (SNPs), and gene expression from multi-omics data.

PubMed

Chae, Heejoon; Lee, Sangseon; Seo, Seokjun; Jung, Daekyoung; Chang, Hyeonsook; Nephew, Kenneth P; Kim, Sun

2016-12-01

Measuring gene expression, DNA sequence variation, and DNA methylation status is routinely done using high throughput sequencing technologies. To analyze such multi-omics data and explore relationships, reliable bioinformatics systems are much needed. Existing systems are either for exploring curated data or for processing omics data in the form of a library such as R. Thus scientists have much difficulty in investigating relationships among gene expression, DNA sequence variation, and DNA methylation using multi-omics data. In this study, we report a system called BioVLAB-mCpG-SNP-EXPRESS for the integrated analysis of DNA methylation, sequence variation (SNPs), and gene expression for distinguishing cellular phenotypes at the pairwise and multiple phenotype levels. The system can be deployed on either the Amazon cloud or a publicly available high-performance computing node, and the data analysis and exploration of the analysis result can be conveniently done using a web-based interface. In order to alleviate analysis complexity, all the process are fully automated, and graphical workflow system is integrated to represent real-time analysis progression. The BioVLAB-mCpG-SNP-EXPRESS system works in three stages. First, it processes and analyzes multi-omics data as input in the form of the raw data, i.e., FastQ files. Second, various integrated analyses such as methylation vs. gene expression and mutation vs. methylation are performed. Finally, the analysis result can be explored in a number of ways through a web interface for the multi-level, multi-perspective exploration. Multi-level interpretation can be done by either gene, gene set, pathway or network level and multi-perspective exploration can be explored from either gene expression, DNA methylation, sequence variation, or their relationship perspective. The utility of the system is demonstrated by performing analysis of phenotypically distinct 30 breast cancer cell line data set. BioVLAB-mCpG-SNP-EXPRESS is available at http://biohealth.snu.ac.kr/software/biovlab_mcpg_snp_express/. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of DNA Sequences by an Optical Time-Integrating Correlator: Proposal

DTIC Science & Technology

1991-11-01

OF THE PROBLEM AND CURRENT TECHNOLOGY 2 3.0 TIME-INTEGRATING CORRELATOR 2 4.0 REPRESENTATIONS OF THE DNA BASES 8 5.0 DNA ANALYSIS STRATEGY 8 6.0... DNA bases where each base is represented by a 7-bits long pseudorandom sequence. 9 Figure 5: The flow of data in a DNA analysis system based on an...logarithmic scale and a linear scale. 15 x LIST OF TABLES PAGE Table 1: Short representations of the DNA bases where each base is represented by 7-bits

Computational method and system for modeling, analyzing, and optimizing DNA amplification and synthesis

DOEpatents

Vandersall, Jennifer A.; Gardner, Shea N.; Clague, David S.

2010-05-04

A computational method and computer-based system of modeling DNA synthesis for the design and interpretation of PCR amplification, parallel DNA synthesis, and microarray chip analysis. The method and system include modules that address the bioinformatics, kinetics, and thermodynamics of DNA amplification and synthesis. Specifically, the steps of DNA selection, as well as the kinetics and thermodynamics of DNA hybridization and extensions, are addressed, which enable the optimization of the processing and the prediction of the products as a function of DNA sequence, mixing protocol, time, temperature and concentration of species.

Evaluation of Methods for the Extraction of DNA from Drinking Water Distribution System Biofilms

PubMed Central

Hwang, Chiachi; Ling, Fangqiong; Andersen, Gary L.; LeChevallier, Mark W.; Liu, Wen-Tso

2012-01-01

While drinking water biofilms have been characterized in various drinking water distribution systems (DWDS), little is known about the impact of different DNA extraction methods on the subsequent analysis of microbial communities in drinking water biofilms. Since different DNA extraction methods have been shown to affect the outcome of microbial community analysis in other environments, it is necessary to select a DNA extraction method prior to the application of molecular tools to characterize the complex microbial ecology of the DWDS. This study compared the quantity and quality of DNA yields from selected DWDS bacteria with different cell wall properties using five widely used DNA extraction methods. These were further selected and evaluated for their efficiency and reproducibility of DNA extraction from DWDS samples. Terminal restriction fragment length analysis and the 454 pyrosequencing technique were used to interpret the differences in microbial community structure and composition, respectively, from extracted DNA. Such assessments serve as a concrete step towards the determination of an optimal DNA extraction method for drinking water biofilms, which can then provide a reliable comparison of the meta-analysis results obtained in different laboratories. PMID:22075624

Preparation and Analysis of Positioned Mononucleosomes

PubMed Central

Kulaeva, Olga; Studitsky, Vasily M.

2016-01-01

Short DNA fragments containing single nucleosomes have been extensively employed as simple model experimental systems for analysis of many intranuclear processes, including binding of proteins to nucleosomes, covalent histone modifications, transcription, DNA repair and ATP-dependent chromatin remodeling. Here we describe several recently developed procedures for obtaining and analysis of mononucleosomes assembled on 200–350-bp DNA fragments. PMID:25827872

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 28 Judicial Administration 1 2010-07-01 2010-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 28 Judicial Administration 1 2011-07-01 2011-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 28 Judicial Administration 1 2012-07-01 2012-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 28 Judicial Administration 1 2014-07-01 2014-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

28 CFR 28.12 - Collection of DNA samples.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 28 Judicial Administration 1 2013-07-01 2013-07-01 false Collection of DNA samples. 28.12 Section 28.12 Judicial Administration DEPARTMENT OF JUSTICE DNA IDENTIFICATION SYSTEM DNA Sample Collection, Analysis, and Indexing § 28.12 Collection of DNA samples. (a) The Bureau of Prisons shall collect a DNA...

Computational Study on Full-length Human Ku70 with Double Stranded DNA: Dynamics, Interactions and Functional Implications

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Cucinotta, Francis A.

2009-01-01

The Ku70/80 heterodimer is the first repair protein in the initial binding of double-strand break (DSB) ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. In this study we constructed a full-length human Ku70 structure based on its crystal structure, and performed 20 ns conventional molecular dynamic (CMD) simulations on this protein and several other complexes with short DNA duplexes of different sequences. The trajectories of these simulations indicated that, without the topological support of Ku80, the residues in the bridge and C-terminal arm of Ku70 are more flexible than other experimentally identified domains. We studied the two missing loops in the crystal structure and predicted that they are also very flexible. Simulations revealed that they make an important contribution to the Ku70 interaction with DNA. Dislocation of the previously studied SAP domain was observed in several systems, implying its role in DNA binding. Targeted molecular dynamic (TMD) simulation was also performed for one system with a far-away 14bp DNA duplex. The TMD trajectory and energetic analysis disclosed detailed interactions of the DNA-binding residues during the DNA dislocation, and revealed a possible conformational transition for a DSB end when encountering Ku70 in solution. Compared to experimentally based analysis, this study identified more detailed interactions between DNA and Ku70. Free energy analysis indicated Ku70 alone is able to bind DNA with relatively high affinity, with consistent contributions from various domains of Ku70 in different systems. The functional implications of these domains in the processes of Ku heterodimerization and DNA damage recognition and repair can be characterized in detail based upon this analysis.

MiniX-STR multiplex system population study in Japan and application to degraded DNA analysis.

PubMed

Asamura, H; Sakai, H; Kobayashi, K; Ota, M; Fukushima, H

2006-05-01

We sought to evaluate a more effective system for analyzing X-chromosomal short tandem repeats (X-STRs) in highly degraded DNA. To generate smaller amplicon lengths, we designed new polymerase chain reaction (PCR) primers for DXS7423, DXS6789, DXS101, GATA31E08, DXS8378, DXS7133, DXS7424, and GATA165B12 at X-linked short tandem repeat (STR) loci, devising two miniX-multiplex PCR systems. Among 333 Japanese individuals, these X-linked loci were detected in amplification products ranging in length from 76 to 169 bp, and statistical analyses of the eight loci indicated a high usefulness for the Japanese forensic practice. Results of tests on highly degraded DNA indicated the miniX-STR multiplex strategies to be an effective system for analyzing degraded DNA. We conclude that analysis by the current miniX-STR multiplex systems offers high effectiveness for personal identification from degraded DNA samples.

Plant genotoxicity: a molecular cytogenetic approach in plant bioassays.

PubMed

Maluszynska, Jolanta; Juchimiuk, Jolanta

2005-06-01

It is important for the prevention of DNA changes caused by environment to understand the biological consequences of DNA damages and their molecular modes of action that lead to repair or alterations of the genetic material. Numerous genotoxicity assay systems have been developed to identify DNA reactive compounds. The available data show that plant bioassays are important tests in the detection of genotoxic contamination in the environment and the establishment of controlling systems. Plant system can detect a wide range of genetic damage, including gene mutations and chromosome aberrations. Recently introduced molecular cytogenetic methods allow analysis of genotoxicity, both at the chromosomal and DNA level. FISH gives a new possibility of the detection and analysis of chromosomal rearrangements in a great detail. DNA fragmentation can be estimated using the TUNEL test and the single cell gel electrophoresis (Comet assay).

Short Tandem Repeat DNA Internet Database

National Institute of Standards and Technology Data Gateway

SRD 130 Short Tandem Repeat DNA Internet Database (Web, free access)   Short Tandem Repeat DNA Internet Database is intended to benefit research and application of short tandem repeat DNA markers for human identity testing. Facts and sequence information on each STR system, population data, commonly used multiplex STR systems, PCR primers and conditions, and a review of various technologies for analysis of STR alleles have been included.

Rapid quantification and sex determination of forensic evidence materials.

PubMed

Andréasson, Hanna; Allen, Marie

2003-11-01

DNA quantification of forensic evidence is very valuable for an optimal use of the available biological material. Moreover, sex determination is of great importance as additional information in criminal investigations as well as in identification of missing persons, no suspect cases, and ancient DNA studies. While routine forensic DNA analysis based on short tandem repeat markers includes a marker for sex determination, analysis of samples containing scarce amounts of DNA is often based on mitochondrial DNA, and sex determination is not performed. In order to allow quantification and simultaneous sex determination on minute amounts of DNA, an assay based on real-time PCR analysis of a marker within the human amelogenin gene has been developed. The sex determination is based on melting curve analysis, while an externally standardized kinetic analysis allows quantification of the nuclear DNA copy number in the sample. This real-time DNA quantification assay has proven to be highly sensitive, enabling quantification of single DNA copies. Although certain limitations were apparent, the system is a rapid, cost-effective, and flexible assay for analysis of forensic casework samples.

Screening and analysis of bioactive compounds with biofingerprinting chromatogram analysis of traditional Chinese medicines targeting DNA by microdialysis/HPLC.

PubMed

Su, Xingye; Kong, Liang; Li, Xin; Chen, Xueguo; Guo, Ming; Zou, Hanfa

2005-05-27

Biofingerprinting chromatogram analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein, cell, etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophoraflavescens Ait. (Sophora) to bind to ct-DNA, respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated.

Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

PubMed

Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

1986-02-01

Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations.

Detection of herpes simplex virus-specific DNA sequences in latently infected mice and in humans.

PubMed Central

Efstathiou, S; Minson, A C; Field, H J; Anderson, J R; Wildy, P

1986-01-01

Herpes simplex virus-specific DNA sequences have been detected by Southern hybridization analysis in both central and peripheral nervous system tissues of latently infected mice. We have detected virus-specific sequences corresponding to the junction fragment but not the genomic termini, an observation first made by Rock and Fraser (Nature [London] 302:523-525, 1983). This "endless" herpes simplex virus DNA is both qualitatively and quantitatively stable in mouse neural tissue analyzed over a 4-month period. In addition, examination of DNA extracted from human trigeminal ganglia has shown herpes simplex virus DNA to be present in an "endless" form similar to that found in the mouse model system. Further restriction enzyme analysis of latently infected mouse brainstem and human trigeminal DNA has shown that this "endless" herpes simplex virus DNA is present in all four isomeric configurations. Images PMID:3003377

Molecular Dynamics Simulations of DNA-Free and DNA-Bound TAL Effectors

PubMed Central

Wan, Hua; Hu, Jian-ping; Li, Kang-shun; Tian, Xu-hong; Chang, Shan

2013-01-01

TAL (transcriptional activator-like) effectors (TALEs) are DNA-binding proteins, containing a modular central domain that recognizes specific DNA sequences. Recently, the crystallographic studies of TALEs revealed the structure of DNA-recognition domain. In this article, molecular dynamics (MD) simulations are employed to study two crystal structures of an 11.5-repeat TALE, in the presence and absence of DNA, respectively. The simulated results indicate that the specific binding of RVDs (repeat-variable diresidues) with DNA leads to the markedly reduced fluctuations of tandem repeats, especially at the two ends. In the DNA-bound TALE system, the base-specific interaction is formed mainly by the residue at position 13 within a TAL repeat. Tandem repeats with weak RVDs are unfavorable for the TALE-DNA binding. These observations are consistent with experimental studies. By using principal component analysis (PCA), the dominant motions are open-close movements between the two ends of the superhelical structure in both DNA-free and DNA-bound TALE systems. The open-close movements are found to be critical for the recognition and binding of TALE-DNA based on the analysis of free energy landscape (FEL). The conformational analysis of DNA indicates that the 5′ end of DNA target sequence has more remarkable structural deformability than the other sites. Meanwhile, the conformational change of DNA is likely associated with the specific interaction of TALE-DNA. We further suggest that the arrangement of N-terminal repeats with strong RVDs may help in the design of efficient TALEs. This study provides some new insights into the understanding of the TALE-DNA recognition mechanism. PMID:24130757

A coarse-grained model for DNA origami.

PubMed

Reshetnikov, Roman V; Stolyarova, Anastasia V; Zalevsky, Arthur O; Panteleev, Dmitry Y; Pavlova, Galina V; Klinov, Dmitry V; Golovin, Andrey V; Protopopova, Anna D

2018-02-16

Modeling tools provide a valuable support for DNA origami design. However, current solutions have limited application for conformational analysis of the designs. In this work we present a tool for a thorough study of DNA origami structure and dynamics. The tool is based on a novel coarse-grained model dedicated to geometry optimization and conformational analysis of DNA origami. We explored the ability of the model to predict dynamic behavior, global shapes, and fine details of two single-layer systems designed in hexagonal and square lattices using atomic force microscopy, Förster resonance energy transfer spectroscopy, and all-atom molecular dynamic simulations for validation of the results. We also examined the performance of the model for multilayer systems by simulation of DNA origami with published cryo-electron microscopy and atomic force microscopy structures. A good agreement between the simulated and experimental data makes the model suitable for conformational analysis of DNA origami objects. The tool is available at http://vsb.fbb.msu.ru/cosm as a web-service and as a standalone version.

A coarse-grained model for DNA origami

PubMed Central

Stolyarova, Anastasia V; Zalevsky, Arthur O; Panteleev, Dmitry Y; Pavlova, Galina V; Klinov, Dmitry V; Golovin, Andrey V; Protopopova, Anna D

2018-01-01

Abstract Modeling tools provide a valuable support for DNA origami design. However, current solutions have limited application for conformational analysis of the designs. In this work we present a tool for a thorough study of DNA origami structure and dynamics. The tool is based on a novel coarse-grained model dedicated to geometry optimization and conformational analysis of DNA origami. We explored the ability of the model to predict dynamic behavior, global shapes, and fine details of two single-layer systems designed in hexagonal and square lattices using atomic force microscopy, Förster resonance energy transfer spectroscopy, and all-atom molecular dynamic simulations for validation of the results. We also examined the performance of the model for multilayer systems by simulation of DNA origami with published cryo-electron microscopy and atomic force microscopy structures. A good agreement between the simulated and experimental data makes the model suitable for conformational analysis of DNA origami objects. The tool is available at http://vsb.fbb.msu.ru/cosm as a web-service and as a standalone version. PMID:29267876

Integrated microfluidic systems for cell lysis, mixing/pumping and DNA amplification

NASA Astrophysics Data System (ADS)

Lee, Chia-Yen; Lee, Gwo-Bin; Lin, Jr-Lung; Huang, Fu-Chun; Liao, Chia-Sheng

2005-06-01

The present paper reports a fully automated microfluidic system for the DNA amplification process by integrating an electroosmotic pump, an active micromixer and an on-chip temperature control system. In this DNA amplification process, the cell lysis is initially performed in a micro cell lysis reactor. Extracted DNA samples, primers and reagents are then driven electroosmotically into a mixing region where they are mixed by the active micromixer. The homogeneous mixture is then thermally cycled in a micro-PCR (polymerase chain reaction) chamber to perform DNA amplification. Experimental results show that the proposed device can successfully automate the sample pretreatment operation for DNA amplification, thereby delivering significant time and effort savings. The new microfluidic system, which facilitates cell lysis, sample driving/mixing and DNA amplification, could provide a significant contribution to ongoing efforts to miniaturize bio-analysis systems by utilizing a simple fabrication process and cheap materials.

East Asian mtDNA haplogroup determination in Koreans: haplogroup-level coding region SNP analysis and subhaplogroup-level control region sequence analysis.

PubMed

Lee, Hwan Young; Yoo, Ji-Eun; Park, Myung Jin; Chung, Ukhee; Kim, Chong-Youl; Shin, Kyoung-Jin

2006-11-01

The present study analyzed 21 coding region SNP markers and one deletion motif for the determination of East Asian mitochondrial DNA (mtDNA) haplogroups by designing three multiplex systems which apply single base extension methods. Using two multiplex systems, all 593 Korean mtDNAs were allocated into 15 haplogroups: M, D, D4, D5, G, M7, M8, M9, M10, M11, R, R9, B, A, and N9. As the D4 haplotypes occurred most frequently in Koreans, the third multiplex system was used to further define D4 subhaplogroups: D4a, D4b, D4e, D4g, D4h, and D4j. This method allowed the complementation of coding region information with control region mutation motifs and the resultant findings also suggest reliable control region mutation motifs for the assignment of East Asian mtDNA haplogroups. These three multiplex systems produce good results in degraded samples as they contain small PCR products (101-154 bp) for single base extension reactions. SNP scoring was performed in 101 old skeletal remains using these three systems to prove their utility in degraded samples. The sequence analysis of mtDNA control region with high incidence of haplogroup-specific mutations and the selective scoring of highly informative coding region SNPs using the three multiplex systems are useful tools for most applications involving East Asian mtDNA haplogroup determination and haplogroup-directed stringent quality control.

High-throughput STR analysis for DNA database using direct PCR.

PubMed

Sim, Jeong Eun; Park, Su Jeong; Lee, Han Chul; Kim, Se-Yong; Kim, Jong Yeol; Lee, Seung Hwan

2013-07-01

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high-throughput and cost-effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter-loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time- and cost-saving manner. © 2013 American Academy of Forensic Sciences Published 2013. This article is a U.S. Government work and is in the public domain in the U.S.A.

[Study of Cervical Exfoliated Cell's DNA Quantitative Analysis Based on Multi-Spectral Imaging Technology].

PubMed

Wu, Zheng; Zeng, Li-bo; Wu, Qiong-shui

2016-02-01

The conventional cervical cancer screening methods mainly include TBS (the bethesda system) classification method and cellular DNA quantitative analysis, however, by using multiple staining method in one cell slide, which is staining the cytoplasm with Papanicolaou reagent and the nucleus with Feulgen reagent, the study of achieving both two methods in the cervical cancer screening at the same time is still blank. Because the difficulty of this multiple staining method is that the absorbance of the non-DNA material may interfere with the absorbance of DNA, so that this paper has set up a multi-spectral imaging system, and established an absorbance unmixing model by using multiple linear regression method based on absorbance's linear superposition character, and successfully stripped out the absorbance of DNA to run the DNA quantitative analysis, and achieved the perfect combination of those two kinds of conventional screening method. Through a series of experiment we have proved that between the absorbance of DNA which is calculated by the absorbance unmixxing model and the absorbance of DNA which is measured there is no significant difference in statistics when the test level is 1%, also the result of actual application has shown that there is no intersection between the confidence interval of the DNA index of the tetraploid cells which are screened by using this paper's analysis method when the confidence level is 99% and the DNA index's judging interval of cancer cells, so that the accuracy and feasibility of the quantitative DNA analysis with multiple staining method expounded by this paper have been verified, therefore this analytical method has a broad application prospect and considerable market potential in early diagnosis of cervical cancer and other cancers.

Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis.

PubMed

Howard, Christopher; Gilmore, Simon; Robertson, James; Peakall, Rod

2008-09-01

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.

Microfluidic Devices for Forensic DNA Analysis: A Review.

PubMed

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-08-05

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10-20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook.

Acceptance of Domestic Cat Mitochondrial DNA in a Criminal Proceeding

PubMed Central

Lyons, Leslie A.; Grahn, Robert A.; Kun, Teri J.; Netzel, Linda R.; Wictum, Elizabeth E.; Halverson, Joy L.

2014-01-01

Shed hair from domestic animals readily adheres to clothing and other contact items, providing a source of transfer evidence for criminal investigations. Mitochondrial DNA is often the only option for DNA analysis of shed hair. Human mitochondrial DNA analysis has been accepted in the US court system since 1996. The murder trial of the State of Missouri versus Henry L. Polk, Jr. represents the first legal proceeding where cat mitochondrial DNA analysis was introduced into evidence. The mitochondrial DNA evidence was initially considered inadmissible due to concerns about the cat dataset and the scientific acceptance of the marker. Those concerns were subsequently addressed, and the evidence was deemed admissible. This report reviews the case in regards to the cat biological evidence and its ultimate admission as generally accepted and reliable. Expansion and saturation analysis of the cat mitochondrial DNA control region dataset supported the initial interpretation of the evidence. PMID:25086413

A study of deoxyribonucleotide metabolism and its relation to DNA synthesis. Supercomputer simulation and model-system analysis.

PubMed

Heinmets, F; Leary, R H

1991-06-01

A model system (1) was established to analyze purine and pyrimidine metabolism. This system has been expanded to include macrosimulation of DNA synthesis and the study of its regulation by terminal deoxynucleoside triphosphates (dNTPs) via a complex set of interactions. Computer experiments reveal that our model exhibits adequate and reasonable sensitivity in terms of dNTP pool levels and rates of DNA synthesis when inputs to the system are varied. These simulation experiments reveal that in order to achieve maximum DNA synthesis (in terms of purine metabolism), a proper balance is required in guanine and adenine input into this metabolic system. Excessive inputs will become inhibitory to DNA synthesis. In addition, studies are carried out on rates of DNA synthesis when various parameters are changed quantitatively. The current system is formulated by 110 differential equations.

DNA:DNA hybridization studies on the pink-pigmented facultative methylotrophs.

PubMed

Hood, D W; Dow, C S; Green, P N

1987-03-01

The genomic relatedness among 36 strains of pink-pigmented facultatively methylotrophic bacteria (PPFMs) was estimated by determination of DNA base composition and by DNA:DNA hybridization studies. A reproducible hybridization system was developed for the rapid analysis of multiple DNA samples. Results indicated that the PPFMs comprise four major and several minor homology groups, and that they should remain grouped in a single genus, Methylobacterium.

DNA Fingerprint Analysis of Three Short Tandem Repeat (STR) Loci for Biochemistry and Forensic Science Laboratory Courses

ERIC Educational Resources Information Center

McNamara-Schroeder, Kathleen; Olonan, Cheryl; Chu, Simon; Montoya, Maria C.; Alviri, Mahta; Ginty, Shannon; Love, John J.

2006-01-01

We have devised and implemented a DNA fingerprinting module for an upper division undergraduate laboratory based on the amplification and analysis of three of the 13 short tandem repeat loci that are required by the Federal Bureau of Investigation Combined DNA Index System (FBI CODIS) data base. Students first collect human epithelial (cheek)…

Using eDNA to estimate distribution of fish species in the St. Louis River

EPA Science Inventory

Environmental DNA (eDNA) analysis of extracellular material shed by aquatic organisms is a noninvasive genetic tool that can improve efficiency and reduce costs associated with species detection in aquatic systems. eDNA methods are widely used to assess presence/absence of a targ...

Development of an efficient fungal DNA extraction method to be used in random amplified polymorphic DNA-PCR analysis to differentiate cyclopiazonic acid mold producers.

PubMed

Sánchez, Beatriz; Rodríguez, Mar; Casado, Eva M; Martín, Alberto; Córdoba, Juan J

2008-12-01

A variety of previously established mechanical and chemical treatments to achieve fungal cell lysis combined with a semiautomatic system operated by a vacuum pump were tested to obtain DNA extract to be directly used in randomly amplified polymorphic DNA (RAPD)-PCR to differentiate cyclopiazonic acid-producing and -nonproducing mold strains. A DNA extraction method that includes digestion with proteinase K and lyticase prior to using a mortar and pestle grinding and a semiautomatic vacuum system yielded DNA of high quality in all the fungal strains and species tested, at concentrations ranging from 17 to 89 ng/microl in 150 microl of the final DNA extract. Two microliters of DNA extracted with this method was directly used for RAPD-PCR using primer (GACA)4. Reproducible RAPD fingerprints showing high differences between producer and nonproducer strains were observed. These differences in the RAPD patterns did not differentiate all the strains tested in clusters by cyclopiazonic acid production but may be very useful to distinguish cyclopiazonic acid producer strains from nonproducer strains by a simple RAPD analysis. Thus, the DNA extracts obtained could be used directly without previous purification and quantification for RAPD analysis to differentiate cyclopiazonic acid producer from nonproducer mold strains. This combined analysis could be adaptable to other toxigenic fungal species to enable differentiation of toxigenic and non-toxigenic molds, a procedure of great interest in food safety.

Purification of crime scene DNA extracts using centrifugal filter devices

PubMed Central

2013-01-01

Background The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. Methods Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. Results Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K. Conclusions Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used. PMID:23618387

Microfluidic Devices for Forensic DNA Analysis: A Review

PubMed Central

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-01-01

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10–20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook. PMID:27527231

Biochip microsystem for bioinformatics recognition and analysis

NASA Technical Reports Server (NTRS)

Lue, Jaw-Chyng (Inventor); Fang, Wai-Chi (Inventor)

2011-01-01

A system with applications in pattern recognition, or classification, of DNA assay samples. Because DNA reference and sample material in wells of an assay may be caused to fluoresce depending upon dye added to the material, the resulting light may be imaged onto an embodiment comprising an array of photodetectors and an adaptive neural network, with applications to DNA analysis. Other embodiments are described and claimed.

Residual eDNA detection sensitivity assessed by quantitative real-time PCR in a river ecosystem.

PubMed

Balasingham, Katherine D; Walter, Ryan P; Heath, Daniel D

2017-05-01

Several studies have demonstrated that environmental DNA (eDNA) can be used to detect the presence of aquatic species, days to weeks after the target species has been removed. However, most studies used eDNA analysis in lentic systems (ponds or lakes), or in controlled laboratory experiments. While eDNA degrades rapidly in all aquatic systems, it also undergoes dilution effects and physical destruction in flowing systems, complicating detection in rivers. However, some eDNA (i.e. residual eDNA) can be retained in aquatic systems, even those subject to high flow regimes. Our goal was to determine residual eDNA detection sensitivity using quantitative real-time polymerase chain reaction (qRT-PCR), in a flowing, uncontrolled river after the eDNA source was removed from the system; we repeated the experiment over 2 years. Residual eDNA had the strongest signal strength at the original source site and was detectable there up to 11.5 h after eDNA source removal. Residual eDNA signal strength decreased as sampling distance downstream from the eDNA source site increased, and was no longer detectable at the source site 48 h after the eDNA source water was exhausted in both experiments. This experiment shows that residual eDNA sampled in surface water can be mapped quantitatively using qRT-PCR, which allows a more accurate spatial identification of the target species location in lotic systems, and relative residual eDNA signal strength may allow the determination of the timing of the presence of target species. © 2016 John Wiley & Sons Ltd.

DNA barcode-based molecular identification system for fish species.

PubMed

Kim, Sungmin; Eo, Hae-Seok; Koo, Hyeyoung; Choi, Jun-Kil; Kim, Won

2010-12-01

In this study, we applied DNA barcoding to identify species using short DNA sequence analysis. We examined the utility of DNA barcoding by identifying 53 Korean freshwater fish species, 233 other freshwater fish species, and 1339 saltwater fish species. We successfully developed a web-based molecular identification system for fish (MISF) using a profile hidden Markov model. MISF facilitates efficient and reliable species identification, overcoming the limitations of conventional taxonomic approaches. MISF is freely accessible at http://bioinfosys.snu.ac.kr:8080/MISF/misf.jsp .

Development of an electrochemical detection system for measuring DNA methylation levels using methyl CpG-binding protein and glucose dehydrogenase-fused zinc finger protein.

PubMed

Lee, Jinhee; Yoshida, Wataru; Abe, Koichi; Nakabayashi, Kazuhiko; Wakeda, Hironobu; Hata, Kenichiro; Marquette, Christophe A; Blum, Loïc J; Sode, Koji; Ikebukuro, Kazunori

2017-07-15

DNA methylation level at a certain gene region is considered as a new type of biomarker for diagnosis and its miniaturized and rapid detection system is required for diagnosis. Here we have developed a simple electrochemical detection system for DNA methylation using methyl CpG-binding domain (MBD) and a glucose dehydrogenase (GDH)-fused zinc finger protein. This analytical system consists of three steps: (1) methylated DNA collection by MBD, (2) PCR amplification of a target genomic region among collected methylated DNA, and (3) electrochemical detection of the PCR products using a GDH-fused zinc finger protein. With this system, we have successfully measured the methylation levels at the promoter region of the androgen receptor gene in 10 6 copies of genomic DNA extracted from PC3 and TSU-PR1 cancer cell lines. Since no sequence analysis or enzymatic digestion is required for this detection system, DNA methylation levels can be measured within 3h with a simple procedure. Copyright © 2016 Elsevier B.V. All rights reserved.

DNAzymes in DNA Nanomachines and DNA Analysis

NASA Astrophysics Data System (ADS)

He, Yu; Tian, Ye; Chen, Yi; Mao, Chengde

This chapter discusses our efforts in using DNAzymes in DNA nano-machines and DNA analysis systems. 10-23 DNAzymes can cleave specific phos-phodiester bonds in RNA. We use them to construct an autonomous DNA-RNA chimera nanomotor, which constantly extracts chemical energy from RNA substrates and transduces the energy into a mechanical motion: cycles of contraction and extension. The motor's motion can be reversibly turned on and off by a DNA analogue (brake) of the RNA substrate. Addition and removal of the brake stops and restarts, respectively, the motor's motion. Furthermore, when the RNA substrates are preorganized into a one-dimensional track, a DNAzyme can continuously move along the track so long as there are substrates available ahead. Based on a similar mechanism, a novel DNA detection system has been developed. A target DNA activates a DNAzyme to cleave RNA-containing molecular beacons (MB), which generates an enhanced fluorescence signal. A following work integrates two steps of signal amplifications: a rolling-circle amplification (RCA) to synthesize multiple copies of DNAzymes, and the DNAzymes catalyze a chemical reaction to generate a colorimetric signal. This method allows detection of DNA analytes whose concentration is as low as 1 pM.

Bona fide colour: DNA prediction of human eye and hair colour from ancient and contemporary skeletal remains

PubMed Central

2013-01-01

Background DNA analysis of ancient skeletal remains is invaluable in evolutionary biology for exploring the history of species, including humans. Contemporary human bones and teeth, however, are relevant in forensic DNA analyses that deal with the identification of perpetrators, missing persons, disaster victims or family relationships. They may also provide useful information towards unravelling controversies that surround famous historical individuals. Retrieving information about a deceased person’s externally visible characteristics can be informative in both types of DNA analyses. Recently, we demonstrated that human eye and hair colour can be reliably predicted from DNA using the HIrisPlex system. Here we test the feasibility of the novel HIrisPlex system at establishing eye and hair colour of deceased individuals from skeletal remains of various post-mortem time ranges and storage conditions. Methods Twenty-one teeth between 1 and approximately 800 years of age and 5 contemporary bones were subjected to DNA extraction using standard organic protocol followed by analysis using the HIrisPlex system. Results Twenty-three out of 26 bone DNA extracts yielded the full 24 SNP HIrisPlex profile, therefore successfully allowing model-based eye and hair colour prediction. HIrisPlex analysis of a tooth from the Polish general Władysław Sikorski (1881 to 1943) revealed blue eye colour and blond hair colour, which was positively verified from reliable documentation. The partial profiles collected in the remaining three cases (two contemporary samples and a 14th century sample) were sufficient for eye colour prediction. Conclusions Overall, we demonstrate that the HIrisPlex system is suitable, sufficiently sensitive and robust to successfully predict eye and hair colour from ancient and contemporary skeletal remains. Our findings, therefore, highlight the HIrisPlex system as a promising tool in future routine forensic casework involving skeletal remains, including ancient DNA studies, for the prediction of eye and hair colour of deceased individuals. PMID:23317428

Bona fide colour: DNA prediction of human eye and hair colour from ancient and contemporary skeletal remains.

PubMed

Draus-Barini, Jolanta; Walsh, Susan; Pośpiech, Ewelina; Kupiec, Tomasz; Głąb, Henryk; Branicki, Wojciech; Kayser, Manfred

2013-01-14

DNA analysis of ancient skeletal remains is invaluable in evolutionary biology for exploring the history of species, including humans. Contemporary human bones and teeth, however, are relevant in forensic DNA analyses that deal with the identification of perpetrators, missing persons, disaster victims or family relationships. They may also provide useful information towards unravelling controversies that surround famous historical individuals. Retrieving information about a deceased person's externally visible characteristics can be informative in both types of DNA analyses. Recently, we demonstrated that human eye and hair colour can be reliably predicted from DNA using the HIrisPlex system. Here we test the feasibility of the novel HIrisPlex system at establishing eye and hair colour of deceased individuals from skeletal remains of various post-mortem time ranges and storage conditions. Twenty-one teeth between 1 and approximately 800 years of age and 5 contemporary bones were subjected to DNA extraction using standard organic protocol followed by analysis using the HIrisPlex system. Twenty-three out of 26 bone DNA extracts yielded the full 24 SNP HIrisPlex profile, therefore successfully allowing model-based eye and hair colour prediction. HIrisPlex analysis of a tooth from the Polish general Władysław Sikorski (1881 to 1943) revealed blue eye colour and blond hair colour, which was positively verified from reliable documentation. The partial profiles collected in the remaining three cases (two contemporary samples and a 14th century sample) were sufficient for eye colour prediction. Overall, we demonstrate that the HIrisPlex system is suitable, sufficiently sensitive and robust to successfully predict eye and hair colour from ancient and contemporary skeletal remains. Our findings, therefore, highlight the HIrisPlex system as a promising tool in future routine forensic casework involving skeletal remains, including ancient DNA studies, for the prediction of eye and hair colour of deceased individuals.

Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites.

PubMed

Feng, Zhiyang; Kallifidas, Dimitris; Brady, Sean F

2011-08-02

A single gram of soil is predicted to contain thousands of unique bacterial species. The majority of these species remain recalcitrant to standard culture methods, prohibiting their use as sources of unique bioactive small molecules. The cloning and analysis of DNA extracted directly from environmental samples (environmental DNA, eDNA) provides a means of exploring the biosynthetic capacity of natural bacterial populations. Environmental DNA libraries contain large reservoirs of bacterial genetic diversity from which new secondary metabolite gene clusters can be systematically recovered and studied. The identification and heterologous expression of type II polyketide synthase-containing eDNA clones is reported here. Functional analysis of three soil DNA-derived polyketide synthase systems in Streptomyces albus revealed diverse metabolites belonging to well-known, rare, and previously uncharacterized structural families. The first of these systems is predicted to encode the production of the known antibiotic landomycin E. The second was found to encode the production of a metabolite with a previously uncharacterized pentacyclic ring system. The third was found to encode the production of unique KB-3346-5 derivatives, which show activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis. These results, together with those of other small-molecule-directed metagenomic studies, suggest that culture-independent approaches are capable of accessing biosynthetic diversity that has not yet been extensively explored using culture-based methods. The large-scale functional screening of eDNA clones should be a productive strategy for generating structurally previously uncharacterized chemical entities for use in future drug development efforts.

An enhanced chemiluminescence resonance energy transfer system based on target recycling G-guadruplexes/hemin DNAzyme catalysis and its application in ultrasensitive detection of DNA.

PubMed

Chen, Jia; Huang, Yong; Vdovenko, Marina; Sakharov, Ivan Yu; Su, Guifa; Zhao, Shulin

2015-06-01

An enhanced chemiluminescence resonance energy transfer (CRET) system based on target recycling G-guadruplexes/hemin DNAzyme catalysis was developed for ultrasensitive detection of DNA. CRET system consists of luminol as chemiluminescent donor, and fluorescein isothiocyanate (FITC) as acceptor. The sensitive detection was achieved by using the system consisted of G-riched DNA, blocker DNA, and the Nb.BbvCI biocatalyst. Upon addition of target DNA to the system, target DNA hybridizes with the quasi-circular DNA structure, and forms a DNA duplex. The formation of DNA duplex triggers selective enzymatic cleavage of quasi-circular DNA by Nb.BbvCI, resulting in the release of target DNA and two G-riched DNAzyme segments. Released target DNA then hybridizes with another quasi-circular DNA structure to initiate the cleavage of the quasi-circular DNA structure. Eventually, each target DNA can go through many cycles, resulting in the digestion of many quasi-circular DNA structures, generating many G-riched DNAzyme segments. G-riched DNAzyme segment products assemble with hemin to form stable hemin/G-quadruplexes that exhibit peroxidase-like activity which can catalyze the oxidation of luminol by H2O2 to produce CL signals. In the presence of FITC, CL of luminol can excite FITC molecules, and thus produced CRET between the luminol and FITC. This unique analysis strategy gives a detection limit down to 80 fM, which is at least four orders of magnitude lower than that of unamplified DNA detection methods. Copyright © 2015 Elsevier B.V. All rights reserved.

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system.

PubMed

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

2015-11-19

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database in which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. This database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.

Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells

NASA Astrophysics Data System (ADS)

Zimny, Philip; Juncker, David; Reisner, Walter

Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols.

Developmental validation of the DNAscan™ Rapid DNA Analysis™ instrument and expert system for reference sample processing.

PubMed

Della Manna, Angelo; Nye, Jeffrey V; Carney, Christopher; Hammons, Jennifer S; Mann, Michael; Al Shamali, Farida; Vallone, Peter M; Romsos, Erica L; Marne, Beth Ann; Tan, Eugene; Turingan, Rosemary S; Hogan, Catherine; Selden, Richard F; French, Julie L

2016-11-01

Since the implementation of forensic DNA typing in labs more than 20 years ago, the analysis procedures and data interpretation have always been conducted in a laboratory by highly trained and qualified scientific personnel. Rapid DNA technology has the potential to expand testing capabilities within forensic laboratories and to allow forensic STR analysis to be performed outside the physical boundaries of the traditional laboratory. The developmental validation of the DNAscan/ANDE Rapid DNA Analysis System was completed using a BioChipSet™ Cassette consumable designed for high DNA content samples, such as single source buccal swabs. A total of eight laboratories participated in the testing which totaled over 2300 swabs, and included nearly 1400 unique individuals. The goal of this extensive study was to obtain, document, analyze, and assess DNAscan and its internal Expert System to reliably genotype reference samples in a manner compliant with the FBI's Quality Assurance Standards (QAS) and the NDIS Operational Procedures. The DNAscan System provided high quality, concordant results for reference buccal swabs, including automated data analysis with an integrated Expert System. Seven external laboratories and NetBio, the developer of the technology, participated in the validation testing demonstrating the reproducibility and reliability of the system and its successful use in a variety of settings by numerous operators. The DNAscan System demonstrated limited cross reactivity with other species, was resilient in the presence of numerous inhibitors, and provided reproducible results for both buccal and purified DNA samples with sensitivity at a level appropriate for buccal swabs. The precision and resolution of the system met industry standards for detection of micro-variants and displayed single base resolution. PCR-based studies provided confidence that the system was robust and that the amplification reaction had been optimized to provide high quality results. The DNAscan integrated Expert System was examined as part of the Developmental Validation and successfully interpreted the over 2000 samples tested with over 99.998% concordant alleles. The system appropriately flagged samples for human review and failed both mixed samples and samples with insufficient genetic information. These results demonstrated the integrated Expert System makes correct allele calls without human intervention. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Large mitochondrial DNA deletion in an infant with addison disease.

PubMed

Duran, Gloria P; Martinez-Aguayo, A; Poggi, H; Lagos, M; Gutierrez, D; Harris, P R

2012-01-01

Mitochondrial diseases are a group of disorders caused by mutations in nuclear DNA or mitochondrial DNA, usually involving multiple organ systems. Primary adrenal insufficiency due to mitochondrial disease is extremely infrequent and has been reported in association with mitochondrial DNA deletion syndromes such as Kearns-Sayre syndrome. To report a 3-year-old boy with Addison disease, congenital glaucoma, chronic pancreatitis, and mitochondrial myopathy due to large mitochondrial DNA deletion. Molecular analysis of mitochondrial DNA samples obtained from peripheral blood, oral mucosa, and muscle tissue. A novel large mitochondrial DNA deletion of 7,372bp was identified involving almost all genes on the big arch of mtDNA. This case reaffirms the association of adrenal insufficiency and mitochondrial DNA deletions and presents new evidence that glaucoma is another manifestation of mitochondrial diseases. Due to the genetic and clinical heterogeneity of mitochondrial disorders, molecular analysis is crucial to confirm diagnosis and to allow accurate genetic counseling.

Integrated genomic classification of melanocytic tumors of the central nervous system using mutation analysis, copy number alterations and DNA methylation profiling.

PubMed

Griewank, Klaus; Koelsche, Christian; van de Nes, Johannes A P; Schrimpf, Daniel; Gessi, Marco; Möller, Inga; Sucker, Antje; Scolyer, Richard A; Buckland, Michael E; Murali, Rajmohan; Pietsch, Torsten; von Deimling, Andreas; Schadendorf, Dirk

2018-06-11

In the central nervous system, distinguishing primary leptomeningeal melanocytic tumors from melanoma metastases and predicting their biological behavior solely using histopathologic criteria can be challenging. We aimed to assess the diagnostic and prognostic value of integrated molecular analysis. Targeted next-generation-sequencing, array-based genome-wide methylation analysis and BAP1 immunohistochemistry was performed on the largest cohort of central nervous system melanocytic tumors analyzed to date, incl. 47 primary tumors of the central nervous system, 16 uveal melanomas. 13 cutaneous melanoma metastasis and 2 blue nevus-like melanomas. Gene mutation, DNA-methylation and copy-number profiles were correlated with clinicopathological features. Combining mutation, copy-number and DNA-methylation profiles clearly distinguished cutaneous melanoma metastases from other melanocytic tumors. Primary leptomeningeal melanocytic tumors, uveal melanomas and blue nevus-like melanoma showed common DNA-methylation, copy-number alteration and gene mutation signatures. Notably, tumors demonstrating chromosome 3 monosomy and BAP1 alterations formed a homogeneous subset within this group. Integrated molecular profiling aids in distinguishing primary from metastatic melanocytic tumors of the central nervous system. Primary leptomeningeal melanocytic tumors, uveal melanoma and blue nevus-like melanoma share molecular similarity with chromosome 3 and BAP1 alterations markers of poor prognosis. Copyright ©2018, American Association for Cancer Research.

FBIS: A regional DNA barcode archival & analysis system for Indian fishes.

PubMed

Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

2012-01-01

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. The database is available for free at http://mail.nbfgr.res.in/fbis/

A transmission imaging spectrograph and microfabricated channel system for DNA analysis.

PubMed

Simpson, J W; Ruiz-Martinez, M C; Mulhern, G T; Berka, J; Latimer, D R; Ball, J A; Rothberg, J M; Went, G T

2000-01-01

In this paper we present the development of a DNA analysis system using a microfabricated channel device and a novel transmission imaging spectrograph which can be efficiently incorporated into a high throughput genomics facility for both sizing and sequencing of DNA fragments. The device contains 48 channels etched on a glass substrate. The channels are sealed with a flat glass plate which also provides a series of apertures for sample loading and contact with buffer reservoirs. Samples can be easily loaded in volumes up to 640 nL without band broadening because of an efficient electrokinetic stacking at the electrophoresis channel entrance. The system uses a dual laser excitation source and a highly sensitive charge-coupled device (CCD) detector allowing for simultaneous detection of many fluorescent dyes. The sieving matrices for the separation of single-stranded DNA fragments are polymerized in situ in denaturing buffer systems. Examples of separation of single-stranded DNA fragments up to 500 bases in length are shown, including accurate sizing of GeneCalling fragments, and sequencing samples prepared with a reduced amount of dye terminators. An increase in sample throughput has been achieved by color multiplexing.

End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

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

Linshiz, Gregory; Jensen, Erik; Stawski, Nina

Synthetic biology aims to engineer biological systems for desired behaviors. The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening. Here, we present a programmable, multipurpose microfluidic platform and associated software and apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis. We show the platform’s capabilities for multiple automated DNA assembly methods, including a new method for Isothermal Hierarchical DNA Construction, and for Escherichia coli and Saccharomyces cerevisiae transformation. The platform enables the automated control of cellular growth, gene expression induction, andmore » proteogenic and metabolic output analysis. Finally, taken together, we demonstrate the microfluidic platform’s potential to provide end-to-end solutions for synthetic biology research, from design to functional analysis.« less

End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

DOE PAGES

Linshiz, Gregory; Jensen, Erik; Stawski, Nina; ...

2016-02-02

Synthetic biology aims to engineer biological systems for desired behaviors. The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening. Here, we present a programmable, multipurpose microfluidic platform and associated software and apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis. We show the platform’s capabilities for multiple automated DNA assembly methods, including a new method for Isothermal Hierarchical DNA Construction, and for Escherichia coli and Saccharomyces cerevisiae transformation. The platform enables the automated control of cellular growth, gene expression induction, andmore » proteogenic and metabolic output analysis. Finally, taken together, we demonstrate the microfluidic platform’s potential to provide end-to-end solutions for synthetic biology research, from design to functional analysis.« less

The Israel DNA database--the establishment of a rapid, semi-automated analysis system.

PubMed

Zamir, Ashira; Dell'Ariccia-Carmon, Aviva; Zaken, Neomi; Oz, Carla

2012-03-01

The Israel Police DNA database, also known as IPDIS (Israel Police DNA Index System), has been operating since February 2007. During that time more than 135,000 reference samples have been uploaded and more than 2000 hits reported. We have developed an effective semi-automated system that includes two automated punchers, three liquid handler robots and four genetic analyzers. An inhouse LIMS program enables full tracking of every sample through the entire process of registration, pre-PCR handling, analysis of profiles, uploading to the database, hit reports and ultimately storage. The LIMS is also responsible for the future tracking of samples and their profiles to be expunged from the database according to the Israeli DNA legislation. The database is administered by an in-house developed software program, where reference and evidentiary profiles are uploaded, stored, searched and matched. The DNA database has proven to be an effective investigative tool which has gained the confidence of the Israeli public and on which the Israel National Police force has grown to rely. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Development of a rapid 21-plex autosomal STR typing system for forensic applications.

PubMed

Yang, Meng; Yin, Caiyong; Lv, Yuexin; Yang, Yaran; Chen, Jing; Yu, Zailiang; Liu, Xu; Xu, Meibo; Chen, Feng; Wu, Huijuan; Yan, Jiangwei

2016-10-01

DNA-STR genotyping technology has been widely used in forensic investigations. Even with such success, there is a great need to reduce the analysis time. In this study, we established a new rapid 21-plex STR typing system, including 13 CODIS loci, Penta D, Penta E, D12S391, D2S1338, D6S1043, D19S433, D2S441 and Amelogenin loci. This system could shorten the amplification time to a minimum of 90 min and does not require DNA extraction from the samples. Validation of the typing system complied with the Scientific Working Group on DNA Analysis Methods (SWGDAM) and the Chinese National Standard (GA/T815-2009) guidelines. The results demonstrated that this 21-plex STR typing system was a valuable tool for rapid criminal investigation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome-Wide Analysis of the Core DNA Replication Machinery in the Higher Plants Arabidopsis and Rice1[W][OA

PubMed Central

Shultz, Randall W.; Tatineni, Vinaya M.; Hanley-Bowdoin, Linda; Thompson, William F.

2007-01-01

Core DNA replication proteins mediate the initiation, elongation, and Okazaki fragment maturation functions of DNA replication. Although this process is generally conserved in eukaryotes, important differences in the molecular architecture of the DNA replication machine and the function of individual subunits have been reported in various model systems. We have combined genome-wide bioinformatic analyses of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) with published experimental data to provide a comprehensive view of the core DNA replication machinery in plants. Many components identified in this analysis have not been studied previously in plant systems, including the GINS (go ichi ni san) complex (PSF1, PSF2, PSF3, and SLD5), MCM8, MCM9, MCM10, NOC3, POLA2, POLA3, POLA4, POLD3, POLD4, and RNASEH2. Our results indicate that the core DNA replication machinery from plants is more similar to vertebrates than single-celled yeasts (Saccharomyces cerevisiae), suggesting that animal models may be more relevant to plant systems. However, we also uncovered some important differences between plants and vertebrate machinery. For example, we did not identify geminin or RNASEH1 genes in plants. Our analyses also indicate that plants may be unique among eukaryotes in that they have multiple copies of numerous core DNA replication genes. This finding raises the question of whether specialized functions have evolved in some cases. This analysis establishes that the core DNA replication machinery is highly conserved across plant species and displays many features in common with other eukaryotes and some characteristics that are unique to plants. PMID:17556508

Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients: Toward a New Approach for Diagnosis and Patient Stratification.

PubMed

Poulsen, Nicklas N; Pedersen, Morten E; Østergaard, Jesper; Petersen, Nickolaj J; Nielsen, Christoffer T; Heegaard, Niels H H; Jensen, Henrik

2016-09-20

Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.

Circulating tumor DNA: a promising biomarker in the liquid biopsy of cancer.

PubMed

Cheng, Feifei; Su, Li; Qian, Cheng

2016-07-26

Tissue biopsy is the standard diagnostic procedure for cancers and also provides a material for genotyping, which can assist in the targeted therapies of cancers. However, tissue biopsy-based cancer diagnostic procedures have limitations in their assessment of cancer development, prognosis and genotyping, due to tumor heterogeneity and evolution. Circulating tumor DNA (ctDNA) is single- or double-stranded DNA released by the tumor cells into the blood and it thus harbors the mutations of the original tumor. In recent years, liquid biopsy based on ctDNA analysis has shed a new light on the molecular diagnosis and monitoring of cancer. Studies found that the screening of genetic mutations using ctDNA is highly sensitive and specific, suggesting that ctDNA analysis may significantly improve current systems of tumor diagnosis, even facilitating early-stage detection. Moreover, ctDNA analysis is capable of accurately determining the tumor progression, prognosis and assisting in targeted therapy. Therefore, using ctDNA as a liquid biopsy may herald a revolution for tumor management. Herein, we review the biology of ctDNA, its detection methods and potential applications in tumor diagnosis, treatment and prognosis.

Circulating tumor DNA: a promising biomarker in the liquid biopsy of cancer

PubMed Central

Cheng, Feifei; Su, Li; Qian, Cheng

2016-01-01

Tissue biopsy is the standard diagnostic procedure for cancers and also provides a material for genotyping, which can assist in the targeted therapies of cancers. However, tissue biopsy-based cancer diagnostic procedures have limitations in their assessment of cancer development, prognosis and genotyping, due to tumor heterogeneity and evolution. Circulating tumor DNA (ctDNA) is single- or double-stranded DNA released by the tumor cells into the blood and it thus harbors the mutations of the original tumor. In recent years, liquid biopsy based on ctDNA analysis has shed a new light on the molecular diagnosis and monitoring of cancer. Studies found that the screening of genetic mutations using ctDNA is highly sensitive and specific, suggesting that ctDNA analysis may significantly improve current systems of tumor diagnosis, even facilitating early-stage detection. Moreover, ctDNA analysis is capable of accurately determining the tumor progression, prognosis and assisting in targeted therapy. Therefore, using ctDNA as a liquid biopsy may herald a revolution for tumor management. Herein, we review the biology of ctDNA, its detection methods and potential applications in tumor diagnosis, treatment and prognosis. PMID:27223063

Isotachophoresis for fractionation and recovery of cytoplasmic RNA and nucleus from single cells.

PubMed

Kuriyama, Kentaro; Shintaku, Hirofumi; Santiago, Juan G

2015-07-01

There is a substantial need for simultaneous analyses of RNA and DNA from individual single cells. Such analysis provides unique evidence of cell-to-cell differences and the correlation between gene expression and genomic mutation in highly heterogeneous cell populations. We present a novel microfluidic system that leverages isotachophoresis to fractionate and isolate cytoplasmic RNA and genomic DNA (gDNA) from single cells. The system uniquely enables independent, sequence-specific analyses of these critical markers. Our system uses a microfluidic chip with a simple geometry and four end-channel electrodes, and completes the entire process in <5 min, including lysis, purification, fractionation, and delivery to DNA and RNA output reservoirs, each containing high quality and purity aliquots with no measurable cross-contamination of cytoplasmic RNA versus gDNA. We demonstrate our system with simultaneous, sequence-specific quantitation using off-chip RT-qPCR and qPCR for simultaneous cytoplasmic RNA and gDNA analyses, respectively. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of an Automated DNA Detection System Using an Electrochemical DNA Chip Technology

NASA Astrophysics Data System (ADS)

Hongo, Sadato; Okada, Jun; Hashimoto, Koji; Tsuji, Koichi; Nikaido, Masaru; Gemma, Nobuhiro

A new compact automated DNA detection system Genelyzer™ has been developed. After injecting a sample solution into a cassette with a built-in electrochemical DNA chip, processes from hybridization reaction to detection and analysis are all operated fully automatically. In order to detect a sample DNA, electrical currents from electrodes due to an oxidization reaction of electrochemically active intercalator molecules bound to hybridized DNAs are detected. The intercalator is supplied as a reagent solution by a fluid supply unit of the system. The feasibility test proved that the simultaneous typing of six single nucleotide polymorphisms (SNPs) associated with a rheumatoid arthritis (RA) was carried out within two hours and that all the results were consistent with those by conventional typing methods. It is expected that this system opens a new way to a DNA testing such as a test for infectious diseases, a personalized medicine, a food inspection, a forensic application and any other applications.

Molecular Genetic Characterization of Mutagenesis Using a Highly Sensitive Single-Stranded DNA Reporter System in Budding Yeast.

PubMed

Chan, Kin

2018-01-01

Mutations are permanent alterations to the coding content of DNA. They are starting material for the Darwinian evolution of species by natural selection, which has yielded an amazing diversity of life on Earth. Mutations can also be the fundamental basis of serious human maladies, most notably cancers. In this chapter, I describe a highly sensitive reporter system for the molecular genetic analysis of mutagenesis, featuring controlled generation of long stretches of single-stranded DNA in budding yeast cells. This system is ~100- to ~1000-fold more susceptible to mutation than conventional double-stranded DNA reporters, and is well suited for generating large mutational datasets to investigate the properties of mutagens.

Building biochips: a protein production pipeline

NASA Astrophysics Data System (ADS)

de Carvalho-Kavanagh, Marianne G. S.; Albala, Joanna S.

2004-06-01

Protein arrays are emerging as a practical format in which to study proteins in high-throughput using many of the same techniques as that of the DNA microarray. The key advantage to array-based methods for protein study is the potential for parallel analysis of thousands of samples in an automated, high-throughput fashion. Building protein arrays capable of this analysis capacity requires a robust expression and purification system capable of generating hundreds to thousands of purified recombinant proteins. We have developed a method to utilize LLNL-I.M.A.G.E. cDNAs to generate recombinant protein libraries using a baculovirus-insect cell expression system. We have used this strategy to produce proteins for analysis of protein/DNA and protein/protein interactions using protein microarrays in order to understand the complex interactions of proteins involved in homologous recombination and DNA repair. Using protein array techniques, a novel interaction between the DNA repair protein, Rad51B, and histones has been identified.

Advances in on-chip photodetection for applications in miniaturized genetic analysis systems

NASA Astrophysics Data System (ADS)

Namasivayam, Vijay; Lin, Rongsheng; Johnson, Brian; Brahmasandra, Sundaresh; Razzacki, Zafar; Burke, David T.; Burns, Mark A.

2004-01-01

Microfabrication techniques have become increasingly popular in the development of next generation DNA analysis devices. Improved on-chip fluorescence detection systems may have applications in developing portable hand-held instruments for point-of-care diagnostics. Miniaturization of fluorescence detection involves construction of ultra-sensitive photodetectors that can be integrated onto a fluidic platform combined with the appropriate optical emission filters. We have previously demonstrated integration PIN photodiodes onto a microfabricated electrophoresis channel for separation and detection of DNA fragments. In this work, we present an improved detector structure that uses a PINN+ photodiode with an on-chip interference filter and a robust liquid barrier layer. This new design yields high sensitivity (detection limit of 0.9 ng µl-1 of DNA), low-noise (S/N ~ 100/1) and enhanced quantum efficiencies (>80%) over the entire visible spectrum. Applications of these photodiodes in various areas of DNA analysis such as microreactions (PCR), separations (electrophoresis) and microfluidics (drop sensing) are presented.

Electron Microscopic Analysis of the Products of DNA Synthesis by DNA Polymerases from Calf Thymus and Herpes Simplex Virus Type I

DTIC Science & Technology

1988-10-03

DNA replication showed an average of 2.5 primers per M13 DNA circle. The measurement of the double stranded length from individual replicative intermediates by electron microscopy was within the accuracy of 10% standard deviation. The product length distribution obtained from the HSV-1 DNA polymerase catalyzed replication of M13 DNA primed with a specific pentadecamer and in the presence of E. Coli SSB protein showed a near Poisson distribution. Replication of the same primer-template system or DNA primase primed M13 DNA template by calf thymus DNA polymerase a showed a

A method for determining the actual rate of orientation switching of DNA self-assembled monolayers using optical and electrochemical frequency response analysis.

PubMed

Casanova-Moreno, J; Bizzotto, D

2015-02-17

Electrostatic control of the orientation of fluorophore-labeled DNA strands immobilized on an electrode surface has been shown to be an effective bioanalytical tool. Modulation techniques and later time-resolved measurements were used to evaluate the kinetics of the switching between lying and standing DNA conformations. These measurements, however, are the result of a convolution between the DNA "switching" response time and the other frequency limited responses in the measurement. In this work, a method for analyzing the response of a potential driven DNA sensor is presented by calculating the potential effectively dropped across the electrode interface (using electrochemical impedance spectroscopy) as opposed to the potential applied to the electrochemical cell. This effectively deconvolutes the effect of the charging time on the observed frequency response. The corrected response shows that DNA is able to switch conformation faster than previously reported using modulation techniques. This approach will ensure accurate measurements independent of the electrochemical system, removing the uncertainty in the analysis of the switching response, enabling comparison between samples and measurement systems.

The Optimization of Electrophoresis on a Glass Microfluidic Chip and its Application in Forensic Science.

PubMed

Han, Jun P; Sun, Jing; Wang, Le; Liu, Peng; Zhuang, Bin; Zhao, Lei; Liu, Yao; Li, Cai X

2017-11-01

Microfluidic chips offer significant speed, cost, and sensitivity advantages, but numerous parameters must be optimized to provide microchip electrophoresis detection. Experiments were conducted to study the factors, including sieving matrices (the concentration and type), surface modification, analysis temperature, and electric field strengths, which all impact the effectiveness of microchip electrophoresis detection of DNA samples. Our results showed that the best resolution for ssDNA was observed using 4.5% w/v (7 M urea) lab-fabricated LPA gel, dynamic wall coating of the microchannel, electrophoresis temperatures between 55 and 60°C, and electrical fields between 350 and 450 V/cm on the microchip-based capillary electrophoresis (μCE) system. One base-pair resolution could be achieved in the 19-cm-length microchannel. Furthermore, both 9947A standard genomic DNA and DNA extracted from blood spots were demonstrated to be successfully separated with well-resolved DNA peaks in 8 min. Therefore, the microchip electrophoresis system demonstrated good potential for rapid forensic DNA analysis. © 2017 American Academy of Forensic Sciences.

A new model for ancient DNA decay based on paleogenomic meta-analysis

PubMed Central

Ware, Roselyn; Smith, Oliver; Collins, Matthew

2017-01-01

Abstract The persistence of DNA over archaeological and paleontological timescales in diverse environments has led to a revolutionary body of paleogenomic research, yet the dynamics of DNA degradation are still poorly understood. We analyzed 185 paleogenomic datasets and compared DNA survival with environmental variables and sample ages. We find cytosine deamination follows a conventional thermal age model, but we find no correlation between DNA fragmentation and sample age over the timespans analyzed, even when controlling for environmental variables. We propose a model for ancient DNA decay wherein fragmentation rapidly reaches a threshold, then subsequently slows. The observed loss of DNA over time may be due to a bulk diffusion process in many cases, highlighting the importance of tissues and environments creating effectively closed systems for DNA preservation. This model of DNA degradation is largely based on mammal bone samples due to published genomic dataset availability. Continued refinement to the model to reflect diverse biological systems and tissue types will further improve our understanding of ancient DNA breakdown dynamics. PMID:28486705

Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices.

PubMed

Fraiture, Marie-Alice; Herman, Philippe; Lefèvre, Loic; Taverniers, Isabel; De Loose, Marc; Deforce, Dieter; Roosens, Nancy H

2015-08-14

In order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element. Food/feed matrices containing transgenic crops (Bt rice or MON863 maize) were analysed using the integrated DNA walking system. First, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples. Due to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix.

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

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

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database inmore » which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.« less

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

DOE PAGES

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

2015-11-19

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database inmore » which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.« less

FBIS: A regional DNA barcode archival & analysis system for Indian fishes

PubMed Central

Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

2012-01-01

DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. Availability The database is available for free at http://mail.nbfgr.res.in/fbis/ PMID:22715304

An Optimized DNA Analysis Workflow for the Sampling, Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints.

PubMed

Solomon, April D; Hytinen, Madison E; McClain, Aryn M; Miller, Marilyn T; Dawson Cruz, Tracey

2018-01-01

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints-touch DNA "sandwiched" between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post-amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri-Sep™ columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7-100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases. © 2017 American Academy of Forensic Sciences.

Genotyping tumour DNA in cerebrospinal fluid and plasma of a HER2-positive breast cancer patient with brain metastases

PubMed Central

Siravegna, Giulia; Geuna, Elena; Mussolin, Benedetta; Crisafulli, Giovanni; Bartolini, Alice; Galizia, Danilo; Casorzo, Laura; Sarotto, Ivana; Scaltriti, Maurizio; Sapino, Anna; Bardelli, Alberto; Montemurro, Filippo

2017-01-01

Background Central nervous system (CNS) involvement contributes to significant morbidity and mortality in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer (mBC) and represents a major challenge for clinicians. Liquid biopsy of cerebrospinal fluid (CSF)-derived circulating tumour DNA (ctDNA) harbours clinically relevant genomic alterations in patients with CNS metastases and could be effective in tracking tumour evolution. Methods In a HER2-positive mBC patient with brain metastases, we applied droplet digital PCR (ddPCR) and next-generation whole exome sequencing (WES) analysis to measure ctDNA dynamic changes in CSF and plasma collected during treatment. Results Baseline CSF-derived ctDNA analysis revealed TP53 and PIK3CA mutations as well as ERBB2 and cMYC amplification. Post-treatment ctDNA analysis showed decreased markers level in plasma, consistent with extra-CNS disease control, while increased in the CSF, confirming poor treatment benefit in the CNS. Discussion Analysis of ctDNA in the CSF of HER2-positive mBC is feasible and could represent a useful companion for clinical management of brain metastases. PMID:29067216

A novel chaotic image encryption scheme using DNA sequence operations

NASA Astrophysics Data System (ADS)

Wang, Xing-Yuan; Zhang, Ying-Qian; Bao, Xue-Mei

2015-10-01

In this paper, we propose a novel image encryption scheme based on DNA (Deoxyribonucleic acid) sequence operations and chaotic system. Firstly, we perform bitwise exclusive OR operation on the pixels of the plain image using the pseudorandom sequences produced by the spatiotemporal chaos system, i.e., CML (coupled map lattice). Secondly, a DNA matrix is obtained by encoding the confused image using a kind of DNA encoding rule. Then we generate the new initial conditions of the CML according to this DNA matrix and the previous initial conditions, which can make the encryption result closely depend on every pixel of the plain image. Thirdly, the rows and columns of the DNA matrix are permuted. Then, the permuted DNA matrix is confused once again. At last, after decoding the confused DNA matrix using a kind of DNA decoding rule, we obtain the ciphered image. Experimental results and theoretical analysis show that the scheme is able to resist various attacks, so it has extraordinarily high security.

High throughput DNA damage quantification of human tissue with home-based collection device

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

Costes, Sylvain V.; Tang, Jonathan; Yannone, Steven M.

Kits, methods and systems for providing a service to provide a subject with information regarding the state of a subject's DNA damage. Collection, processing and analysis of samples are also described.

Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories.

PubMed

Jäger, Anne C; Alvarez, Michelle L; Davis, Carey P; Guzmán, Ernesto; Han, Yonmee; Way, Lisa; Walichiewicz, Paulina; Silva, David; Pham, Nguyen; Caves, Glorianna; Bruand, Jocelyne; Schlesinger, Felix; Pond, Stephanie J K; Varlaro, Joe; Stephens, Kathryn M; Holt, Cydne L

2017-05-01

Human DNA profiling using PCR at polymorphic short tandem repeat (STR) loci followed by capillary electrophoresis (CE) size separation and length-based allele typing has been the standard in the forensic community for over 20 years. Over the last decade, Next-Generation Sequencing (NGS) matured rapidly, bringing modern advantages to forensic DNA analysis. The MiSeq FGx™ Forensic Genomics System, comprised of the ForenSeq™ DNA Signature Prep Kit, MiSeq FGx™ Reagent Kit, MiSeq FGx™ instrument and ForenSeq™ Universal Analysis Software, uses PCR to simultaneously amplify up to 231 forensic loci in a single multiplex reaction. Targeted loci include Amelogenin, 27 common, forensic autosomal STRs, 24 Y-STRs, 7 X-STRs and three classes of single nucleotide polymorphisms (SNPs). The ForenSeq™ kit includes two primer sets: Amelogenin, 58 STRs and 94 identity informative SNPs (iiSNPs) are amplified using DNA Primer Set A (DPMA; 153 loci); if a laboratory chooses to generate investigative leads using DNA Primer Set B, amplification is targeted to the 153 loci in DPMA plus 22 phenotypic informative (piSNPs) and 56 biogeographical ancestry SNPs (aiSNPs). High-resolution genotypes, including detection of intra-STR sequence variants, are semi-automatically generated with the ForenSeq™ software. This system was subjected to developmental validation studies according to the 2012 Revised SWGDAM Validation Guidelines. A two-step PCR first amplifies the target forensic STR and SNP loci (PCR1); unique, sample-specific indexed adapters or "barcodes" are attached in PCR2. Approximately 1736 ForenSeq™ reactions were analyzed. Studies include DNA substrate testing (cotton swabs, FTA cards, filter paper), species studies from a range of nonhuman organisms, DNA input sensitivity studies from 1ng down to 7.8pg, two-person human DNA mixture testing with three genotype combinations, stability analysis of partially degraded DNA, and effects of five commonly encountered PCR inhibitors. Calculations from ForenSeq™ STR and SNP repeatability and reproducibility studies (1ng template) indicate 100.0% accuracy of the MiSeq FGx™ System in allele calling relative to CE for STRs (1260 samples), and >99.1% accuracy relative to bead array typing for SNPs (1260 samples for iiSNPs, 310 samples for aiSNPs and piSNPs), with >99.0% and >97.8% precision, respectively. Call rates of >99.0% were observed for all STRs and SNPs amplified with both ForenSeq™ primer mixes. Limitations of the MiSeq FGx™ System are discussed. Results described here demonstrate that the MiSeq FGx™ System meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

The Use and Effectiveness of Triple Multiplex System for Coding Region Single Nucleotide Polymorphism in Mitochondrial DNA Typing of Archaeologically Obtained Human Skeletons from Premodern Joseon Tombs of Korea

PubMed Central

Oh, Chang Seok; Lee, Soong Deok; Kim, Yi-Suk; Shin, Dong Hoon

2015-01-01

Previous study showed that East Asian mtDNA haplogroups, especially those of Koreans, could be successfully assigned by the coupled use of analyses on coding region SNP markers and control region mutation motifs. In this study, we tried to see if the same triple multiplex analysis for coding regions SNPs could be also applicable to ancient samples from East Asia as the complementation for sequence analysis of mtDNA control region. By the study on Joseon skeleton samples, we know that mtDNA haplogroup determined by coding region SNP markers successfully falls within the same haplogroup that sequence analysis on control region can assign. Considering that ancient samples in previous studies make no small number of errors in control region mtDNA sequencing, coding region SNP analysis can be used as good complimentary to the conventional haplogroup determination, especially of archaeological human bone samples buried underground over long periods. PMID:26345190

Microfluidic Arrayed Lab-On-A-Chip for Electrochemical Capacitive Detection of DNA Hybridization Events.

PubMed

Ben-Yoav, Hadar; Dykstra, Peter H; Bentley, William E; Ghodssi, Reza

2017-01-01

A microfluidic electrochemical lab-on-a-chip (LOC) device for DNA hybridization detection has been developed. The device comprises a 3 × 3 array of microelectrodes integrated with a dual layer microfluidic valved manipulation system that provides controlled and automated capabilities for high throughput analysis of microliter volume samples. The surface of the microelectrodes is functionalized with single-stranded DNA (ssDNA) probes which enable specific detection of complementary ssDNA targets. These targets are detected by a capacitive technique which measures dielectric variation at the microelectrode-electrolyte interface due to DNA hybridization events. A quantitative analysis of the hybridization events is carried out based on a sensing modeling that includes detailed analysis of energy storage and dissipation components. By calculating these components during hybridization events the device is able to demonstrate specific and dose response sensing characteristics. The developed microfluidic LOC for DNA hybridization detection offers a technology for real-time and label-free assessment of genetic markers outside of laboratory settings, such as at the point-of-care or in-field environmental monitoring.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

PubMed

Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

2017-01-05

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of DNA interactions using single-molecule force spectroscopy.

PubMed

Ritzefeld, Markus; Walhorn, Volker; Anselmetti, Dario; Sewald, Norbert

2013-06-01

Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given.

Introducing W.A.T.E.R.S.: a workflow for the alignment, taxonomy, and ecology of ribosomal sequences.

PubMed

Hartman, Amber L; Riddle, Sean; McPhillips, Timothy; Ludäscher, Bertram; Eisen, Jonathan A

2010-06-12

For more than two decades microbiologists have used a highly conserved microbial gene as a phylogenetic marker for bacteria and archaea. The small-subunit ribosomal RNA gene, also known as 16 S rRNA, is encoded by ribosomal DNA, 16 S rDNA, and has provided a powerful comparative tool to microbial ecologists. Over time, the microbial ecology field has matured from small-scale studies in a select number of environments to massive collections of sequence data that are paired with dozens of corresponding collection variables. As the complexity of data and tool sets have grown, the need for flexible automation and maintenance of the core processes of 16 S rDNA sequence analysis has increased correspondingly. We present WATERS, an integrated approach for 16 S rDNA analysis that bundles a suite of publicly available 16 S rDNA analysis software tools into a single software package. The "toolkit" includes sequence alignment, chimera removal, OTU determination, taxonomy assignment, phylogentic tree construction as well as a host of ecological analysis and visualization tools. WATERS employs a flexible, collection-oriented 'workflow' approach using the open-source Kepler system as a platform. By packaging available software tools into a single automated workflow, WATERS simplifies 16 S rDNA analyses, especially for those without specialized bioinformatics, programming expertise. In addition, WATERS, like some of the newer comprehensive rRNA analysis tools, allows researchers to minimize the time dedicated to carrying out tedious informatics steps and to focus their attention instead on the biological interpretation of the results. One advantage of WATERS over other comprehensive tools is that the use of the Kepler workflow system facilitates result interpretation and reproducibility via a data provenance sub-system. Furthermore, new "actors" can be added to the workflow as desired and we see WATERS as an initial seed for a sizeable and growing repository of interoperable, easy-to-combine tools for asking increasingly complex microbial ecology questions.

RNA-seq Analysis Reveals Unique Transcriptome Signatures in Systemic Lupus Erythematosus Patients with Distinct Autoantibody Specificities

PubMed Central

Rai, Richa; Chauhan, Sudhir Kumar; Singh, Vikas Vikram; Rai, Madhukar; Rai, Geeta

2016-01-01

Systemic lupus erythematosus (SLE) patients exhibit immense heterogeneity which is challenging from the diagnostic perspective. Emerging high throughput sequencing technologies have been proved to be a useful platform to understand the complex and dynamic disease processes. SLE patients categorised based on autoantibody specificities are reported to have differential immuno-regulatory mechanisms. Therefore, we performed RNA-seq analysis to identify transcriptomics of SLE patients with distinguished autoantibody specificities. The SLE patients were segregated into three subsets based on the type of autoantibodies present in their sera (anti-dsDNA+ group with anti-dsDNA autoantibody alone; anti-ENA+ group having autoantibodies against extractable nuclear antigens (ENA) only, and anti-dsDNA+ENA+ group having autoantibodies to both dsDNA and ENA). Global transcriptome profiling for each SLE patients subsets was performed using Illumina® Hiseq-2000 platform. The biological relevance of dysregulated transcripts in each SLE subsets was assessed by ingenuity pathway analysis (IPA) software. We observed that dysregulation in the transcriptome expression pattern was clearly distinct in each SLE patients subsets. IPA analysis of transcripts uniquely expressed in different SLE groups revealed specific biological pathways to be affected in each SLE subsets. Multiple cytokine signaling pathways were specifically dysregulated in anti-dsDNA+ patients whereas Interferon signaling was predominantly dysregulated in anti-ENA+ patients. In anti-dsDNA+ENA+ patients regulation of actin based motility by Rho pathway was significantly affected. The granulocyte gene signature was a common feature to all SLE subsets; however, anti-dsDNA+ group showed relatively predominant expression of these genes. Dysregulation of Plasma cell related transcripts were higher in anti-dsDNA+ and anti-ENA+ patients as compared to anti-dsDNA+ ENA+. Association of specific canonical pathways with the uniquely expressed transcripts in each SLE subgroup indicates that specific immunological disease mechanisms are operative in distinct SLE patients’ subsets. This ‘sub-grouping’ approach could further be useful for clinical evaluation of SLE patients and devising targeted therapeutics. PMID:27835693

Development and validation of a multiplex reaction analyzing eight miniSTRs of the X chromosome for identity and kinship testing with degraded DNA.

PubMed

Castañeda, María; Odriozola, Adrián; Gómez, Javier; Zarrabeitia, María T

2013-07-01

We report the development of an effective system for analyzing X chromosome-linked mini short tandem repeat loci with reduced-size amplicons (less than 220 bp), useful for analyzing highly degraded DNA samples. To generate smaller amplicons, we redesigned primers for eight X-linked microsatellites (DXS7132, DXS10079, DXS10074, DXS10075, DXS6801, DXS6809, DXS6789, and DXS6799) and established efficient conditions for a multiplex PCR system (miniX). The validation tests confirmed that it has good sensitivity, requiring as little as 20 pg of DNA, and performs well with DNA from paraffin-embedded tissues, thus showing potential for improved analysis and identification of highly degraded and/or very limited DNA samples. Consequently, this system may help to solve complex forensic cases, particularly when autosomal markers convey insufficient information.

Automated high-throughput purification of genomic DNA from plant leaf or seed using MagneSil paramagnetic particles

NASA Astrophysics Data System (ADS)

Bitner, Rex M.; Koller, Susan C.

2004-06-01

Three different methods of automated high throughput purification of genomic DNA from plant materials processed in 96 well plates are described. One method uses MagneSil paramagnetic particles to purify DNA present in single leaf punch samples or small seed samples, using 320ul capacity 96 well plates which minimizes reagent and plate costs. A second method uses 2.2 ml and 1.2 ml capacity plates and allows the purification of larger amounts of DNA from 5-6 punches of materials or larger amounts of seeds. The third method uses the MagneSil ONE purification system to purify a fixed amount of DNA, thus simplifying the processing of downstream applications by normalizing the amounts of DNA so they do not require quantitation. Protocols for the purification of a fixed yield of DNA, e.g. 1 ug, from plant leaf or seed samples using MagneSil paramagnetic particles and a Beckman-Coulter BioMek FX robot are described. DNA from all three methods is suitable for applications such as PCR, RAPD, STR, READIT SNP analysis, and multiplexed PCR systems. The MagneSil ONE system is also suitable for use with SNP detection systems such as Third Wave Technology"s Invader methods.

Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

2014-11-01

A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

Digital DNA detection based on a compact optofluidic laser with ultra-low sample consumption.

PubMed

Lee, Wonsuk; Chen, Qiushu; Fan, Xudong; Yoon, Dong Ki

2016-11-29

DNA lasers self-amplify optical signals from a DNA analyte as well as thermodynamic differences between sequences, allowing quasi-digital DNA detection. However, these systems have drawbacks, such as relatively large sample consumption and complicated dye labelling. Moreover, although the lasing signal can detect the target DNA, it is superimposed on an unintended fluorescence background, which persists for non-target DNA samples as well. From an optical point of view, it is thus not truly digital detection and requires spectral analysis to identify the target. In this work, we propose and demonstrate an optofluidic laser that has a single layer of DNA molecules as the gain material. A target DNA produces intensive laser emission comparable to existing DNA lasers, while any unnecessary fluorescence background is successfully suppressed. As a result, the target DNA can be detected with a single laser pulse, in a truly digital manner. Since the DNA molecules cover only a single layer on the surface of the laser microcavity, the DNA sample consumption is a few orders of magnitude lower than that of existing DNA lasers. Furthermore, the DNA molecules are stained by simply immersing the microcavity in the intercalating dye solution, and thus the proposed DNA laser is free of any complex dye-labelling process prior to analysis.

Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

NASA Astrophysics Data System (ADS)

Bitner, Rex M.; Koller, Susan C.

2002-06-01

The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

A new model for ancient DNA decay based on paleogenomic meta-analysis.

PubMed

Kistler, Logan; Ware, Roselyn; Smith, Oliver; Collins, Matthew; Allaby, Robin G

2017-06-20

The persistence of DNA over archaeological and paleontological timescales in diverse environments has led to a revolutionary body of paleogenomic research, yet the dynamics of DNA degradation are still poorly understood. We analyzed 185 paleogenomic datasets and compared DNA survival with environmental variables and sample ages. We find cytosine deamination follows a conventional thermal age model, but we find no correlation between DNA fragmentation and sample age over the timespans analyzed, even when controlling for environmental variables. We propose a model for ancient DNA decay wherein fragmentation rapidly reaches a threshold, then subsequently slows. The observed loss of DNA over time may be due to a bulk diffusion process in many cases, highlighting the importance of tissues and environments creating effectively closed systems for DNA preservation. This model of DNA degradation is largely based on mammal bone samples due to published genomic dataset availability. Continued refinement to the model to reflect diverse biological systems and tissue types will further improve our understanding of ancient DNA breakdown dynamics. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

DNA confinement in nanochannels: physics and biological applications

NASA Astrophysics Data System (ADS)

Reisner, Walter; Pedersen, Jonas N.; Austin, Robert H.

2012-10-01

DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement—including the effect of varying ionic strength—and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

Mechanisms of Enhanced Catalysis in Enzyme-DNA Nanostructures Revealed through Molecular Simulations and Experimental Analysis.

PubMed

Gao, Yingning; Roberts, Christopher C; Toop, Aaron; Chang, Chia-En A; Wheeldon, Ian

2016-08-03

Understanding and controlling the molecular interactions between enzyme substrates and DNA nanostructures has important implications in the advancement of enzyme-DNA technologies as solutions in biocatalysis. Such hybrid nanostructures can be used to create enzyme systems with enhanced catalysis by controlling the local chemical and physical environments and the spatial organization of enzymes. Here we have used molecular simulations with corresponding experiments to describe a mechanism of enhanced catalysis due to locally increased substrate concentrations. With a series of DNA nanostructures conjugated to horseradish peroxidase, we show that binding interactions between substrates and the DNA structures can increase local substrate concentrations. Increased local substrate concentrations in HRP(DNA) nanostructures resulted in 2.9- and 2.4-fold decreases in the apparent Michaelis constants of tetramethylbenzidine and 4-aminophenol, substrates of HRP with tunable binding interactions to DNA nanostructures with dissociation constants in the micromolar range. Molecular simulations and kinetic analysis also revealed that increased local substrate concentrations enhanced the rates of substrate association. Identification of the mechanism of increased local concentration of substrates in close proximity to enzymes and their active sites adds to our understanding of nanostructured biocatalysis from which we can develop guidelines for enhancing catalysis in rationally designed systems. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA confinement in nanochannels: physics and biological applications.

PubMed

Reisner, Walter; Pedersen, Jonas N; Austin, Robert H

2012-10-01

DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement--including the effect of varying ionic strength--and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

Systematic prediction of control proteins and their DNA binding sites

PubMed Central

Sorokin, Valeriy; Severinov, Konstantin; Gelfand, Mikhail S.

2009-01-01

We present here the results of a systematic bioinformatics analysis of control (C) proteins, a class of DNA-binding regulators that control time-delayed transcription of their own genes as well as restriction endonuclease genes in many type II restriction-modification systems. More than 290 C protein homologs were identified and DNA-binding sites for ∼70% of new and previously known C proteins were predicted by a combination of phylogenetic footprinting and motif searches in DNA upstream of C protein genes. Additional analysis revealed that a large proportion of C protein genes are translated from leaderless RNA, which may contribute to time-delayed nature of genetic switches operated by these proteins. Analysis of genetic contexts of newly identified C protein genes revealed that they are not exclusively associated with restriction-modification genes; numerous instances of associations with genes originating from mobile genetic elements were observed. These instances might be vestiges of ancient horizontal transfers and indicate that during evolution ancestral restriction-modification system genes were the sites of mobile elements insertions. PMID:19056824

The development of miniplex primer sets for the analysis of degraded DNA

NASA Astrophysics Data System (ADS)

McCord, Bruce; Opel, Kerry; Chung, Denise; Drabek, Jiri; Tatarek, Nancy; Meadows Jantz, Lee; Butler, John

2005-05-01

In this project, a new set of multiplexed PCR reactions has been developed for the analysis of degraded DNA. These DNA markers, known as Miniplexes, utilize primers that have shorter amplicons for use in short tandem repeat (STR) analysis of degraded DNA. In our work we have defined six of these new STR multiplexes, each of which consists of 3 to 4 reduced size STR loci, and each labeled with a different fluorescent dye. When compared to commercially available STR systems, reductions in size of up to 300 basepairs are possible. In addition, these newly designed amplicons consist of loci that are fully compatible with the the national computer DNA database known as CODIS. To demonstrate compatibility with commercial STR kits, a concordance study of 532 DNA samples of Caucasian, African American, and Hispanic origin was undertaken There was 99.77% concordance between allele calls with the two methods. Of these 532 samples, only 15 samples showed discrepancies at one of 12 loci. These occurred predominantly at 2 loci, vWA and D13S317. DNA sequencing revealed that these locations had deletions between the two primer binding sites. Uncommon deletions like these can be expected in certain samples and will not affect the utility of the Miniplexes as tools for degraded DNA analysis. The Miniplexes were also applied to enzymatically digested DNA to assess their potential in degraded DNA analysis. The results demonstrated a greatly improved efficiency in the analysis of degraded DNA when compared to commercial STR genotyping kits. A series of human skeletal remains that had been exposed to a variety of environmental conditions were also examined. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only sixteen percent of the samples tested generated full profiles with a commercial kit. In addition, complete profiles were obtained for eleven of the twelve Miniplex loci which had amplicon size ranges less than 200 base pairs. These data clearly demonstrate that smaller PCR amplicons provide an attractive alternative to mitochondrial DNA for forensic analysis of degraded DNA.

New applications of CRISPR/Cas9 system on mutant DNA detection.

PubMed

Jia, Chenqiang; Huai, Cong; Ding, Jiaqi; Hu, Lingna; Su, Bo; Chen, Hongyan; Lu, Daru

2018-01-30

The detection of mutant DNA is critical for precision medicine, but low-frequency DNA mutation is very hard to be determined. CRISPR/Cas9 is a robust tool for in vivo gene editing, and shows the potential for precise in vitro DNA cleavage. Here we developed a DNA mutation detection system based on CRISPR/Cas9 that can detect gene mutation efficiently even in a low-frequency condition. The system of CRISPR/Cas9 cleavage in vitro showed a high accuracy similar to traditional T7 endonuclease I (T7E1) assay in estimating mutant DNA proportion in the condition of normal frequency. The technology was further used for low-frequency mutant DNA detection of EGFR and HBB somatic mutations. To the end, Cas9 was employed to cleave the wild-type (WT) DNA and to enrich the mutant DNA. Using amplified fragment length polymorphism analysis (AFLPA) and Sanger sequencing, we assessed the sensitivity of CRISPR/Cas9 cleavage-based PCR, in which mutations at 1%-10% could be enriched and detected. When combined with blocker PCR, its sensitivity reached up to 0.1%. Our results suggested that this new application of CRISPR/Cas9 system is a robust and potential method for heterogeneous specimens in the clinical diagnosis and treatment management. Copyright © 2017 Elsevier B.V. All rights reserved.

High-density fiber-optic DNA random microsphere array.

PubMed

Ferguson, J A; Steemers, F J; Walt, D R

2000-11-15

A high-density fiber-optic DNA microarray sensor was developed to monitor multiple DNA sequences in parallel. Microarrays were prepared by randomly distributing DNA probe-functionalized 3.1-microm-diameter microspheres in an array of wells etched in a 500-microm-diameter optical imaging fiber. Registration of the microspheres was performed using an optical encoding scheme and a custom-built imaging system. Hybridization was visualized using fluorescent-labeled DNA targets with a detection limit of 10 fM. Hybridization times of seconds are required for nanomolar target concentrations, and analysis is performed in minutes.

Clinical Applications of Circulating Tumor Cells and Circulating Tumor DNA as Liquid Biopsy.

PubMed

Alix-Panabières, Catherine; Pantel, Klaus

2016-05-01

"Liquid biopsy" focusing on the analysis of circulating tumor cells (CTC) and circulating cell-free tumor DNA (ctDNA) in the blood of patients with cancer has received enormous attention because of its obvious clinical implications for personalized medicine. Analyses of CTCs and ctDNA have paved new diagnostic avenues and are, to date, the cornerstones of liquid biopsy diagnostics. The present review focuses on key areas of clinical applications of CTCs and ctDNA, including detection of cancer, prediction of prognosis in patients with curable disease, monitoring systemic therapies, and stratification of patients based on the detection of therapeutic targets or resistance mechanisms. The application of CTCs and ctDNA for the early detection of cancer is of high public interest, but it faces serious challenges regarding specificity and sensitivity of the current assays. Prediction of prognosis in patients with curable disease can already be achieved in several tumor entities, particularly in breast cancer. Monitoring the success or failure of systemic therapies (i.e., chemotherapy, hormonal therapy, or other targeted therapies) by sequential measurements of CTCs or ctDNA is also feasible. Interventional studies on treatment stratification based on the analysis of CTCs and ctDNA are needed to implement liquid biopsy into personalized medicine. Cancer Discov; 6(5); 479-91. ©2016 AACR. ©2016 American Association for Cancer Research.

DNA

ERIC Educational Resources Information Center

Stent, Gunther S.

1970-01-01

This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

Comparison of methods of DNA extraction for real-time PCR in a model of pleural tuberculosis.

PubMed

Santos, Ana; Cremades, Rosa; Rodríguez, Juan Carlos; García-Pachón, Eduardo; Ruiz, Montserrat; Royo, Gloria

2010-01-01

Molecular methods have been reported to have different sensitivities in the diagnosis of pleural tuberculosis and this may in part be caused by the use of different methods of DNA extraction. Our study compares nine DNA extraction systems in an experimental model of pleural tuberculosis. An inoculum of Mycobacterium tuberculosis was added to 23 pleural liquid samples with different characteristics. DNA was subsequently extracted using nine different methods (seven manual and two automatic) for analysis with real-time PCR. Only two methods were able to detect the presence of M. tuberculosis DNA in all the samples: extraction using columns (Qiagen) and automated extraction with the TNAI system (Roche). The automatic method is more expensive, but requires less time. Almost all the false negatives were because of the difficulty involved in extracting M. tuberculosis DNA, as in general, all the methods studied are capable of eliminating inhibitory substances that block the amplification reaction. The method of M. tuberculosis DNA extraction used affects the results of the diagnosis of pleural tuberculosis by molecular methods. DNA extraction systems that have been shown to be effective in pleural liquid should be used.

Using DNA-labelled nano- and microparticles to track particle transport in the environment

NASA Astrophysics Data System (ADS)

McNew, Coy; Wang, Chaozi; Dahlke, Helen; Lyon, Steve; Walter, Todd

2017-04-01

By utilizing bio-molecular nanotechnology developed for nano-medicines and drug delivery, we are able to produce DNA-labelled nano- and microparticle tracers for use in a myriad of environmental systems. The use of custom sequenced DNA allows for the fabrication of an enormous number of uniquely labelled tracers with identical transport properties (approximately 1.61 x 1060 unique sequences), each independently quantifiable, that can be applied simultaneously in any hydrologic system. By controlling the fabrication procedure to produce particles of custom size and charge, we are able to tag each size-charge combination uniquely in order to directly probe the effect of these variables on the transport properties of the particles. Here we present our methods for fabrication, extraction, and analysis of the DNA nano- and microparticle tracers, along with results from several successful applications of the tracers, including transport and retention analysis at the lab, continuum, and field scales. To date, our DNA-labelled nano- and microparticle tracers have proved useful in surface and subsurface water applications, soil retention, and even subglacial flow pathways. The range of potential applications continue to prove nearly limitless.

Post-conviction DNA testing: the UK's first ‘exoneration’ case?

PubMed Central

Johnson, Paul; Williams, Robin

2005-01-01

The routine incorporation of forensic DNA profiling into the criminal justice systems of the United Kingdom has been widely promoted as a device for improving the quality of investigative and prosecutorial processes. From its first uses in the 1980s, in cases of serious crime, to the now daily collection, analysis and comparison of genetic samples in the National DNA Database, DNA profiling has become a standard instrument of policing and a powerful evidential resource for prosecutors. However, the use of post-conviction DNA testing has, until recently, been uncommon in the United Kingdom. This paper explores the first case, in England, of the contribution of DNA profiling to a successful appeal against conviction by an imprisoned offender. Analysis of the details of this case is used to emphasise the ways in which novel forms of scientific evidence remain subject to traditional and heterogeneous tests of relevance and credibility. PMID:15112595

Internal validation of the DNAscan/ANDE™ Rapid DNA Analysis™ platform and its associated PowerPlex® 16 high content DNA biochip cassette for use as an expert system with reference buccal swabs.

PubMed

Moreno, Lilliana I; Brown, Alice L; Callaghan, Thomas F

2017-07-01

Rapid DNA platforms are fully integrated systems capable of producing and analyzing short tandem repeat (STR) profiles from reference sample buccal swabs in less than two hours. The technology requires minimal user interaction and experience making it possible for high quality profiles to be generated outside an accredited laboratory. The automated production of point of collection reference STR profiles could eliminate the time delay for shipment and analysis of arrestee samples at centralized laboratories. Furthermore, point of collection analysis would allow searching against profiles from unsolved crimes during the normal booking process once the infrastructure to immediately search the Combined DNA Index System (CODIS) database from the booking station is established. The DNAscan/ANDE™ Rapid DNA Analysis™ System developed by Network Biosystems was evaluated for robustness and reliability in the production of high quality reference STR profiles for database enrollment and searching applications. A total of 193 reference samples were assessed for concordance of the CODIS 13 loci. Studies to evaluate contamination, reproducibility, precision, stutter, peak height ratio, noise and sensitivity were also performed. The system proved to be robust, consistent and dependable. Results indicated an overall success rate of 75% for the 13 CODIS core loci and more importantly no incorrect calls were identified. The DNAscan/ANDE™ could be confidently used without human interaction in both laboratory and non-laboratory settings to generate reference profiles. Published by Elsevier B.V.

Design and analysis of linear cascade DNA hybridization chain reactions using DNA hairpins

NASA Astrophysics Data System (ADS)

Bui, Hieu; Garg, Sudhanshu; Miao, Vincent; Song, Tianqi; Mokhtar, Reem; Reif, John

2017-01-01

DNA self-assembly has been employed non-conventionally to construct nanoscale structures and dynamic nanoscale machines. The technique of hybridization chain reactions by triggered self-assembly has been shown to form various interesting nanoscale structures ranging from simple linear DNA oligomers to dendritic DNA structures. Inspired by earlier triggered self-assembly works, we present a system for controlled self-assembly of linear cascade DNA hybridization chain reactions using nine distinct DNA hairpins. NUPACK is employed to assist in designing DNA sequences and Matlab has been used to simulate DNA hairpin interactions. Gel electrophoresis and ensemble fluorescence reaction kinetics data indicate strong evidence of linear cascade DNA hybridization chain reactions. The half-time completion of the proposed linear cascade reactions indicates a linear dependency on the number of hairpins.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

PubMed Central

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-01-01

Abstract Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

Alignment of high-throughput sequencing data inside in-memory databases.

PubMed

Firnkorn, Daniel; Knaup-Gregori, Petra; Lorenzo Bermejo, Justo; Ganzinger, Matthias

2014-01-01

In times of high-throughput DNA sequencing techniques, performance-capable analysis of DNA sequences is of high importance. Computer supported DNA analysis is still an intensive time-consuming task. In this paper we explore the potential of a new In-Memory database technology by using SAP's High Performance Analytic Appliance (HANA). We focus on read alignment as one of the first steps in DNA sequence analysis. In particular, we examined the widely used Burrows-Wheeler Aligner (BWA) and implemented stored procedures in both, HANA and the free database system MySQL, to compare execution time and memory management. To ensure that the results are comparable, MySQL has been running in memory as well, utilizing its integrated memory engine for database table creation. We implemented stored procedures, containing exact and inexact searching of DNA reads within the reference genome GRCh37. Due to technical restrictions in SAP HANA concerning recursion, the inexact matching problem could not be implemented on this platform. Hence, performance analysis between HANA and MySQL was made by comparing the execution time of the exact search procedures. Here, HANA was approximately 27 times faster than MySQL which means, that there is a high potential within the new In-Memory concepts, leading to further developments of DNA analysis procedures in the future.

Nanoscale Bio-engineering Solutions for Space Exploration: The Nanopore Sequencer

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Cozmuta, Ioana

2004-01-01

Characterization of biological systems at the molecular level and extraction of essential information for nano-engineering design to guide the nano-fabrication of solid-state sensors and molecular identification devices is a computational challenge. The alpha hemolysin protein ion channel is used as a model system for structural analysis of nucleic acids like DNA. Applied voltage draws a DNA strand and surrounding ionic solution through the biological nanopore. The subunits in the DNA strand block ion flow by differing amounts. Atomistic scale simulations are employed using NASA supercomputers to study DNA translocation, with the aim to enhance single DNA subunit identification. Compared to protein channels, solid-state nanopores offer a better temporal control of the translocation of DNA and the possibility to easily tune its chemistry to increase the signal resolution. Potential applications for NASA missions, besides real-time genome sequencing include astronaut health, life detection and decoding of various genomes.

Nanoscale Bioengineering Solutions for Space Exploration the Nanopore Sequencer

NASA Technical Reports Server (NTRS)

Ioana, Cozmuta; Viktor, Stoic

2005-01-01

Characterization of biological systems at the molecular level and extraction of essential information for nano-engineering design to guide the nano-fabrication of solid-state sensors and molecular identification devices is a computational challenge. The alpha hemolysin protein ion channel is used as a model system for structural analysis of nucleic acids like DNA. Applied voltage draws a DNA strand and surrounding ionic solution through the biological nanopore. The subunits in the DNA strand block ion flow by differing amounts. Atomistic scale simulations are employed using NASA supercomputers to study DNA translocation. with the aim to enhance single DNA subunit identification. Compared to protein channels, solid-state nanopores offer a better temporal control of the translocation of DNA and the possibility to easily tune its chemistry to increase the signal resolution. Potential applications for NASA missions, besides real-time genome sequencing include astronaut health, life detection and decoding of various genomes. http://phenomrph.arc.nasa.gov/index.php

Forensic genetic analysis of bone remain samples.

PubMed

Siriboonpiputtana, T; Rinthachai, T; Shotivaranon, J; Peonim, V; Rerkamnuaychoke, B

2018-03-01

DNA typing from degraded human remains is still challenging forensic DNA scientists not only in the prospective of DNA purification but also in the interpretation of established DNA profiles and data manipulation, especially in mass fatalities. In this report, we presented DNA typing protocol to investigate many skeletal remains in different degrees of decomposing. In addition, we established the grading system aiming for prior determination of the association between levels of decomposing and overall STR amplification efficacy. A total of 80 bone samples were subjected to DNA isolation using the modified DNA IQ™ System (Promega, USA) for bone extraction following with STR analysis using the AmpFLSTR Identifiler ® (Thermo Fisher Scientific, USA). In low destruction group, complete STR profiles were observed as 84.4% whereas partial profiles and non-amplified were found as 9.4% and 6.2%, respectively. Moreover, in medium destruction group, both complete and partial STR profiles were observed as 31.2% while 37.5% of this group was unable to amplify. Nevertheless, we could not purify DNA and were unable to generate STR profile in any sample from the high destroyed bone samples. Compact bones such as femur and humerus have high successful amplification rate superior than loose/spongy bones. Furthermore, costal cartilage could be a designate specimen for DNA isolation in a case of the body that was discovered approximately to 3 days after death which enabled to isolate high quality and quantity of DNA, reduce time and cost, and do not require special tools such as freezer mill. Copyright © 2018 Elsevier B.V. All rights reserved.

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs

PubMed Central

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10−13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs. PMID:27355212

Multiplex APLP System for High-Resolution Haplogrouping of Extremely Degraded East-Asian Mitochondrial DNAs.

PubMed

Kakuda, Tsuneo; Shojo, Hideki; Tanaka, Mayumi; Nambiar, Phrabhakaran; Minaguchi, Kiyoshi; Umetsu, Kazuo; Adachi, Noboru

2016-01-01

Mitochondrial DNA (mtDNA) serves as a powerful tool for exploring matrilineal phylogeographic ancestry, as well as for analyzing highly degraded samples, because of its polymorphic nature and high copy numbers per cell. The recent advent of complete mitochondrial genome sequencing has led to improved techniques for phylogenetic analyses based on mtDNA, and many multiplex genotyping methods have been developed for the hierarchical analysis of phylogenetically important mutations. However, few high-resolution multiplex genotyping systems for analyzing East-Asian mtDNA can be applied to extremely degraded samples. Here, we present a multiplex system for analyzing mitochondrial single nucleotide polymorphisms (mtSNPs), which relies on a novel amplified product-length polymorphisms (APLP) method that uses inosine-flapped primers and is specifically designed for the detailed haplogrouping of extremely degraded East-Asian mtDNAs. We used fourteen 6-plex polymerase chain reactions (PCRs) and subsequent electrophoresis to examine 81 haplogroup-defining SNPs and 3 insertion/deletion sites, and we were able to securely assign the studied mtDNAs to relevant haplogroups. Our system requires only 1×10-13 g (100 fg) of crude DNA to obtain a full profile. Owing to its small amplicon size (<110 bp), this new APLP system was successfully applied to extremely degraded samples for which direct sequencing of hypervariable segments using mini-primer sets was unsuccessful, and proved to be more robust than conventional APLP analysis. Thus, our new APLP system is effective for retrieving reliable data from extremely degraded East-Asian mtDNAs.

Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

PubMed

Grasso, Chiara; Trevisan, Morena; Fiano, Valentina; Tarallo, Valentina; De Marco, Laura; Sacerdote, Carlotta; Richiardi, Lorenzo; Merletti, Franco; Gillio-Tos, Anna

2016-01-01

Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

NASA Astrophysics Data System (ADS)

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-09-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology.

A Thiazole Coumarin (TC) Turn-On Fluorescence Probe for AT-Base Pair Detection and Multipurpose Applications in Different Biological Systems

PubMed Central

Narayanaswamy, Nagarjun; Kumar, Manoj; Das, Sadhan; Sharma, Rahul; Samanta, Pralok K.; Pati, Swapan K.; Dhar, Suman K.; Kundu, Tapas K.; Govindaraju, T.

2014-01-01

Sequence-specific recognition of DNA by small turn-on fluorescence probes is a promising tool for bioimaging, bioanalytical and biomedical applications. Here, the authors report a novel cell-permeable and red fluorescent hemicyanine-based thiazole coumarin (TC) probe for DNA recognition, nuclear staining and cell cycle analysis. TC exhibited strong fluorescence enhancement in the presence of DNA containing AT-base pairs, but did not fluoresce with GC sequences, single-stranded DNA, RNA and proteins. The fluorescence staining of HeLa S3 and HEK 293 cells by TC followed by DNase and RNase digestion studies depicted the selective staining of DNA in the nucleus over the cytoplasmic region. Fluorescence-activated cell sorting (FACS) analysis by flow cytometry demonstrated the potential application of TC in cell cycle analysis in HEK 293 cells. Metaphase chromosome and malaria parasite DNA imaging studies further confirmed the in vivo diagnostic and therapeutic applications of probe TC. Probe TC may find multiple applications in fluorescence spectroscopy, diagnostics, bioimaging and molecular and cell biology. PMID:25252596

Multiple-Locus VNTR Analysis (MLVA) for Bacterial Strain Identification - Quarterly Progress Report for the period 7/1/00 to 10/30/00

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

Dr. Paul Keim

2000-11-07

Multiple locus VNTR analysis (MLVA) systems are being developed for B. anthracis, Y. pestis and F. tularensis. These are high resolution DNA fingerprinting systems that will allow for molecular epidemiology and forensic analysis of these pathogens.

Multiple-Locus VNTR Analysis (MLVA) for Bacterial Strain Identification - Quarterly Progress Report for the Period 4/1/00 to 6/30/00

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

Dr. Paul Keim

2000-11-07

Multiple locus VNTR analysis (MLVA) systems are being developed for B. anthracis, Y. pestis and F. tularensis. These are high resolution DNA fingerprinting systems that will allow for molecular epidemiology and forensic analysis of these pathogens.

DNA accumulation on ventilation system filters in university buildings in Singapore

PubMed Central

Luhung, Irvan; Wu, Yan; Xu, Siyu; Yamamoto, Naomichi; Nazaroff, William W.

2017-01-01

Introduction Biological particles deposit on air handling system filters as they process air. This study reports and interprets abundance and diversity information regarding biomass accumulation on ordinarily used filters acquired from several locations in a university environment. Methods DNA-based analysis was applied both to quantify (via DNA fluorometry and qPCR) and to characterize (via high-throughput sequencing) the microbial material on filters, which mainly processed recirculated indoor air. Results were interpreted in relation to building occupancy and ventilation system operational parameters. Results Based on accumulated biomass, average DNA concentrations per AHU filter surface area across nine indoor locations after twelve weeks of filter use were in the respective ranges 1.1 to 41 ng per cm2 for total DNA, 0.02 to 3.3 ng per cm2 for bacterial DNA and 0.2 to 2.0 ng DNA per cm2 for fungal DNA. The most abundant genera detected on the AHU filter samples were Clostridium, Streptophyta, Bacillus, Acinetobacter and Ktedonobacter for bacteria and Aspergillus, Cladosporium, Nigrospora, Rigidoporus and Lentinus for fungi. Conditional indoor airborne DNA concentrations (median (range)) were estimated to be 13 (2.6–107) pg/m3 for total DNA, 0.4 (0.05–8.4) pg/m3 for bacterial DNA and 2.3 (1.0–5.1) pg/m3 for fungal DNA. Conclusion Conditional airborne concentrations and the relative abundances of selected groups of genera correlate well with occupancy level. Bacterial DNA was found to be more responsive than fungal DNA to differences in occupancy level and indoor environmental conditions. PMID:29023520

Systemic Lupus Erythematosus with and without Anti-dsDNA Antibodies: Analysis from a Large Monocentric Cohort.

PubMed

Conti, Fabrizio; Ceccarelli, Fulvia; Perricone, Carlo; Massaro, Laura; Marocchi, Elisa; Miranda, Francesca; Spinelli, Francesca Romana; Truglia, Simona; Alessandri, Cristiano; Valesini, Guido

2015-01-01

The anti-dsDNA antibodies are a marker for Systemic Lupus Erythematosus (SLE) and 70-98% of patients test positive. We evaluated the demographic, clinical, laboratory, and therapeutical features of a monocentric SLE cohort according to the anti-dsDNA status. We identified three groups: anti-dsDNA + (persistent positivity); anti-dsDNA ± (initial positivity and subsequent negativity during disease course); anti-dsDNA - (persistent negativity). Disease activity was assessed by the European Consensus Lupus Activity Measurement (ECLAM). We evaluated 393 patients (anti-dsDNA +: 62.3%; anti-dsDNA ±: 13.3%; anti-dsDNA -: 24.4%). The renal involvement was significantly more frequent in anti-dsDNA + (30.2%), compared with anti-dsDNA ± and anti-dsDNA - (21.1% and 18.7%, resp.; P = 0.001). Serositis resulted significantly more frequent in anti-dsDNA - (82.3%) compared to anti-dsDNA + and anti-dsDNA ± (20.8% and 13.4%, resp.; P < 0.0001). The reduction of C4 serum levels was identified significantly more frequently in anti-dsDNA + and anti-dsDNA ± (40.0% and 44.2%, resp.) compared with anti-dsDNA - (21.8%, P = 0.005). We did not identify significant differences in the mean ECLAM values before and after modification of anti-dsDNA status (P = 0.7). Anti-dsDNA status influences the clinical and immunological features of SLE patients. Nonetheless, it does not appear to affect disease activity.

Validation of a rapid DNA process with the RapidHIT® ID system using GlobalFiler® Express chemistry, a platform optimized for decentralized testing environments.

PubMed

Salceda, Susana; Barican, Arnaldo; Buscaino, Jacklyn; Goldman, Bruce; Klevenberg, Jim; Kuhn, Melissa; Lehto, Dennis; Lin, Frank; Nguyen, Phong; Park, Charles; Pearson, Francesca; Pittaro, Rick; Salodkar, Sayali; Schueren, Robert; Smith, Corey; Troup, Charles; Tsou, Dean; Vangbo, Mattias; Wunderle, Justus; King, David

2017-05-01

The RapidHIT ® ID is a fully automated sample-to-answer system for short tandem repeat (STR)-based human identification. The RapidHIT ID has been optimized for use in decentralized environments and processes presumed single source DNA samples, generating Combined DNA Index System (CODIS)-compatible DNA profiles in less than 90min. The system is easy to use, requiring less than one minute of hands-on time. Profiles are reviewed using centralized linking software, RapidLINK™ (IntegenX, Pleasanton, CA), a software tool designed to collate DNA profiles from single or multiple RapidHIT ID systems at different geographic locations. The RapidHIT ID has been designed to employ GlobalFiler ® Express and AmpFLSTR ® NGMSElect™, Thermo Fisher Scientific (Waltham, MA) STR chemistries. The Developmental Validation studies were performed using GlobalFiler ® Express with single source reference samples according to Scientific Working Group for DNA Analysis Methods guidelines. These results show that multiple RapidHIT ID systems networked with RapidLINK software form a highly reliable system for wide-scale deployment in locations such as police booking stations and border crossings enabling real-time testing of arrestees, potential human trafficking victims, and other instances where rapid turnaround is essential. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

[Multilocus genotyping of polymorphous STR-loci of chromosomal DNA in individual cells: technical difficulties].

PubMed

Ivanov, P L; Leonov, S N; Zemskova, E Iu; Kobylianskiĭ, A G; Dziubenko, E V

2013-01-01

This study was designed to estimate the effectiveness of special technical procedures for the enhancement of sensitivity of multiplex analysis of DNA, such as the use of low-plexity PCR systems and the whole genome preamplification technology, and the possibility of their application for the purpose of forensic medical genotyping of polymorphous STR-loci of chromosomal DNA in individual cells. The authors refused to use the imitation model (equivalent DNA dilutions) for the sake of obtaining the maximally informative data and chose to work with real preparations of solitary buccal epithelial cells isolated by the laser microdissection technique. It was shown that neither the use of the low-plexity multilocus PCR systems nor the whole genome pre-amplification technology makes possible reliable genotyping of STR-loci of chromosomal DNA in individual cells. The proposed techniques allow for DNA genotyping in preparations consisting of 10 diploid cells whereas the methods for reliable genotyping of STR-loci of chromosomal DNA in individual cells remains to be developed.

Systemically administered gp100 encoding DNA vaccine for melanoma using water-in-oil-in-water multiple emulsion delivery systems.

PubMed

Kalariya, Mayurkumar; Amiji, Mansoor M

2013-09-10

The purpose of this study was to develop a water-in-oil-in-water (W/O/W) multiple emulsions-based vaccine delivery system for plasmid DNA encoding the gp100 peptide antigen for melanoma immunotherapy. The gp100 encoding plasmid DNA was encapsulated in the inner-most aqueous phase of squalane oil containing W/O/W multiple emulsions using a two-step emulsification method. In vitro transfection ability of the encapsulated plasmid DNA was investigated in murine dendritic cells by transgene expression analysis using fluorescence microscopy and ELISA methods. Prophylactic immunization using the W/O/W multiple emulsions encapsulated the gp100 encoding plasmid DNA vaccine significantly reduced tumor volume in C57BL/6 mice during subsequent B16-F10 tumor challenge. In addition, serum Th1 cytokine levels and immuno-histochemistry of excised tumor tissues indicated activation of cytotoxic T-lymphocytes mediated anti-tumor immunity causing tumor growth suppression. The W/O/W multiple emulsions-based vaccine delivery system efficiently delivers the gp100 plasmid DNA to induce cell-mediated anti-tumor immunity. Copyright © 2013 Elsevier B.V. All rights reserved.

Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

PubMed

Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

2016-02-01

A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Intricate and Cell Type-Specific Populations of Endogenous Circular DNA (eccDNA) in Caenorhabditis elegans and Homo sapiens.

PubMed

Shoura, Massa J; Gabdank, Idan; Hansen, Loren; Merker, Jason; Gotlib, Jason; Levene, Stephen D; Fire, Andrew Z

2017-10-05

Investigations aimed at defining the 3D configuration of eukaryotic chromosomes have consistently encountered an endogenous population of chromosome-derived circular genomic DNA, referred to as extrachromosomal circular DNA (eccDNA). While the production, distribution, and activities of eccDNAs remain understudied, eccDNA formation from specific regions of the linear genome has profound consequences on the regulatory and coding capabilities for these regions. Here, we define eccDNA distributions in Caenorhabditis elegans and in three human cell types, utilizing a set of DNA topology-dependent approaches for enrichment and characterization. The use of parallel biophysical, enzymatic, and informatic approaches provides a comprehensive profiling of eccDNA robust to isolation and analysis methodology. Results in human and nematode systems provide quantitative analysis of the eccDNA loci at both unique and repetitive regions. Our studies converge on and support a consistent picture, in which endogenous genomic DNA circles are present in normal physiological states, and in which the circles come from both coding and noncoding genomic regions. Prominent among the coding regions generating DNA circles are several genes known to produce a diversity of protein isoforms, with mucin proteins and titin as specific examples. Copyright © 2017 Shoura et al.

Quantum entanglement and quantum information in biological systems (DNA)

NASA Astrophysics Data System (ADS)

Hubač, Ivan; Švec, Miloslav; Wilson, Stephen

2017-12-01

Recent studies of DNA show that the hydrogen bonds between given base pairs can be treated as diabatic systems with spin-orbit coupling. For solid state systems strong diabaticity and spin-orbit coupling the possibility of forming Majorana fermions has been discussed. We analyze the hydrogen bonds in the base pairs in DNA from this perspective. Our analysis is based on a quasiparticle supersymmetric transformation which couples electronic and vibrational motion and includes normal coordinates and the corresponding momenta. We define qubits formed by Majorana fermions in the hydrogen bonds and also discuss the entangled states in base pairs. Quantum information and quantum entropy are introduced. In addition to the well-known classical information connected with the DNA base pairs, we also consider quantum information and show that the classical and quantum information are closely connected.

HLA genotyping by next-generation sequencing of complementary DNA.

PubMed

Segawa, Hidenobu; Kukita, Yoji; Kato, Kikuya

2017-11-28

Genotyping of the human leucocyte antigen (HLA) is indispensable for various medical treatments. However, unambiguous genotyping is technically challenging due to high polymorphism of the corresponding genomic region. Next-generation sequencing is changing the landscape of genotyping. In addition to high throughput of data, its additional advantage is that DNA templates are derived from single molecules, which is a strong merit for the phasing problem. Although most currently developed technologies use genomic DNA, use of cDNA could enable genotyping with reduced costs in data production and analysis. We thus developed an HLA genotyping system based on next-generation sequencing of cDNA. Each HLA gene was divided into 3 or 4 target regions subjected to PCR amplification and subsequent sequencing with Ion Torrent PGM. The sequence data were then subjected to an automated analysis. The principle of the analysis was to construct candidate sequences generated from all possible combinations of variable bases and arrange them in decreasing order of the number of reads. Upon collecting candidate sequences from all target regions, 2 haplotypes were usually assigned. Cases not assigned 2 haplotypes were forwarded to 4 additional processes: selection of candidate sequences applying more stringent criteria, removal of artificial haplotypes, selection of candidate sequences with a relaxed threshold for sequence matching, and countermeasure for incomplete sequences in the HLA database. The genotyping system was evaluated using 30 samples; the overall accuracy was 97.0% at the field 3 level and 98.3% at the G group level. With one sample, genotyping of DPB1 was not completed due to short read size. We then developed a method for complete sequencing of individual molecules of the DPB1 gene, using the molecular barcode technology. The performance of the automatic genotyping system was comparable to that of systems developed in previous studies. Thus, next-generation sequencing of cDNA is a viable option for HLA genotyping.

BioBarcode: a general DNA barcoding database and server platform for Asian biodiversity resources.

PubMed

Lim, Jeongheui; Kim, Sang-Yoon; Kim, Sungmin; Eo, Hae-Seok; Kim, Chang-Bae; Paek, Woon Kee; Kim, Won; Bhak, Jong

2009-12-03

DNA barcoding provides a rapid, accurate, and standardized method for species-level identification using short DNA sequences. Such a standardized identification method is useful for mapping all the species on Earth, particularly when DNA sequencing technology is cheaply available. There are many nations in Asia with many biodiversity resources that need to be mapped and registered in databases. We have built a general DNA barcode data processing system, BioBarcode, with open source software - which is a general purpose database and server. It uses mySQL RDBMS 5.0, BLAST2, and Apache httpd server. An exemplary database of BioBarcode has around 11,300 specimen entries (including GenBank data) and registers the biological species to map their genetic relationships. The BioBarcode database contains a chromatogram viewer which improves the performance in DNA sequence analyses. Asia has a very high degree of biodiversity and the BioBarcode database server system aims to provide an efficient bioinformatics protocol that can be freely used by Asian researchers and research organizations interested in DNA barcoding. The BioBarcode promotes the rapid acquisition of biological species DNA sequence data that meet global standards by providing specialized services, and provides useful tools that will make barcoding cheaper and faster in the biodiversity community such as standardization, depository, management, and analysis of DNA barcode data. The system can be downloaded upon request, and an exemplary server has been constructed with which to build an Asian biodiversity system http://www.asianbarcode.org.

Quantitative fluorescence correlation spectroscopy on DNA in living cells

NASA Astrophysics Data System (ADS)

Hodges, Cameron; Kafle, Rudra P.; Meiners, Jens-Christian

2017-02-01

FCS is a fluorescence technique conventionally used to study the kinetics of fluorescent molecules in a dilute solution. Being a non-invasive technique, it is now drawing increasing interest for the study of more complex systems like the dynamics of DNA or proteins in living cells. Unlike an ordinary dye solution, the dynamics of macromolecules like proteins or entangled DNA in crowded environments is often slow and subdiffusive in nature. This in turn leads to longer residence times of the attached fluorophores in the excitation volume of the microscope and artifacts from photobleaching abound that can easily obscure the signature of the molecular dynamics of interest and make quantitative analysis challenging.We discuss methods and procedures to make FCS applicable to quantitative studies of the dynamics of DNA in live prokaryotic and eukaryotic cells. The intensity autocorrelation is computed function from weighted arrival times of the photons on the detector that maximizes the information content while simultaneously correcting for the effect of photobleaching to yield an autocorrelation function that reflects only the underlying dynamics of the sample. This autocorrelation function in turn is used to calculate the mean square displacement of the fluorophores attached to DNA. The displacement data is more amenable to further quantitative analysis than the raw correlation functions. By using a suitable integral transform of the mean square displacement, we can then determine the viscoelastic moduli of the DNA in its cellular environment. The entire analysis procedure is extensively calibrated and validated using model systems and computational simulations.

GenePublisher: Automated analysis of DNA microarray data.

PubMed

Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, Thomas; Friis, Carsten

2003-07-01

GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with a specification of the data. The server performs normalization, statistical analysis and visualization of the data. The results are run against databases of signal transduction pathways, metabolic pathways and promoter sequences in order to extract more information. The results of the entire analysis are summarized in report form and returned to the user.

Utilizing DNA analysis to combat the world wide plague of present day slavery – trafficking in persons

PubMed Central

Palmbach, Timothy; Blom, Jeffrey; Hoynes, Emily; Primorac, Dragan; Gaboury, Mario

2014-01-01

A study was conducted to determine if modern forensic DNA typing methods can be properly employed throughout the world with a final goal of increasing arrests, prosecutions, and convictions of perpetrators of modern day trafficking in persons while concurrently reducing the burden of victim testimony in legal proceedings. Without interruption of investigations, collection of samples containing DNA was conducted in a variety of settings. Evidentiary samples were analyzed on the ANDE Rapid DNA system. Many of the collected swabs yielded informative short tandem repeat profiles with Rapid DNA technology. PMID:24577820

Utilizing DNA analysis to combat the world wide plague of present day slavery--trafficking in persons.

PubMed

Palmbach, Timothy M; Blom, Jeffrey; Hoynes, Emily; Primorac, Dragan; Gaboury, Mario

2014-02-01

A study was conducted to determine if modern forensic DNA typing methods can be properly employed throughout the world with a final goal of increasing arrests, prosecutions, and convictions of perpetrators of modern day trafficking in persons while concurrently reducing the burden of victim testimony in legal proceedings. Without interruption of investigations, collection of samples containing DNA was conducted in a variety of settings. Evidentiary samples were analyzed on the ANDE Rapid DNA system. Many of the collected swabs yielded informative short tandem repeat profiles with Rapid DNA technology.

Developmental validation of the PowerPlex(®) ESI 16 and PowerPlex(®) ESI 17 Systems: STR multiplexes for the new European standard.

PubMed

Tucker, Valerie C; Hopwood, Andrew J; Sprecher, Cynthia J; McLaren, Robert S; Rabbach, Dawn R; Ensenberger, Martin G; Thompson, Jonelle M; Storts, Douglas R

2011-11-01

In response to the ENFSI and EDNAP groups' call for new STR multiplexes for Europe, Promega(®) developed a suite of four new DNA profiling kits. This paper describes the developmental validation study performed on the PowerPlex(®) ESI 16 (European Standard Investigator 16) and the PowerPlex(®) ESI 17 Systems. The PowerPlex(®) ESI 16 System combines the 11 loci compatible with the UK National DNA Database(®), contained within the AmpFlSTR(®) SGM Plus(®) PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to reduce the amplicon size of the loci found in the AmpFlSTR(®) SGM Plus(®) kit. This design facilitates increased robustness and amplification success for the loci used in the national DNA databases created in many countries, when analyzing degraded DNA samples. The PowerPlex(®) ESI 17 System amplifies the same loci as the PowerPlex(®) ESI 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR(®) SGM Plus(®) kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex(®) ESI 16 and ESI 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5pg of a fully heterozygous single source DNA template. This high level of sensitivity was found to impact on mixture analyses, where 54-86% of unique minor contributor alleles were routinely observed in a 1:19 mixture ratio. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of data obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

QPSO-Based Adaptive DNA Computing Algorithm

PubMed Central

Karakose, Mehmet; Cigdem, Ugur

2013-01-01

DNA (deoxyribonucleic acid) computing that is a new computation model based on DNA molecules for information storage has been increasingly used for optimization and data analysis in recent years. However, DNA computing algorithm has some limitations in terms of convergence speed, adaptability, and effectiveness. In this paper, a new approach for improvement of DNA computing is proposed. This new approach aims to perform DNA computing algorithm with adaptive parameters towards the desired goal using quantum-behaved particle swarm optimization (QPSO). Some contributions provided by the proposed QPSO based on adaptive DNA computing algorithm are as follows: (1) parameters of population size, crossover rate, maximum number of operations, enzyme and virus mutation rate, and fitness function of DNA computing algorithm are simultaneously tuned for adaptive process, (2) adaptive algorithm is performed using QPSO algorithm for goal-driven progress, faster operation, and flexibility in data, and (3) numerical realization of DNA computing algorithm with proposed approach is implemented in system identification. Two experiments with different systems were carried out to evaluate the performance of the proposed approach with comparative results. Experimental results obtained with Matlab and FPGA demonstrate ability to provide effective optimization, considerable convergence speed, and high accuracy according to DNA computing algorithm. PMID:23935409

A homozygote splice site PMS2 mutation as cause of Turcot syndrome gives rise to two different abnormal transcripts.

PubMed

Sjursen, Wenche; Bjørnevoll, Inga; Engebretsen, Lars F; Fjelland, Kristine; Halvorsen, Tore; Myrvold, Helge E

2009-01-01

Turcot syndrome is a rare, inherited disease predisposing of tumours in the central nerve system and in the colorectal system. This report describes a Turcot patient with an extraordinary clinical history. The patient is still alive at the age of 43. She was operated at the age of 10 by brain tumour and at the age of 16 by colorectal cancer. She has since then been treated for multiple cancers (gastrointestinal, endometrial, basal cell carcinomas), and removal of adenomatous polyps at several occasions. The aim of this work was to investigate if there was any specific genotype that explains her remarkable clinical history. Microsatellite instability and immunohistochemistry analysis for four DNA mismatch repair proteins were performed. DNA mutation analysis was done for genes involved in polyposis and mismatch repair by denaturing high performance liquid chromatography and sequencing. cDNA analysis was carried out for the mismatch repair gene PMS2. The patients genotype was found to be a homozygous splice site mutation in the PMS2 gene, c.989-1G

Premature aging and cancer in nucleotide excision repair-disorders

PubMed Central

Diderich, K.; Alanazi, M.; Hoeijmakers, J.H.J.

2014-01-01

During past decades the major impact of DNA damage on cancer as ‘disease of the genes’ has become abundantly apparent. In addition to cancer recent years have also uncovered a very strong association of DNA damage with many features of (premature) aging. The notion that DNA repair systems not only protect against cancer but equally against too fast aging has become evident from a systematic, integral analysis of a variety of mouse mutants carrying defects in e.g. transcription-coupled repair with or without an additional impairment of global genome nucleotide excision repair and the corresponding segmental premature aging syndromes in man. A striking correlation between the degree of the DNA repair deficiency and the acceleration of specific progeroid symptoms has been discovered for those repair systems that primarily protect from the cytotoxic and cytostatic effects of DNA damage. These observations are explained from the perspective of nucleotide excision repair mouse mutant and human syndromes. However, similar principles likely apply to other DNA repair pathways including interstrand crosslink repair and double strand break repair and genome maintenance systems in general, supporting the notion that DNA damage constitutes an important intermediate in the process of aging. PMID:21680258

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.

Pediatric Glioblastoma Therapies Based on Patient-Derived Stem Cell Resources

DTIC Science & Technology

2014-11-01

genomic DNA and then subjected to Illumina high-throughput sequencing . In this analysis, shRNAs lost in the GSC population represent candidate gene...and genomic DNA and then subjected to Illumina high-throughput sequencing . In this analysis, shRNAs lost in the GSC population represent candidate...PRISM 7900 Sequence Detection System ( Genomics Resource, FHCRC). Relative transcript abundance was analyzed using the 2−ΔΔCt method. TRIzol (Invitrogen

Flow through PCR module of BioBriefcase

NASA Astrophysics Data System (ADS)

Arroyo, E.; Wheeler, E. K.; Shediac, R.; Hindson, B.; Nasarabadi, S.; Vrankovich, G.; Bell, P.; Bailey, C.; Sheppod, T.; Christian, A. T.

2005-11-01

The BioBriefcase is an integrated briefcase-sized aerosol collection and analysis system for autonomous monitoring of the environment, which is currently being jointly developed by Lawrence Livermore and Sandia National Laboratories. This poster presents results from the polymerase chain reaction (PCR) module of the system. The DNA must be purified after exiting the aerosol collector to prevent inhibition of the enzymatic reaction. Traditional solid-phase extraction results in a large loss of sample. In this flow-through system, we perform sample purification, concentration and amplification in one reactor, which minimizes the loss of material. The sample from the aerosol collector is mixed with a denaturation solution prior to flowing through a capillary packed with silica beads. The DNA adheres to the silica beads allowing the environmental contaminants to be flushed to waste while effectively concentrating the DNA on the silica matrix. The adhered DNA is amplified while on the surface of the silica beads, resulting in a lower limit of detection than an equivalent eluted sample. Thus, this system is beneficial since more DNA is available for amplification, less reagents are utilized, and contamination risks are reduced.

Development of a photodiode array biochip using a bipolar semiconductor and its application to detection of human papilloma virus.

PubMed

Baek, Taek Jin; Park, Pan Yun; Han, Kwi Nam; Kwon, Ho Taik; Seong, Gi Hun

2008-03-01

We describe a DNA microarray system using a bipolar integrated circuit photodiode array (PDA) chip as a new platform for DNA analysis. The PDA chip comprises an 8 x 6 array of photodiodes each with a diameter of 600 microm. Each photodiode element acts both as a support for an immobilizing probe DNA and as a two-dimensional photodetector. The usefulness of the PDA microarray platform is demonstrated by the detection of high-risk subtypes of human papilloma virus (HPV). The polymerase chain reaction (PCR)-amplified biotinylated HPV target DNA was hybridized with the immobilized probe DNA on the photodiode surface, and the chip was incubated in an anti-biotin antibody-conjugated gold nanoparticle solution. The silver enhancement by the gold nanoparticles bound to the biotin of the HPV target DNA precipitates silver metal particles at the chip surfaces, which block light irradiated from above. The resulting drop in output voltage depends on the amount of target DNA present in the sample solution, which allows the specific detection and the quantitative analysis of the complementary target DNA. The PDA chip showed high relative signal ratios of HPV probe DNA hybridized with complementary target DNA, indicating an excellent capability in discriminating HPV subtypes. The detection limit for the HPV target DNA analysis improved from 1.2 nM to 30 pM by changing the silver development time from 5 to 10 min. Moreover, the enhanced silver development promoted by the gold nanoparticles could be applied to a broader range of target DNA concentration by controlling the silver development time.

Automated analysis of time-lapse fluorescence microscopy images: from live cell images to intracellular foci.

PubMed

Dzyubachyk, Oleh; Essers, Jeroen; van Cappellen, Wiggert A; Baldeyron, Céline; Inagaki, Akiko; Niessen, Wiro J; Meijering, Erik

2010-10-01

Complete, accurate and reproducible analysis of intracellular foci from fluorescence microscopy image sequences of live cells requires full automation of all processing steps involved: cell segmentation and tracking followed by foci segmentation and pattern analysis. Integrated systems for this purpose are lacking. Extending our previous work in cell segmentation and tracking, we developed a new system for performing fully automated analysis of fluorescent foci in single cells. The system was validated by applying it to two common tasks: intracellular foci counting (in DNA damage repair experiments) and cell-phase identification based on foci pattern analysis (in DNA replication experiments). Experimental results show that the system performs comparably to expert human observers. Thus, it may replace tedious manual analyses for the considered tasks, and enables high-content screening. The described system was implemented in MATLAB (The MathWorks, Inc., USA) and compiled to run within the MATLAB environment. The routines together with four sample datasets are available at http://celmia.bigr.nl/. The software is planned for public release, free of charge for non-commercial use, after publication of this article.

Breast Cancer Translational Research Center of Excellence

DTIC Science & Technology

2015-09-01

we will identify partner proteins by either Y2H or pull-down studies using Mass Spectrometry Analysis. Aim 2. Development of DNA aptamers against...CD44 that inhibit breast cancer invasion and metastasis. During the previous period, we developed novel and unique DNA aptamers that specifically...bound to exon v10 of CD44 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) systems. These DNA aptamers inhibited TN breast

Image Analysis of DNA Fiber and Nucleus in Plants.

PubMed

Ohmido, Nobuko; Wako, Toshiyuki; Kato, Seiji; Fukui, Kiichi

2016-01-01

Advances in cytology have led to the application of a wide range of visualization methods in plant genome studies. Image analysis methods are indispensable tools where morphology, density, and color play important roles in the biological systems. Visualization and image analysis methods are useful techniques in the analyses of the detailed structure and function of extended DNA fibers (EDFs) and interphase nuclei. The EDF is the highest in the spatial resolving power to reveal genome structure and it can be used for physical mapping, especially for closely located genes and tandemly repeated sequences. One the other hand, analyzing nuclear DNA and proteins would reveal nuclear structure and functions. In this chapter, we describe the image analysis protocol for quantitatively analyzing different types of plant genome, EDFs and interphase nuclei.

Functional integration of PCR amplification and capillary eletrophoresis in a microfabricated DNA analysis device

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

Woolley, A.T.; deMello, A.J.; Mathies, R.A.

Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10{degree}C/s heating, 2.5{degree}C/s cooling) with the high-speed (<120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an `electrophoretic valve` to couple the PCR and CE devices on-chip. To demonstrate the functionality ofmore » this system, a 15 min PCR amplification of a {Beta}-globin target cloned in m13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. 33 refs., 6 figs.« less

PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats.

PubMed

Guillen, I A; Camacho, H; Tuero, A D; Bacardí, D; Palenzuela, D O; Aguilera, A; Silva, J A; Estrada, R; Gell, O; Suárez, J; Ancizar, J; Brown, E; Colarte, A B; Castro, J; Novoa, L I

2016-09-01

The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies.

A genetic investigation of Korean mummies from the Joseon Dynasty.

PubMed

Kim, Na Young; Lee, Hwan Young; Park, Myung Jin; Yang, Woo Ick; Shin, Kyoung-Jin

2011-01-01

Two Korean mummies (Danwoong-mirra and Yoon-mirra) found in medieval tombs in the central region of the Korean peninsula were genetically investigated by analysis of mitochondrial DNA (mtDNA), Y-chromosomal short tandem repeat (Y-STR) and the ABO gene. Danwoong-mirra is a male child mummy and Yoon-mirra is a pregnant female mummy, dating back about 550 and 450 years, respectively. DNA was extracted from soft tissues or bones. mtDNA, Y-STR and the ABO gene were amplified using a small size amplicon strategy and were analyzed according to the criteria of ancient DNA analysis to ensure that authentic DNA typing results were obtained from these ancient samples. Analysis of mtDNA hypervariable region sequence and coding region single nucleotide polymorphism (SNP) information revealed that Danwoong-mirra and Yoon-mirra belong to the East Asian mtDNA haplogroups D4 and M7c, respectively. The Y-STRs were analyzed in the male child mummy (Danwoong-mirra) using the AmpFlSTR® Yfiler PCR Amplification Kit and an in-house Y-miniplex plus system, and could be characterized in 4 loci with small amplicon size. The analysis of ABO gene SNPs using multiplex single base extension methods revealed that the ABO blood types of Danwoong-mirra and Yoon-mirra are AO01 and AB, respectively. The small size amplicon strategy and the authentication process in the present study will be effectively applicable to future genetic analyses of various forensic and ancient samples.

Cloning of rat MLH1 and expression analysis of MSH2, MSH3, MSH6, and MLH1 during spermatogenesis.

PubMed

Geeta Vani, R; Varghese, C M; Rao, M R

1999-12-15

The mismatch repair system has been highly conserved in various species. In eukaryotic cells, the Mut S and Mut L homologues play crucial roles in both DNA mismatch repair and meiotic recombination. A full-length rat cDNA clone for rat MLH1 has been constructed using the RT-PCR method. The cDNA has an open reading frame of 2274 nucleotides for a protein of 757 amino acids. We have also obtained partial cDNA clones for MSH3 and MSH6. Northern blot analysis of rat MLH1, MSH2, MSH3, and MSH6 in the testes of rats of different ages showed differential expression of these genes as a function of developmental maturation of the testes. The expression analysis suggests that MSH3 may have a more predominant role in the meiotic recombination process. Copyright 1999 Academic Press.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

PubMed

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-06-02

Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Examples of kinship analysis where Profiler Plus™ was not discriminatory enough for the identification of victims using DNA identification.

PubMed

Hartman, D; Benton, L; Morenos, L; Beyer, J; Spiden, M; Stock, A

2011-02-25

The identification of the victims of the 2009 Victorian bushfires disaster, as in other mass disasters, relied on a number of scientific disciplines - including DNA analysis. As part of the DVI response, DNA analysis was performed to assist in the identification of victims through kinship (familial matching to relatives) or direct (self source of sample) matching of DNA profiles. The majority of the DNA identifications made (82%) were achieved through kinship matching of familial reference samples to post mortem (PM) samples obtained from the victims. Although each location affected by the bushfires could be treated as a mini-disaster (having a small closed-set of victims), with many such sites spread over vast areas, DNA analysis requires that the short tandem repeat (STR) system used be able to afford enough discrimination between all the DVI cases to assign a match. This publication highlights that although a 9-loci multiplex was sufficient for a DVI of this nature, there were instances that brought to light the short comings of using a 9-loci multiplex for kinship matching--particularly where multiple family members are victims. Moreso it serves to reinforce the recommendation that a minimum of 12 autosomal STR markers (plus Amelogenin) be used for DNA identification of victims which relies heavily on kinship matching. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Non-biased and efficient global amplification of a single-cell cDNA library

PubMed Central

Huang, Huan; Goto, Mari; Tsunoda, Hiroyuki; Sun, Lizhou; Taniguchi, Kiyomi; Matsunaga, Hiroko; Kambara, Hideki

2014-01-01

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively. PMID:24141095

The antiviral defense mechanisms in mandarin fish induced by DNA vaccination against a rhabdovirus.

PubMed

Chen, Zhong-Yuan; Lei, Xiao-Ying; Zhang, Qi-Ya

2012-06-15

Plasmid DNAs containing Siniperca chuatsi rhabdovirus (SCRV) glycoprotein gene (pcDNA-G) and nucleoprotein gene (pcDNA-N) were constructed, and used to determine the antiviral immune response elicited by DNA vaccination in mandarin fish. In vitro and in vivo expression of the plasmid constructs was confirmed in transfected cells and muscle tissues of vaccinated fish by Western blot, indirect immunofluorescence or RT-PCR analysis. Fish injected with pcDNA-G exhibited protective effect against SCRV challenge with a relative percent survival (RPS) of 77.5%, but no significant protection (RPS of 2.5%) was observed in fish vaccinated with pcDNA-N. Immunohistochemical analysis showed that vaccination with pcDNA-G decreased histological lesions and suppressed the virus replication in fish target organs, e.g. kidney, liver, spleen, gill and heart. Transcriptional analysis further revealed that the expression levels of type I IFN system genes including interferon regulation factor-7 (IRF-7) gene, myxovirus resistance (Mx) gene and virus inhibitory protein (Viperin) gene were strongly up-regulated after injection with pcDNA-G, whereas the level of transcription of immunoglobulin M (IgM) gene did not show a statistically significant change. These results reveal that type I IFN antiviral immune response is rapidly triggered by the plasmid DNA containing rhabdovirus glycoprotein gene in fish, which offers an explanation of molecular mechanisms for DNA vaccination inducing mandarin fish resist to SCRV disease. Copyright © 2011 Elsevier B.V. All rights reserved.

A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis

PubMed Central

2017-01-01

Amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) is a potential method for use in forensic DNA analyses. In this application, PCR-MPS may supplement or replace other instrumental analysis methods such as capillary electrophoresis and Sanger sequencing for STR and mitochondrial DNA typing, respectively. PCR-MPS also may enable the expansion of forensic DNA analysis methods to include new marker systems such as single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) that currently are assayable using various instrumental analysis methods including microarray and quantitative PCR. Acceptance of PCR-MPS as a forensic method will depend in part upon developing protocols and criteria that define the limitations of a method, including a defensible analytical threshold or method detection limit. This paper describes an approach to establish objective analytical thresholds suitable for multiplexed PCR-MPS methods. A definition is proposed for PCR-MPS method background noise, and an analytical threshold based on background noise is described. PMID:28542338

A technique for setting analytical thresholds in massively parallel sequencing-based forensic DNA analysis.

PubMed

Young, Brian; King, Jonathan L; Budowle, Bruce; Armogida, Luigi

2017-01-01

Amplicon (targeted) sequencing by massively parallel sequencing (PCR-MPS) is a potential method for use in forensic DNA analyses. In this application, PCR-MPS may supplement or replace other instrumental analysis methods such as capillary electrophoresis and Sanger sequencing for STR and mitochondrial DNA typing, respectively. PCR-MPS also may enable the expansion of forensic DNA analysis methods to include new marker systems such as single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) that currently are assayable using various instrumental analysis methods including microarray and quantitative PCR. Acceptance of PCR-MPS as a forensic method will depend in part upon developing protocols and criteria that define the limitations of a method, including a defensible analytical threshold or method detection limit. This paper describes an approach to establish objective analytical thresholds suitable for multiplexed PCR-MPS methods. A definition is proposed for PCR-MPS method background noise, and an analytical threshold based on background noise is described.

Synthesis, characterization, crystal structure, DNA/BSA binding ability and antibacterial activity of asymmetric europium complex based on 1,10- phenanthroline

NASA Astrophysics Data System (ADS)

Alfi, Nafiseh; Khorasani-Motlagh, Mozhgan; Rezvani, Ali Reza; Noroozifar, Meissam; Molčanov, Krešimir

2017-06-01

A heteroleptic europium coordination compound formulated as [Eu(phen)2(OH2)2(Cl)2](Cl)(H2O) (phen = 1,10-phenanthroline), has been synthesized and characterized by elemental analysis, FT-IR spectroscopy, and single-crystal X-ray diffractometer. Crystal structure analysis reveals the complex is crystallized in orthorhombic system with Pca21 space group. Electronic absorption and various emission methods for investigation of the binding system of europium(III) complex to Fish Salmon deoxyribonucleic acid (FS-DNA) and Bovamin Serum Albumin (BSA) have been explored. Furthermore, the binding constants, binding sites and the corresponding thermodynamic parameters of the interaction system based on the van't Hoff equation for FS-DNA and BSA were calculated. The thermodynamic parameters reflect the exothermic nature of emission process (ΔH°<0 and ΔS°<0). The experimental results seem to indicate that the [Eu(phen)2(OH2)2(Cl)2](Cl)(H2O) bound to FS-DNA by non-intercalative mode which the groove binding is preferable mode. Also, the complex exhibits a brilliant antimicrobial activity in vitro against standard bacterial strains.

Sexual dimorphic floral development in dioecious plants revealed by transcriptome, phytohormone, and DNA methylation analysis in Populus tomentosa.

PubMed

Song, Yuepeng; Ma, Kaifeng; Ci, Dong; Chen, Qingqing; Tian, Jiaxing; Zhang, Deqiang

2013-12-01

Dioecious plants have evolved sex-specific floral development mechanisms. However, the precise gene expression patterns in dioecious plant flower development remain unclear. Here, we used andromonoecious poplar, an exceptional model system, to eliminate the confounding effects of genetic background of dioecious plants. Comparative transcriptome and physiological analysis allowed us to characterize sex-specific development of female and male flowers. Transcriptome analysis identified genes significantly differentially expressed between the sexes, including genes related to floral development, phytohormone synthesis and metabolism, and DNA methylation. Correlation analysis revealed a significant correlation between phytohormone signaling and gene expression, identifying specific phytohormone-responsive genes and their cis-regulatory elements. Two genes related to DNA methylation, METHYLTRANSFERASE1 (MET1) and DECREASED DNA METHYLATION 1 (DDM1), which are located in the sex determination region of Chromosome XIX, have differential expression between female and male flowers. A time-course analysis revealed that MET1 and DDM1 expression may produce different DNA methylation levels in female and male flowers. Understanding the interactions of phytohormone signaling, DNA methylation and target gene expression should lead to a better understanding of sexual differences in floral development. Thus, this study identifies a set of candidate genes for further studies of poplar sexual dimorphism and relates sex-specific floral development to physiological and epigenetic changes.

AmyI-1-18, a cationic α-helical antimicrobial octadecapeptide derived from α-amylase in rice, inhibits the translation and folding processes in a protein synthesis system.

PubMed

Taniguchi, Masayuki; Ochiai, Akihito; Fukuda, Shun; Sato, Teppei; Saitoh, Eiichi; Kato, Tetsuo; Tanaka, Takaaki

2016-10-01

In our previous study, we used a cell-free rapid translation system (RTS), which is an in vitro protein synthesis system based on Escherichia coli lysate, for evaluating the inhibition of green fluorescent protein (GFP) synthesis by pyrrhocoricin. In this study, using an RTS, we evaluated the inhibition of GFP synthesis by AmyI-1-18, an antimicrobial octadecapeptide. We found that, similarly to pyrrhocoricin, AmyI-1-18 inhibited GFP synthesis in the RTS in a concentration-dependent manner. In addition, the blockage of transcription and translation steps in the RTS was individually estimated using RT-PCR after gene expression to determine the mRNA products and using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine the amounts of GFP expressed from purified mRNA, respectively. The results demonstrated that the inhibition of GFP synthesis by AmyI-1-18 did not occur at the transcription step but rather at the translation step. Furthermore, we assessed the inhibition of DnaK-mediated refolding of chemically denatured luciferase by AmyI-1-18; AmyI-1-18 inhibited the protein folding activity of the ATP-dependent DnaK/DnaJ molecular chaperone system in a concentration-dependent manner. Surface plasmon resonance (SPR) analysis showed that AmyI-1-18 strongly bound to RNA with a KD value of 1.4 × 10(-8) M but not to DNA and that AmyI-1-18 specifically bound to DnaK with a KD value of 4.4 × 10(-6) M. These SPR analysis results supported the results obtained in both the RTS and the molecular chaperone system. These results demonstrated that both RNA and DnaK are most likely the target of AmyI-1-18 in the protein synthesis system. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA.

PubMed

Iyer, Lakshminarayan M; Zhang, Dapeng; Burroughs, A Maxwell; Aravind, L

2013-09-01

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel 'readers' of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology.

Computational identification of novel biochemical systems involved in oxidation, glycosylation and other complex modifications of bases in DNA

PubMed Central

Iyer, Lakshminarayan M.; Zhang, Dapeng; Maxwell Burroughs, A.; Aravind, L.

2013-01-01

Discovery of the TET/JBP family of dioxygenases that modify bases in DNA has sparked considerable interest in novel DNA base modifications and their biological roles. Using sensitive sequence and structure analyses combined with contextual information from comparative genomics, we computationally characterize over 12 novel biochemical systems for DNA modifications. We predict previously unidentified enzymes, such as the kinetoplastid J-base generating glycosyltransferase (and its homolog GREB1), the catalytic specificity of bacteriophage TET/JBP proteins and their role in complex DNA base modifications. We also predict the enzymes involved in synthesis of hypermodified bases such as alpha-glutamylthymine and alpha-putrescinylthymine that have remained enigmatic for several decades. Moreover, the current analysis suggests that bacteriophages and certain nucleo-cytoplasmic large DNA viruses contain an unexpectedly diverse range of DNA modification systems, in addition to those using previously characterized enzymes such as Dam, Dcm, TET/JBP, pyrimidine hydroxymethylases, Mom and glycosyltransferases. These include enzymes generating modified bases such as deazaguanines related to queuine and archaeosine, pyrimidines comparable with lysidine, those derived using modified S-adenosyl methionine derivatives and those using TET/JBP-generated hydroxymethyl pyrimidines as biosynthetic starting points. We present evidence that some of these modification systems are also widely dispersed across prokaryotes and certain eukaryotes such as basidiomycetes, chlorophyte and stramenopile alga, where they could serve as novel epigenetic marks for regulation or discrimination of self from non-self DNA. Our study extends the role of the PUA-like fold domains in recognition of modified nucleic acids and predicts versions of the ASCH and EVE domains to be novel ‘readers’ of modified bases in DNA. These results open opportunities for the investigation of the biology of these systems and their use in biotechnology. PMID:23814188

Engineering bacteria to solve the Burnt Pancake Problem

PubMed Central

Haynes, Karmella A; Broderick, Marian L; Brown, Adam D; Butner, Trevor L; Dickson, James O; Harden, W Lance; Heard, Lane H; Jessen, Eric L; Malloy, Kelly J; Ogden, Brad J; Rosemond, Sabriya; Simpson, Samantha; Zwack, Erin; Campbell, A Malcolm; Eckdahl, Todd T; Heyer, Laurie J; Poet, Jeffrey L

2008-01-01

Background We investigated the possibility of executing DNA-based computation in living cells by engineering Escherichia coli to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA. Results Inversions (or "flips") of the DNA fragment pancakes are driven by the Salmonella typhimurium Hin/hix DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in E. coli. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; E. coli cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of in vivo flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes. Conclusion The Hin/hix system is a proof-of-concept demonstration of in vivo computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/hix may provide a flexible new tool for manipulating transgenic DNA in vivo. PMID:18492232

Functional Characterization of Two Novel Human Prostate Cancer Metastasis Related Genes

DTIC Science & Technology

2008-02-01

systems (27-29), a major leap in functional genomic investigation would be the ability to perform genetic subtractive analysis with in vivo-derived...been designed to detect and isolate different DNA sequences present in one complimentary (31) or genomic (32) DNA library but absent in another. The...many disorders if applied correctly, the use of control specimens different from the native tissue for subtractive genomic analysis in some studies has

Design of a Clinical Information Management System to Support DNA Analysis Laboratory Operation

PubMed Central

Dubay, Christopher J.; Zimmerman, David; Popovich, Bradley

1995-01-01

The LabDirector system has been developed at the Oregon Health Sciences University to support the operation of our clinical DNA analysis laboratory. Through an iterative design process which has spanned two years, we have produced a system that is both highly tailored to a clinical genetics production laboratory and flexible in its implementation, to support the rapid growth and change of protocols and methodologies in use in the field. The administrative aspects of the system are integrated with an enterprise schedule management system. The laboratory side of the system is driven by a protocol modeling and execution system. The close integration between these two aspects of the clinical laboratory facilitates smooth operations, and allows management to accurately measure costs and performance. The entire application has been designed and documented to provide utility to a wide range of clinical laboratory environments.

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

PubMed

Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

2015-08-01

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

CRISPR-Cas systems exploit viral DNA injection to establish and maintain adaptive immunity.

PubMed

Modell, Joshua W; Jiang, Wenyan; Marraffini, Luciano A

2017-04-06

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide protection against viral and plasmid infection by capturing short DNA sequences from these invaders and integrating them into the CRISPR locus of the prokaryotic host. These sequences, known as spacers, are transcribed into short CRISPR RNA guides that specify the cleavage site of Cas nucleases in the genome of the invader. It is not known when spacer sequences are acquired during viral infection. Here, to investigate this, we tracked spacer acquisition in Staphylococcus aureus cells harbouring a type II CRISPR-Cas9 system after infection with the staphylococcal bacteriophage ϕ12. We found that new spacers were acquired immediately after infection preferentially from the cos site, the viral free DNA end that is first injected into the cell. Analysis of spacer acquisition after infection with mutant phages demonstrated that most spacers are acquired during DNA injection, but not during other stages of the viral cycle that produce free DNA ends, such as DNA replication or packaging. Finally, we showed that spacers acquired from early-injected genomic regions, which direct Cas9 cleavage of the viral DNA immediately after infection, provide better immunity than spacers acquired from late-injected regions. Our results reveal that CRISPR-Cas systems exploit the phage life cycle to generate a pattern of spacer acquisition that ensures a successful CRISPR immune response.

Light emitting diode, photodiode-based fluorescence detection system for DNA analysis with microchip electrophoresis.

PubMed

Hall, Gordon H; Glerum, D Moira; Backhouse, Christopher J

2016-02-01

Electrophoretic separation of fluorescently end-labeled DNA after a PCR serves as a gold standard in genetic diagnostics. Because of their size and cost, instruments for this type of analysis have had limited market uptake, particularly for point-of-care applications. This might be changed through a higher level of system integration and lower instrument costs that can be realized through the use of LEDs for excitation and photodiodes for detection--if they provide sufficient sensitivity. Here, we demonstrate an optimized microchip electrophoresis instrument using polymeric fluidic chips with fluorescence detection of end-labeled DNA with a LOD of 0.15 nM of Alexa Fluor 532. This represents orders of magnitude improvement over previously reported instruments of this type. We demonstrate the system with an electrophoretic separation of two PCR products and their respective primers. We believe that this is the first LED-induced fluorescence microchip electrophoresis system with photodiode-based detection that could be used for standard applications of PCR and electrophoresis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

BioBarcode: a general DNA barcoding database and server platform for Asian biodiversity resources

PubMed Central

2009-01-01

Background DNA barcoding provides a rapid, accurate, and standardized method for species-level identification using short DNA sequences. Such a standardized identification method is useful for mapping all the species on Earth, particularly when DNA sequencing technology is cheaply available. There are many nations in Asia with many biodiversity resources that need to be mapped and registered in databases. Results We have built a general DNA barcode data processing system, BioBarcode, with open source software - which is a general purpose database and server. It uses mySQL RDBMS 5.0, BLAST2, and Apache httpd server. An exemplary database of BioBarcode has around 11,300 specimen entries (including GenBank data) and registers the biological species to map their genetic relationships. The BioBarcode database contains a chromatogram viewer which improves the performance in DNA sequence analyses. Conclusion Asia has a very high degree of biodiversity and the BioBarcode database server system aims to provide an efficient bioinformatics protocol that can be freely used by Asian researchers and research organizations interested in DNA barcoding. The BioBarcode promotes the rapid acquisition of biological species DNA sequence data that meet global standards by providing specialized services, and provides useful tools that will make barcoding cheaper and faster in the biodiversity community such as standardization, depository, management, and analysis of DNA barcode data. The system can be downloaded upon request, and an exemplary server has been constructed with which to build an Asian biodiversity system http://www.asianbarcode.org. PMID:19958506

Investigating the epigenetic effects of a prototype smoke-derived carcinogen in human cells.

PubMed

Tommasi, Stella; Kim, Sang-in; Zhong, Xueyan; Wu, Xiwei; Pfeifer, Gerd P; Besaratinia, Ahmad

2010-05-12

Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease.

Investigating the Epigenetic Effects of a Prototype Smoke-Derived Carcinogen in Human Cells

PubMed Central

Tommasi, Stella; Kim, Sang-in; Zhong, Xueyan; Wu, Xiwei; Pfeifer, Gerd P.; Besaratinia, Ahmad

2010-01-01

Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease. PMID:20485678

Improving efficiency of a small forensic DNA laboratory: validation of robotic assays and evaluation of microcapillary array device.

PubMed

Crouse, Cecelia A; Yeung, Stephanie; Greenspoon, Susan; McGuckian, Amy; Sikorsky, Julie; Ban, Jeff; Mathies, Richard

2005-08-01

To present validation studies performed for the implementation of existing and new technologies to increase the efficiency in the forensic DNA Section of the Palm Beach County Sheriff's Office (PBSO) Crime Laboratory. Using federally funded grants, internal support, and an external Process Mapping Team, the PBSO collaborated with forensic vendors, universities, and other forensic laboratories to enhance DNA testing procedures, including validation of the DNA IQ magnetic bead extraction system, robotic DNA extraction using the BioMek2000, the ABI7000 Sequence Detection System, and is currently evaluating a micro Capillary Array Electrophoresis device. The PBSO successfully validated and implemented both manual and automated Promega DNA IQ magnetic bead extractions system, which have increased DNA profile results from samples with low DNA template concentrations. The Beckman BioMek2000 DNA robotic workstation has been validated for blood, tissue, bone, hair, epithelial cells (touch evidence), and mixed stains such as semen. There has been a dramatic increase in the number of samples tested per case since implementation of the robotic extraction protocols. The validation of the ABI7000 real-time quantitative polymerase chain reaction (qPCR) technology and the single multiplex short tandem repeat (STR) PowerPlex16 BIO amplification system has provided both a time and a financial benefit. In addition, the qPCR system allows more accurate DNA concentration data and the PowerPlex 16 BIO multiplex generates DNA profiles data in half the time when compared to PowerPlex1.1 and PowerPlex2.1 STR systems. The PBSO's future efficiency requirements are being addressed through collaboration with the University of California at Berkeley and the Virginia Division of Forensic Science to validate microcapillary array electrophoresis instrumentation. Initial data demonstrated the electrophoresis of 96 samples in less than twenty minutes. The PBSO demonstrated, through the validation of more efficient extraction and quantification technology, an increase in the number of evidence samples tested using robotic/DNA IQ magnetic bead DNA extraction, a decrease in the number of negative samples amplified due to qPCR and implementation of a single multiplex amplification system. In addition, initial studies show the microcapillary array electrophoresis device (microCAE) evaluation results provide greater sensitivity and faster STR analysis output than current platforms.

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

PubMed

Bae, Seunghee; An, In-Sook; An, Sungkwan

2015-09-01

Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

Characterizing the distribution of an endangered salmonid using environmental DNA analysis

USGS Publications Warehouse

Laramie, Matthew B.; Pilliod, David S.; Goldberg, Caren S.

2015-01-01

Determining species distributions accurately is crucial to developing conservation and management strategies for imperiled species, but a challenging task for small populations. We evaluated the efficacy of environmental DNA (eDNA) analysis for improving detection and thus potentially refining the known distribution of Chinook salmon (Oncorhynchus tshawytscha) in the Methow and Okanogan Subbasins of the Upper Columbia River, which span the border between Washington, USA and British Columbia, Canada. We developed an assay to target a 90 base pair sequence of Chinook DNA and used quantitative polymerase chain reaction (qPCR) to quantify the amount of Chinook eDNA in triplicate 1-L water samples collected at 48 stream locations in June and again in August 2012. The overall probability of detecting Chinook with our eDNA method in areas within the known distribution was 0.77 (±0.05 SE). Detection probability was lower in June (0.62, ±0.08 SE) during high flows and at the beginning of spring Chinook migration than during base flows in August (0.93, ±0.04 SE). In the Methow subbasin, mean eDNA concentration was higher in August compared to June, especially in smaller tributaries, probably resulting from the arrival of spring Chinook adults, reduced discharge, or both. Chinook eDNA concentrations did not appear to change in the Okanogan subbasin from June to August. Contrary to our expectations about downstream eDNA accumulation, Chinook eDNA did not decrease in concentration in upstream reaches (0–120 km). Further examination of factors influencing spatial distribution of eDNA in lotic systems may allow for greater inference of local population densities along stream networks or watersheds. These results demonstrate the potential effectiveness of eDNA detection methods for determining landscape-level distribution of anadromous salmonids in large river systems.

Characterization of UVC-induced DNA damage in bloodstains: forensic implications.

PubMed

Hall, Ashley; Ballantyne, Jack

2004-09-01

The ability to detect DNA polymorphisms using molecular genetic techniques has revolutionized the forensic analysis of biological evidence. DNA typing now plays a critical role within the criminal justice system, but one of the limiting factors with the technology is that DNA isolated from biological stains recovered from the crime scene is sometimes so damaged as to be intractable to analysis. Potential remedies for damaged DNA are likely to be dependent upon the precise nature of the DNA damage present in any particular sample but, unfortunately, current knowledge of the biochemical nature, and the extent, of such DNA damage in dried biological stains is rudimentary. As a model for DNA damage assessment in biological stains recovered from crime scenes, we have subjected human bloodstains and naked DNA in the hydrated and dehydrated states to varying doses of UVC radiation. It was possible to damage the DNA sufficiently in a bloodstain to cause a standard autosomal short tandem repeat (STR) profile to be lost. However, a detailed analysis of the process, based upon assays developed to detect bipyrimidine photoproducts (BPPPs), single- and double-strand breaks, and DNA-DNA crosslinks, produced some unexpected findings. Contrary to the situation with living tissues or cells in culture, the predominant UVC-induced damage to DNA in bloodstains appears not to be pyrimidine dimers. Although some evidence for the presence of BPPPs and DNA crosslinks was obtained, the major form of UVC damage causing genetic profile loss appeared to be single-strand breaks. It was not possible, however, to preclude the possibility that a combination of damage types was responsible for the profile loss observed. We demonstrate here that a significant measure of protection against UVC-mediated genetic profile loss in dried biological stain material is afforded by the dehydrated state of the DNA and, to a lesser extent, the DNA cellular milieu.

A DNA network as an information processing system.

PubMed

Santini, Cristina Costa; Bath, Jonathan; Turberfield, Andrew J; Tyrrell, Andy M

2012-01-01

Biomolecular systems that can process information are sought for computational applications, because of their potential for parallelism and miniaturization and because their biocompatibility also makes them suitable for future biomedical applications. DNA has been used to design machines, motors, finite automata, logic gates, reaction networks and logic programs, amongst many other structures and dynamic behaviours. Here we design and program a synthetic DNA network to implement computational paradigms abstracted from cellular regulatory networks. These show information processing properties that are desirable in artificial, engineered molecular systems, including robustness of the output in relation to different sources of variation. We show the results of numerical simulations of the dynamic behaviour of the network and preliminary experimental analysis of its main components.

Probing the structural dynamics of the CRISPR-Cas9 RNA-guided DNA-cleavage system by coarse-grained modeling.

PubMed

Zheng, Wenjun

2017-02-01

In the adaptive immune systems of many bacteria and archaea, the Cas9 endonuclease forms a complex with specific guide/scaffold RNA to identify and cleave complementary target sequences in foreign DNA. This DNA targeting machinery has been exploited in numerous applications of genome editing and transcription control. However, the molecular mechanism of the Cas9 system is still obscure. Recently, high-resolution structures have been solved for Cas9 in different structural forms (e.g., unbound forms, RNA-bound binary complexes, and RNA-DNA-bound tertiary complexes, corresponding to an inactive state, a pre-target-bound state, and a cleavage-competent or product state), which offered key structural insights to the Cas9 mechanism. To further probe the structural dynamics of Cas9 interacting with RNA and DNA at the amino-acid level of details, we have performed systematic coarse-grained modeling using an elastic network model and related analyses. Our normal mode analysis predicted a few key modes of collective motions that capture the observed conformational changes featuring large domain motions triggered by binding of RNA and DNA. Our flexibility analysis identified specific regions with high or low flexibility that coincide with key functional sites (such as DNA/RNA-binding sites, nuclease cleavage sites, and key hinges). We also identified a small set of hotspot residues that control the energetics of functional motions, which overlap with known functional sites and offer promising targets for future mutagenesis efforts to improve the specificity of Cas9. Finally, we modeled the conformational transitions of Cas9 from the unbound form to the binary complex and then the tertiary complex, and predicted a distinct sequence of domain motions. In sum, our findings have offered rich structural and dynamic details relevant to the Cas9 machinery, and will guide future investigation and engineering of the Cas9 systems. Proteins 2017; 85:342-353. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Anisotropic Brownian motion in ordered phases of DNA fragments.

PubMed

Dobrindt, J; Rodrigo Teixeira da Silva, E; Alves, C; Oliveira, C L P; Nallet, F; Andreoli de Oliveira, E; Navailles, L

2012-01-01

Using Fluorescence Recovery After Photobleaching, we investigate the Brownian motion of DNA rod-like fragments in two distinct anisotropic phases with a local nematic symmetry. The height of the measurement volume ensures the averaging of the anisotropy of the in-plane diffusive motion parallel or perpendicular to the local nematic director in aligned domains. Still, as shown in using a model specifically designed to handle such a situation and predicting a non-Gaussian shape for the bleached spot as fluorescence recovery proceeds, the two distinct diffusion coefficients of the DNA particles can be retrieved from data analysis. In the first system investigated (a ternary DNA-lipid lamellar complex), the magnitude and anisotropy of the diffusion coefficient of the DNA fragments confined by the lipid bilayers are obtained for the first time. In the second, binary DNA-solvent system, the magnitude of the diffusion coefficient is found to decrease markedly as DNA concentration is increased from isotropic to cholesteric phase. In addition, the diffusion coefficient anisotropy measured within cholesteric domains in the phase coexistence region increases with concentration, and eventually reaches a high value in the cholesteric phase.

Rapid self-assembly of DNA on a microfluidic chip

PubMed Central

Zheng, Yao; Footz, Tim; Manage, Dammika P; Backhouse, Christopher James

2005-01-01

Background DNA self-assembly methods have played a major role in enabling methods for acquiring genetic information without having to resort to sequencing, a relatively slow and costly procedure. However, even self-assembly processes tend to be very slow when they rely upon diffusion on a large scale. Miniaturisation and integration therefore hold the promise of greatly increasing this speed of operation. Results We have developed a rapid method for implementing the self-assembly of DNA within a microfluidic system by electrically extracting the DNA from an environment containing an uncharged denaturant. By controlling the parameters of the electrophoretic extraction and subsequent analysis of the DNA we are able to control when the hybridisation occurs as well as the degree of hybridisation. By avoiding off-chip processing or long thermal treatments we are able to perform this hybridisation rapidly and can perform hybridisation, sizing, heteroduplex analysis and single-stranded conformation analysis within a matter of minutes. The rapidity of this analysis allows the sampling of transient effects that may improve the sensitivity of mutation detection. Conclusions We believe that this method will aid the integration of self-assembly methods upon microfluidic chips. The speed of this analysis also appears to provide information upon the dynamics of the self-assembly process. PMID:15717935

Scaling in nature: From DNA through heartbeats to weather

NASA Astrophysics Data System (ADS)

Havlin, S.; Buldyrev, S. V.; Bunde, A.; Goldberger, A. L.; Ivanov, P. Ch.; Peng, C.-K.; Stanley, H. E.

1999-12-01

The purpose of this talk is to describe some recent progress in applying scaling concepts to various systems in nature. We review several systems characterized by scaling laws such as DNA sequences, heartbeat rates and weather variations. We discuss the finding that the exponent α quantifying the scaling in DNA in smaller for coding than for noncoding sequences. We also discuss the application of fractal scaling analysis to the dynamics of heartbeat regulation, and report the recent finding that the scaling exponent α is smaller during sleep periods compared to wake periods. We also discuss the recent findings that suggest a universal scaling exponent characterizing the weather fluctuations.

Scaling in nature: from DNA through heartbeats to weather

NASA Technical Reports Server (NTRS)

Havlin, S.; Buldyrev, S. V.; Bunde, A.; Goldberger, A. L.; Peng, C. K.; Stanley, H. E.

1999-01-01

The purpose of this report is to describe some recent progress in applying scaling concepts to various systems in nature. We review several systems characterized by scaling laws such as DNA sequences, heartbeat rates and weather variations. We discuss the finding that the exponent alpha quantifying the scaling in DNA in smaller for coding than for noncoding sequences. We also discuss the application of fractal scaling analysis to the dynamics of heartbeat regulation, and report the recent finding that the scaling exponent alpha is smaller during sleep periods compared to wake periods. We also discuss the recent findings that suggest a universal scaling exponent characterizing the weather fluctuations.

Cell-Free DNA in Metastatic Colorectal Cancer: A Systematic Review and Meta-Analysis.

PubMed

Spindler, Karen-Lise G; Boysen, Anders K; Pallisgård, Niels; Johansen, Julia S; Tabernero, Josep; Sørensen, Morten M; Jensen, Benny V; Hansen, Torben F; Sefrioui, David; Andersen, Rikke F; Brandslund, Ivan; Jakobsen, Anders

2017-09-01

Circulating DNA can be detected and quantified in the blood of cancer patients and used for detection of tumor-specific genetic alterations. The clinical utility has been intensively investigated for the past 10 years. The majority of reports focus on analyzing the clinical potential of tumor-specific mutations, whereas the use of total cell-free DNA (cfDNA) quantification is somehow controversial and sparsely described in the literature, but holds important clinical information in itself. The purpose of the present report was to present a systematic review and meta-analysis of the prognostic value of total cfDNA in patients with metastatic colorectal cancer (mCRC) treated with chemotherapy. In addition, we report on the overall performance of cfDNA as source for KRAS mutation detection. A systematic literature search of PubMed and Embase was performed by two independent investigators. Eligibility criteria were (a) total cfDNA analysis, (b) mCRC, and (c) prognostic value during palliative treatment. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines were followed, and meta-analysis applied on both aggregate data extraction and individual patients' data. Ten eligible cohorts were identified, including a total of 1,076 patients. Seven studies used quantitative polymerase chain reaction methods, two BEAMing [beads, emulsification, amplification, and magnetics] technology, and one study digital droplet polymerase chain reaction. The baseline levels of cfDNA was similar in the presented studies, and all studies reported a clear prognostic value in favor of patients with lowest levels of baseline cfDNA. A meta-analysis revealed a combined estimate of favorable overall survival hazard ratio (HR) in patients with levels below the median cfDNA (HR = 2.39, 95% confidence interval 2.03-2.82, p  < .0001). The total cfDNA levels are high in patients with mCRC and bear strong prognostic information, which should be tested prospectively by using a predefined cut-off value based on normal values in healthy cohorts. Finally, the potential use of cfDNA for detection of tumor-specific mutations was emphasized in a large individual patients' data meta-analysis. Reliable prognostic markers could help to guide patients and treating physicians regarding the relevance and choice of systemic therapy. Small fragments of circulating cell-free DNA (cfDNA) can be measured in a simple blood sample. This report presents the first meta-analysis of the prognostic value of total cfDNA measurement in patients with metastatic colorectal cancer. Data from 1,076 patients confirmed that patients with the lowest pre-treatment levels of cfDNA had a significantly higher chance of longer survival than those with higher levels. Cell-free DNA analysis can also be used for detection of tumor-specific mutations, and hold potential as a valuable tool in colorectal cancer treatment. © AlphaMed Press 2017.

gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group

NASA Technical Reports Server (NTRS)

La Duc, Myron T.; Satomi, Masataka; Agata, Norio; Venkateswaran, Kasthuri

2004-01-01

Bacillus anthracis, the causative agent of the human disease anthrax, Bacillus cereus, a food-borne pathogen capable of causing human illness, and Bacillus thuringiensis, a well-characterized insecticidal toxin producer, all cluster together within a very tight clade (B. cereus group) phylogenetically and are indistinguishable from one another via 16S rDNA sequence analysis. As new pathogens are continually emerging, it is imperative to devise a system capable of rapidly and accurately differentiating closely related, yet phenotypically distinct species. Although the gyrB gene has proven useful in discriminating closely related species, its sequence analysis has not yet been validated by DNA:DNA hybridization, the taxonomically accepted "gold standard". We phylogenetically characterized the gyrB sequences of various species and serotypes encompassed in the "B. cereus group," including lab strains and environmental isolates. Results were compared to those obtained from analyses of phenotypic characteristics, 16S rDNA sequence, DNA:DNA hybridization, and virulence factors. The gyrB gene proved more highly differential than 16S, while, at the same time, as analytical as costly and laborious DNA:DNA hybridization techniques in differentiating species within the B. cereus group.

FOX-superroots of Lotus corniculatus, overexpressing Arabidopsis full-length cDNA, show stable variations in morphological traits.

PubMed

Himuro, Yasuyo; Tanaka, Hidenori; Hashiguchi, Masatsugu; Ichikawa, Takanari; Nakazawa, Miki; Seki, Motoaki; Fujita, Miki; Shinozaki, Kazuo; Matsui, Minami; Akashi, Ryo; Hoffmann, Franz

2011-01-15

Using the full-length cDNA overexpressor (FOX) gene-hunting system, we have generated 130 Arabidopsis FOX-superroot lines in bird's-foot trefoil (Lotus corniculatus) for the systematic functional analysis of genes expressed in roots and for the selection of induced mutants with interesting root growth characteristics. We used the Arabidopsis-FOX Agrobacterium library (constructed by ligating pBIG2113SF) for the Agrobacterium-mediated transformation of superroots (SR) and the subsequent selection of gain-of-function mutants with ectopically expressed Arabidopsis genes. The original superroot culture of L. corniculatus is a unique host system displaying fast root growth in vitro, allowing continuous root cloning, direct somatic embryogenesis and mass regeneration of plants under entirely hormone-free culture conditions. Several of the Arabidopsis FOX-superroot lines show interesting deviations from normal growth and morphology of roots from SR-plants, such as differences in pigmentation, growth rate, length or diameter. Some of these mutations are of potential agricultural interest. Genomic PCR analysis revealed that 100 (76.9%) out of the 130 transgenic lines showed the amplification of single fragments. Sequence analysis of the PCR fragments from these 100 lines identified full-length cDNA in 74 of them. Forty-three out of 74 full-length cDNA carried known genes. The Arabidopsis FOX-superroot lines of L. corniculatus, produced in this study, expand the FOX hunting system and provide a new tool for the genetic analysis and control of root growth in a leguminous forage plant. Copyright © 2010 Elsevier GmbH. All rights reserved.

A multilevel Lab on chip platform for DNA analysis.

PubMed

Marasso, Simone Luigi; Giuri, Eros; Canavese, Giancarlo; Castagna, Riccardo; Quaglio, Marzia; Ferrante, Ivan; Perrone, Denis; Cocuzza, Matteo

2011-02-01

Lab-on-chips (LOCs) are critical systems that have been introduced to speed up and reduce the cost of traditional, laborious and extensive analyses in biological and biomedical fields. These ambitious and challenging issues ask for multi-disciplinary competences that range from engineering to biology. Starting from the aim to integrate microarray technology and microfluidic devices, a complex multilevel analysis platform has been designed, fabricated and tested (All rights reserved-IT Patent number TO2009A000915). This LOC successfully manages to interface microfluidic channels with standard DNA microarray glass slides, in order to implement a complete biological protocol. Typical Micro Electro Mechanical Systems (MEMS) materials and process technologies were employed. A silicon/glass microfluidic chip and a Polydimethylsiloxane (PDMS) reaction chamber were fabricated and interfaced with a standard microarray glass slide. In order to have a high disposable system all micro-elements were passive and an external apparatus provided fluidic driving and thermal control. The major microfluidic and handling problems were investigated and innovative solutions were found. Finally, an entirely automated DNA hybridization protocol was successfully tested with a significant reduction in analysis time and reagent consumption with respect to a conventional protocol.

An integrated strategy combining DNA walking and NGS to detect GMOs.

PubMed

Fraiture, Marie-Alice; Herman, Philippe; Papazova, Nina; De Loose, Marc; Deforce, Dieter; Ruttink, Tom; Roosens, Nancy H

2017-10-01

Recently, we developed a DNA walking system for the detection and characterization of a broad spectrum of GMOs in routine analysis of food/feed matrices. Here, we present a new version with improved throughput and sensitivity by coupling the DNA walking system to Pacific Bioscience® Next-generation sequencing technology. The performance of the new strategy was thoroughly assessed through several assays. First, we tested its detection and identification capability on grains with high or low GMO content. Second, the potential impacts of food processing were investigated using rice noodle samples. Finally, GMO mixtures and a real-life sample were analyzed to illustrate the applicability of the proposed strategy in routine GMO analysis. In all tested samples, the presence of multiple GMOs was unambiguously proven by the characterization of transgene flanking regions and the combinations of elements that are typical for transgene constructs. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mechanical Strength and Stability of DNA-modified Gold Nanoparticle Systems

NASA Astrophysics Data System (ADS)

Lam, Letisha McLaughlin

Systems in which gold nanoparticles (AuNPs) are functionalized with DNA have the potential for a broad range of applications in gene regulation therapies, drug delivery, sensing, innovative biomaterials and material templates. The use of DNA-modified gold nanoparticle (AuNP-DNA) systems is driven by their ease of assembly with bottom-up methods as well as the tunability of the systems' mechanical, optical, and electronic properties by exploiting AuNP characteristics and behavior in a multi-particle arrangement. Periodic arrangements of AuNPs precisely distributed through ligated DNA linkers may be assembled and used on relatively large length scales, on the order of hundreds of nanometers, for use in potential nanoscale technologies and applications. However, because of the size and heterogeneous composition of AuNP-DNA systems, their stability under mechanical loading is not well understood or quantified on relevant physical scales for these applications. Hence, a large-scale specialized finite-element predictive approach with a dislocation-density based crystalline plasticity has been used to investigate the mechanical stability of AuNP-DNA-ligand systems with AuNPs within the physical dimensions required for plasmon resonance. The crystalline formulation for the AuNPs accounts for multiple crystalline slip, dislocation-density evolution, lattice rotations, and large inelastic strains. A hypoelastic formulation was used for the DNA and the ligands. The nonlinear finite-element scheme is based on accounting for finite elastic and inelastic strains. These approaches were employed to predict and understand the fundamental scale-dependent microstructural behavior, the evolving heterogeneous microstructure, and localized phenomena that can contribute to failure initiation and instability. Each system was loaded using quasi-static plane strain tension and compression to simulate application loading conditions, and the elastic and inelastic evolutions were analyzed for evidence of mechanical strengthening as well as possible failure modes. To establish a foundation for AuNP-DNA stability analysis, several different two-particle conformations were investigated, including systems with pentagonally twinned AuNPs, systems with circular AuNPs, systems with non-textured and textured cuboctahedron AuNPs with 6 nm DNA, 12 nm DNA, and 18 nm DNA. In general, the analyses indicated that the systems' stability are mainly affected by large stress gradients at AuNP-ligand interfaces, as well as large dislocation-density, normal stresses, and inelastic accumulations in the region adjacent to these interfaces between the AuNPs and the DNA. The predictions also indicate that highly faceted f.c.c. AuNPs with DNA lengths of approximately 6 nm in biaxial loading conditions were found to have the highest strength and overall stability. Furthermore, periodic AuNP-DNA superlattice composites, which mimic the crystallography of f.c.c. atomic lattices, were investigated for mechanical effectiveness as both a composite material and thin film. This investigation analyzed the stress behavior and inelastic evolution of f.c.c. AuNP-DNA superlattice systems with different Au volume fractions, matrix strengths, intrinsic nanoparticle crystallographic orientations and sizes. These analyses were also extended to superlattice f.c.c. composites on a silicon substrate. The results indicate that f.c.c. AuNP-DNA superlattices have a combination of high strength and toughness due to the ductile nature of the nanoparticles in conjunction with the physical properties of the DNA and matrix materials. The superlattice films also exhibited high strengths and toughness, with the limiting factor being the interrelated aspects of film thickness and delamination. These predictions can be used as guidelines for using these composites, superlattices, and thin films as candidates for innovative building blocks for new material systems.

DNA typing for the identification of old skeletal remains from Korean War victims.

PubMed

Lee, Hwan Young; Kim, Na Young; Park, Myung Jin; Sim, Jeong Eun; Yang, Woo Ick; Shin, Kyoung-Jin

2010-11-01

The identification of missing casualties of the Korean War (1950-1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA-matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y-chromosomal STR, and mtDNA-genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini- and Y-STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains. © 2010 American Academy of Forensic Sciences.

Plasma DNA aberrations in systemic lupus erythematosus revealed by genomic and methylomic sequencing

PubMed Central

Chan, Rebecca W. Y.; Jiang, Peiyong; Peng, Xianlu; Tam, Lai-Shan; Liao, Gary J. W.; Li, Edmund K. M.; Wong, Priscilla C. H.; Sun, Hao; Chan, K. C. Allen; Chiu, Rossa W. K.; Lo, Y. M. Dennis

2014-01-01

We performed a high-resolution analysis of the biological characteristics of plasma DNA in systemic lupus erythematosus (SLE) patients using massively parallel genomic and methylomic sequencing. A number of plasma DNA abnormalities were found. First, aberrations in measured genomic representations (MGRs) were identified in the plasma DNA of SLE patients. The extent of the aberrations in MGRs correlated with anti-double–stranded DNA (anti-dsDNA) antibody level. Second, the plasma DNA of active SLE patients exhibited skewed molecular size-distribution profiles with a significantly increased proportion of short DNA fragments. The extent of plasma DNA shortening in SLE patients correlated with the SLE disease activity index (SLEDAI) and anti-dsDNA antibody level. Third, the plasma DNA of active SLE patients showed decreased methylation densities. The extent of hypomethylation correlated with SLEDAI and anti-dsDNA antibody level. To explore the impact of anti-dsDNA antibody on plasma DNA in SLE, a column-based protein G capture approach was used to fractionate the IgG-bound and non–IgG-bound DNA in plasma. Compared with healthy individuals, SLE patients had higher concentrations of IgG-bound DNA in plasma. More IgG binding occurs at genomic locations showing increased MGRs. Furthermore, the IgG-bound plasma DNA was shorter in size and more hypomethylated than the non–IgG-bound plasma DNA. These observations have enhanced our understanding of the spectrum of plasma DNA aberrations in SLE and may provide new molecular markers for SLE. Our results also suggest that caution should be exercised when interpreting plasma DNA-based noninvasive prenatal testing and cancer testing conducted for SLE patients. PMID:25427797

Automation of DNA and miRNA co-extraction for miRNA-based identification of human body fluids and tissues.

PubMed

Kulstein, Galina; Marienfeld, Ralf; Miltner, Erich; Wiegand, Peter

2016-10-01

In the last years, microRNA (miRNA) analysis came into focus in the field of forensic genetics. Yet, no standardized and recommendable protocols for co-isolation of miRNA and DNA from forensic relevant samples have been developed so far. Hence, this study evaluated the performance of an automated Maxwell® 16 System-based strategy (Promega) for co-extraction of DNA and miRNA from forensically relevant (blood and saliva) samples compared to (semi-)manual extraction methods. Three procedures were compared on the basis of recovered quantity of DNA and miRNA (as determined by real-time PCR and Bioanalyzer), miRNA profiling (shown by Cq values and extraction efficiency), STR profiles, duration, contamination risk and handling. All in all, the results highlight that the automated co-extraction procedure yielded the highest miRNA and DNA amounts from saliva and blood samples compared to both (semi-)manual protocols. Also, for aged and genuine samples of forensically relevant traces the miRNA and DNA yields were sufficient for subsequent downstream analysis. Furthermore, the strategy allows miRNA extraction only in cases where it is relevant to obtain additional information about the sample type. Besides, this system enables flexible sample throughput and labor-saving sample processing with reduced risk of cross-contamination. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a novel forensic STR multiplex for ancestry analysis and extended identity testing.

PubMed

Phillips, Chris; Fernandez-Formoso, Luis; Gelabert-Besada, Miguel; Garcia-Magariños, Manuel; Santos, Carla; Fondevila, Manuel; Carracedo, Angel; Lareu, Maria Victoria

2013-04-01

There is growing interest in developing additional DNA typing techniques to provide better investigative leads in forensic analysis. These include inference of genetic ancestry and prediction of common physical characteristics of DNA donors. To date, forensic ancestry analysis has centered on population-divergent SNPs but these binary loci cannot reliably detect DNA mixtures, common in forensic samples. Furthermore, STR genotypes, forming the principal DNA profiling system, are not routinely combined with forensic SNPs to strengthen frequency data available for ancestry inference. We report development of a 12-STR multiplex composed of ancestry informative marker STRs (AIM-STRs) selected from 434 tetranucleotide repeat loci. We adapted our online Bayesian classifier for AIM-SNPs: Snipper, to handle multiallele STR data using frequency-based training sets. We assessed the ability of the 12-plex AIM-STRs to differentiate CEPH Human Genome Diversity Panel populations, plus their informativeness combined with established forensic STRs and AIM-SNPs. We found combining STRs and SNPs improves the success rate of ancestry assignments while providing a reliable mixture detection system lacking from SNP analysis alone. As the 12 STRs generally show a broad range of alleles in all populations, they provide highly informative supplementary STRs for extended relationship testing and identification of missing persons with incomplete reference pedigrees. Lastly, mixed marker approaches (combining STRs with binary loci) for simple ancestry inference tests beyond forensic analysis bring advantages and we discuss the genotyping options available. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

PubMed

Ota, Hiroyuki; Lim, Tae-Kyu; Tanaka, Tsuyoshi; Yoshino, Tomoko; Harada, Manabu; Matsunaga, Tadashi

2006-09-18

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

DNA biosensors implemented on PNA-functionalized microstructured optical fibers Bragg gratings

NASA Astrophysics Data System (ADS)

Candiani, A.; Giannetti, S.; Cucinotta, A.; Bertucci, A.; Manicardi, A.; Konstantaki, M.; Margulis, W.; Pissadakis, S.; Corradini, R.; Selleri, S.

2013-05-01

A novel DNA sensing platform based on a Peptide Nucleic Acid - functionalized Microstructured Optical Fibers gratings has been demonstrated. The inner surface of different MOFs has been functionalized using PNA probes, OligoNucleotides mimic that are well suited for specific DNA target sequences detection. The hybrid sensing systems were tested for optical DNA detection of targets of relevance in biomedical application, using the cystic fibrosis gene mutation, and food-analysis, using the genomic DNA from genetic modified organism soy flour. After the solutions of DNA molecules has been infiltrated inside the fibers capillaries and hybridization has occurred, oligonucleotidefunctionalized gold nanoparticles were infiltrated and used to form a sandwich-like system to achieve signal amplification. Spectral measurements of the reflected signal reveal a clear wavelength shift of the reflected modes when the infiltrated complementary DNA matches with the PNA probes placed on the inner fiber surface. Measurements have also been made using the mismatched DNA solution for the c, containing a single nucleotide polymorphism, showing no significant changes in the reflected spectrum. Several experiments have been carried out demonstrating the reproducibility of the results and the high selectivity of the sensors, showing the simplicity and the potential of this approach.

Fabrication of high quality cDNA microarray using a small amount of cDNA.

PubMed

Park, Chan Hee; Jeong, Ha Jin; Jung, Jae Jun; Lee, Gui Yeon; Kim, Sang-Chul; Kim, Tae Soo; Yang, Sang Hwa; Chung, Hyun Cheol; Rha, Sun Young

2004-05-01

DNA microarray technology has become an essential part of biological research. It enables the genome-scale analysis of gene expression in various types of model systems. Manufacturing high quality cDNA microarrays of microdeposition type depends on some key factors including a printing device, spotting pins, glass slides, spotting solution, and humidity during spotting. UsingEthe Microgrid II TAS model printing device, this study defined the optimal conditions for producing high density, high quality cDNA microarrays with the least amount of cDNA product. It was observed that aminosilane-modified slides were superior to other types of surface modified-slides. A humidity of 30+/-3% in a closed environment and the overnight drying of the spotted slides gave the best conditions for arraying. In addition, the cDNA dissolved in 30% DMSO gave the optimal conditions for spotting compared to the 1X ArrayIt, 3X SSC and 50% DMSO. Lastly, cDNA in the concentration range of 100-300 ng/ micro l was determined to be best for arraying and post-processing. Currently, the printing system in this study yields reproducible 9000 spots with a spot size 150 mm diameter, and a 200 nm spot spacing.

Design, optimisation and preliminary validation of a human specific loop-mediated amplification assay for the rapid detection of human DNA at forensic crime scenes.

PubMed

Hird, H J; Brown, M K

2017-11-01

The identification of samples at a crime scene which require forensic DNA typing has been the focus of recent research interest. We propose a simple, but sensitive analysis system which can be deployed at a crime scene to identify crime scene stains as human or non-human. The proposed system uses the isothermal amplification of DNA in a rapid assay format, which returns results in as little as 30min from sampling. The assay system runs on the Genie II device, a proven in-field detection system which could be deployed at a crime scene. The results presented here demonstrate that the system was sufficiently specific and sensitive and was able to detect the presence of human blood, semen and saliva on mock forensic samples. Copyright © 2017. Published by Elsevier B.V.

Performance Characteristics and Comparison of Abbott and artus Real-Time Systems for Hepatitis B Virus DNA Quantification ▿

PubMed Central

Ismail, Ashrafali M.; Sivakumar, Jayashree; Anantharam, Raghavendran; Dayalan, Sujitha; Samuel, Prasanna; Fletcher, Gnanadurai J.; Gnanamony, Manu; Abraham, Priya

2011-01-01

Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log10 IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log10 IU/ml and limits of agreement of −0.91 to 1.11 log10 IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B. PMID:21795507

Performance characteristics and comparison of Abbott and artus real-time systems for hepatitis B virus DNA quantification.

PubMed

Ismail, Ashrafali M; Sivakumar, Jayashree; Anantharam, Raghavendran; Dayalan, Sujitha; Samuel, Prasanna; Fletcher, Gnanadurai J; Gnanamony, Manu; Abraham, Priya

2011-09-01

Virological monitoring of hepatitis B virus (HBV) DNA is critical to the management of HBV infection. With several HBV DNA quantification assays available, it is important to use the most efficient testing system for virological monitoring. In this study, we evaluated the performance characteristics and comparability of three HBV DNA quantification systems: Abbott HBV real-time PCR (Abbott PCR), artus HBV real-time PCR with QIAamp DNA blood kit purification (artus-DB), and artus HBV real-time PCR with the QIAamp DSP virus kit purification (artus-DSP). The lower limits of detection of these systems were established against the WHO international standards for HBV DNA and were found to be 1.43, 82, and 9 IU/ml, respectively. The intra-assay and interassay coefficients of variation of plasma samples (1 to 6 log(10) IU/ml) ranged between 0.05 to 8.34% and 0.16 to 3.48% for the Abbott PCR, 1.53 to 26.85% and 0.50 to 12.89% for artus-DB, and 0.29 to 7.42% and 0.94 to 3.01% for artus-DSP, respectively. Ninety HBV clinical samples were used for comparison of assays, and paired quantitative results showed strong correlation by linear regression analysis (artus-DB with Abbott PCR, r = 0.95; Abbott PCR with artus-DSP, r = 0.97; and artus-DSP with artus-DB, r = 0.94). Bland-Altman analysis showed a good level of agreement for Abbott PCR and artus-DSP, with a mean difference of 0.10 log(10) IU/ml and limits of agreement of -0.91 to 1.11 log(10) IU/ml. No genotype-specific bias was seen in all three systems for HBV genotypes A, C, and D, which are predominant in this region. This finding illustrates that the Abbott real-time HBV and artus-DSP systems show more comparable performance than the artus-DB system, meeting the current guidelines for assays to be used in the management of hepatitis B.

Rapid estimation of microbial populations in fish samples by using terminal restriction fragment length polymorphism analysis of 16S rDNA.

PubMed

Tanaka, Yuichiro; Takahashi, Hajime; Kitazawa, Nao; Kimura, Bon

2010-01-01

A rapid system using terminal restriction fragment length polymorphism (T-RFLP) analysis targeting 16S rDNA is described for microbial population analysis in edible fish samples. The defined terminal restriction fragment database was constructed by collecting 102 strains of bacteria representing 53 genera that are associated with fish. Digestion of these 102 strains with two restriction enzymes, HhaI and MspI, formed 54 pattern groups with discrimination to the genus level. This T-RFLP system produced results comparable to those from a culture-based method in six natural fish samples with a qualitative correspondence of 71.4 to 92.3%. Using the T-RFLP system allowed an estimation of the microbial population within 7 h. Rapid assay of the microbial population is advantageous for food manufacturers and testing laboratories; moreover, the strategy presented here allows adaptation to specific testing applications.

Fiber optic chemical sensors: The evolution of high- density fiber-optic DNA microarrays

NASA Astrophysics Data System (ADS)

Ferguson, Jane A.

2001-06-01

Sensors were developed for multianalyte monitoring, fermentation monitoring, lactate analysis, remote oxygen detection for use in bioremediation monitoring and in a fuel spill clean-up project, heavy metal analysis, and high density DNA microarrays. The major focus of this thesis involved creating and improving high-density DNA gene arrays. Fiber optic sensors are created using fluorescent indicators, polymeric supports, and optical fiber substrates. The fluorescent indicator is entrapped in a polymer layer and attached to the tip of the optical fiber. The tip of the fiber bearing the sensing layer (the distal end) is placed in the sample of interest while the other end of the fiber (the proximal end) is connected to an analysis system. Any length of fiber can be used without compromising the integrity or sensitivity of the system. A fiber optic oxygen sensor was designed incorporating an oxygen sensitive fluorescent dye and a gas permeable polymer attached to an optical fiber. The construction simplicity and ruggedness of the sensor enabled its deployment for in situ chemical oxidation and bioremediation studies. Optical fibers were also used as the substrate to detect biomolecules in solution. To monitor bioprocesses, the production of the analyte of interest must be coupled with a species that is optically measurable. For example, oxygen is consumed in many metabolic functions. The fiber optic oxygen sensor is equipped with an additional sensing layer. Upon contact with a specific biochemical in the sample, a reaction occurs in the additional sensing layer that either consumes or produces oxygen. This dual layer system was used to monitor the presence of lactate, an important metabolite for clinical and bioprocess analysis. In many biological and environmental systems, the generation of one species occurs coincidentally with the generation or consumption of another species. A multianalyte sensor was prepared that can monitor the simultaneous activity of pH, CO2 and O2. This sensor is useful for monitoring bioprocesses such as (beer) fermentation and for clinical situations such as blood gas analysis. DNA sensors were created by attaching short single strands of DNA (probes) to the fiber tip. A matching single strand (target) forms a strong interacting pair with the probe upon contact. The target strands in a sample are labeled with a fluorescent dye. When a probe-target pair is formed and excitation light is sent down the fiber, the fiber bearing the pair emits light that is captured and detected. A high density DNA array was created by isolating thousands of discrete DNA sensors on the tip of an imaging optical fiber. This array was made possible by the formation of microwells on the imaging fiber tip. Microspheres functionalized with DNA were placed in the wells of the fiber and each microsphere was independently and simultaneously monitored. (Abstract shortened by UMI.)

Development of a 20-locus fluorescent multiplex system as a valuable tool for national DNA database.

PubMed

Jiang, Xianhua; Guo, Fei; Jia, Fei; Jin, Ping; Sun, Zhu

2013-02-01

The multiplex system allows the detection of 19 autosomal short tandem repeat (STR) loci [including all Combined DNA Index System (CODIS) STR loci as well as D2S1338, D6S1043, D12S391, D19S433, Penta D and Penta E] plus the sex-determining locus Amelogenin in a single reaction, comprising all STR loci in various commercial kits used in the China national DNA database (NDNAD). Primers are designed so that the amplicons are distributed ranging from 90 base pairs (bp) to 450 bp within a five-dye fluorescent design with the fifth dye reserved for the internal size standard. With 30 cycles, 125 pg to 2 ng DNA template showed optimal profiling result, while robust profiles could also be achieved by adjusting the cycle numbers for the DNA template beyond that optimal DNA input range. Mixture studies showed that 83% and 87% of minor alleles were detected at 9:1 and 1:9 ratios, respectively. When 4 ng of degraded DNA was digested by 2-min DNase and 1 ng undegraded DNA was added to 400 μM haematin, the complete profiles were still observed. Polymerase chain reaction (PCR)-based procedures were examined and optimized including the concentrations of primer set, magnesium and the Taq polymerase as well as volume, cycle number and annealing temperature. In addition, the system has been validated by 3000 bloodstain samples and 35 common case samples in line with the Chinese National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The total probability of identity (TPI) can reach to 8×10(-24), where DNA database can be improved at the level of 10 million DNA profiles or more because the number of expected match is far from one person (4×10(-10)) and can be negligible. Further, our system also demonstrates its good performance in case samples and it will be an ideal tool for forensic DNA typing and databasing with potential application. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The structure and intermolecular forces of DNA condensates.

PubMed

Yoo, Jejoong; Aksimentiev, Aleksei

2016-03-18

Spontaneous assembly of DNA molecules into compact structures is ubiquitous in biological systems. Experiment has shown that polycations can turn electrostatic self-repulsion of DNA into attraction, yet the physical mechanism of DNA condensation has remained elusive. Here, we report the results of atomistic molecular dynamics simulations that elucidated the microscopic structure of dense DNA assemblies and the physics of interactions that makes such assemblies possible. Reproducing the setup of the DNA condensation experiments, we measured the internal pressure of DNA arrays as a function of the DNA-DNA distance, showing a quantitative agreement between the results of our simulations and the experimental data. Analysis of the MD trajectories determined the DNA-DNA force in a DNA condensate to be pairwise, the DNA condensation to be driven by electrostatics of polycations and not hydration, and the concentration of bridging cations, not adsorbed cations, to determine the magnitude and the sign of the DNA-DNA force. Finally, our simulations quantitatively characterized the orientational correlations of DNA in DNA arrays as well as diffusive motion of DNA and cations. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Affordable hands-on DNA sequencing and genotyping: an exercise for teaching DNA analysis to undergraduates.

PubMed

Shah, Kushani; Thomas, Shelby; Stein, Arnold

2013-01-01

In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C Sanger sequencing reactions. They prepare and run the gels, perform Southern blots (which require only 10 min), and detect sequencing ladders using a colorimetric detection system. Students enlarge their sequencing ladders from digital images of their small nylon membranes, and read the sequence manually. They compare their reads with the actual DNA sequence using BLAST2. After mastering the DNA sequencing system, students prepare their own DNA from a cheek swab, polymerase chain reaction-amplify a region of their DNA that encompasses a SNP of interest, and perform sequencing to determine their genotype at the SNP position. A family pedigree can also be constructed. The SNP chosen by the instructor was rs17822931, which is in the ABCC11 gene and is the determinant of human earwax type. Genotypes at the rs178229931 site vary in different ethnic populations. © 2013 by The International Union of Biochemistry and Molecular Biology.

A Single-Molecule Barcoding System using Nanoslits for DNA Analysis

NASA Astrophysics Data System (ADS)

Jo, Kyubong; Schramm, Timothy M.; Schwartz, David C.

Single DNA molecule approaches are playing an increasingly central role in the analytical genomic sciences because single molecule techniques intrinsically provide individualized measurements of selected molecules, free from the constraints of bulk techniques, which blindly average noise and mask the presence of minor analyte components. Accordingly, a principal challenge that must be addressed by all single molecule approaches aimed at genome analysis is how to immobilize and manipulate DNA molecules for measurements that foster construction of large, biologically relevant data sets. For meeting this challenge, this chapter discusses an integrated approach for microfabricated and nanofabricated devices for the manipulation of elongated DNA molecules within nanoscale geometries. Ideally, large DNA coils stretch via nanoconfinement when channel dimensions are within tens of nanometers. Importantly, stretched, often immobilized, DNA molecules spanning hundreds of kilobase pairs are required by all analytical platforms working with large genomic substrates because imaging techniques acquire sequence information from molecules that normally exist in free solution as unrevealing random coils resembling floppy balls of yarn. However, nanoscale devices fabricated with sufficiently small dimensions fostering molecular stretching make these devices impractical because of the requirement of exotic fabrication technologies, costly materials, and poor operational efficiencies. In this chapter, such problems are addressed by discussion of a new approach to DNA presentation and analysis that establishes scaleable nanoconfinement conditions through reduction of ionic strength; stiffening DNA molecules thus enabling their arraying for analysis using easily fabricated devices that can also be mass produced. This new approach to DNA nanoconfinement is complemented by the development of a novel labeling scheme for reliable marking of individual molecules with fluorochrome labels, creating molecular barcodes, which are efficiently read using fluorescence resonance energy transfer techniques for minimizing noise from unincorporated labels. As such, our integrative approach for the realization of genomic analysis through nanoconfinement, named nanocoding, was demonstrated through the barcoding and mapping of bacterial artificial chromosomal molecules, thereby providing the basis for a high-throughput platform competent for whole genome investigations.

Detection of irradiated quail meat by using DNA comet assay and evaluation of comets by image analysis

NASA Astrophysics Data System (ADS)

Erel, Yakup; Yazici, Nizamettin; Özvatan, Sumer; Ercin, Demet; Cetinkaya, Nurcan

2009-09-01

A simple technique of microgel electrophoresis of single cells (DNA comet assay) was used to detect DNA comets in irradiated quail meat samples. Obtained DNA comets were evaluated by both photomicrographic and image analysis. Quail meat samples were exposed to radiation doses of 0.52, 1.05, 1.45, 2.00, 2.92 and 4.00 kGy in gamma cell (gammacell 60Co, dose rate 1.31 kGy/h) covering the permissible limits for enzymatic decay and stored at 2 °C. The cells isolated from muscle (chest, thorax) in cold PBS were analyzed using the DNA comet assay on 1, 2, 3, 4, 7, 8 and 11 day post irradiation. The cells were lysed between 2, 5 and 9 min in 2.5% SDS and electrophorosis was carried out at a voltage of 2 V/cm for 2 min. After propidium iodide staining, the slides were evaluated through a fluorescent microscope. In all irradiated samples, fragmented DNA stretched towards the anode and damaged cells appeared as a comet. All measurement data were analyzed using BS 200 ProP with software image analysis (BS 200 ProP, BAB Imaging System, Ankara, Turkey). The density of DNA in the tails increased with increasing radiation dose. However, in non-irradiated samples, the large molecules of DNA remained relatively intact and there was only minor or no migration of DNA; the cells were round or had very short tails only. The values of tail DNA%, tail length and tail moment were significantly different and identical between 0.9 and 4.0 kGy dose exposure, and also among storage times on day 1, 4 and 8. In conclusion, the DNA Comet Assay EN 13784 standard method may be used not only for screening method for detection of irradiated quail meat depending on storage time and condition but also for the quantification of applied dose if it is combined with image analysis. Image analysis may provide a powerful tool for the evaluation of head and tail of comet intensity related with applied doses.

Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.

PubMed

Borysov, Sergiy; Bryant, Victoria L; Alexandrow, Mark G

2015-01-01

Of critical importance to many of the events underlying transcriptional control of gene expression are modifications to core and linker histones that regulate the accessibility of trans-acting factors to the DNA substrate within the context of chromatin. Likewise, control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is known to involve the creation of chromatin accessibility. In the latter case, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility. Here, we outline a novel experimental strategy for addressing the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. Our laboratory has used this powerful system in novel ways to directly address the ability of DNA replication proteins to create chromatin accessibility, and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the mechanisms and associated factors involved in causing chromatin decondensation by a protein of interest. Alternative approaches involving co-expression of other proteins (competitors or stimulators), concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, all of which are illustrative of the utility of this experimental system for extending basic findings to physiologically relevant mechanisms. Although used by our group to analyze mechanisms underlying DNA replication associated chromatin accessibility, this unique and powerful experimental system has the propensity to be a valuable tool for understanding chromatin remodeling mechanisms orchestrated by other cellular processes such as DNA repair, recombination, mitotic chromosome condensation, or other chromosome dynamics involving chromatin alterations and accessibility.

Real-time PCR machine system modeling and a systematic approach for the robust design of a real-time PCR-on-a-chip system.

PubMed

Lee, Da-Sheng

2010-01-01

Chip-based DNA quantification systems are widespread, and used in many point-of-care applications. However, instruments for such applications may not be maintained or calibrated regularly. Since machine reliability is a key issue for normal operation, this study presents a system model of the real-time Polymerase Chain Reaction (PCR) machine to analyze the instrument design through numerical experiments. Based on model analysis, a systematic approach was developed to lower the variation of DNA quantification and achieve a robust design for a real-time PCR-on-a-chip system. Accelerated lift testing was adopted to evaluate the reliability of the chip prototype. According to the life test plan, this proposed real-time PCR-on-a-chip system was simulated to work continuously for over three years with similar reproducibility in DNA quantification. This not only shows the robustness of the lab-on-a-chip system, but also verifies the effectiveness of our systematic method for achieving a robust design.

The Web-Based DNA Vaccine Database DNAVaxDB and Its Usage for Rational DNA Vaccine Design.

PubMed

Racz, Rebecca; He, Yongqun

2016-01-01

A DNA vaccine is a vaccine that uses a mammalian expression vector to express one or more protein antigens and is administered in vivo to induce an adaptive immune response. Since the 1990s, a significant amount of research has been performed on DNA vaccines and the mechanisms behind them. To meet the needs of the DNA vaccine research community, we created DNAVaxDB ( http://www.violinet.org/dnavaxdb ), the first Web-based database and analysis resource of experimentally verified DNA vaccines. All the data in DNAVaxDB, which includes plasmids, antigens, vaccines, and sources, is manually curated and experimentally verified. This chapter goes over the detail of DNAVaxDB system and shows how the DNA vaccine database, combined with the Vaxign vaccine design tool, can be used for rational design of a DNA vaccine against a pathogen, such as Mycobacterium bovis.

Hda Monomerization by ADP Binding Promotes Replicase Clamp-mediated DnaA-ATP Hydrolysis*S⃞

PubMed Central

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-01-01

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only ∼100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain. PMID:18977760

Hda monomerization by ADP binding promotes replicase clamp-mediated DnaA-ATP hydrolysis.

PubMed

Su'etsugu, Masayuki; Nakamura, Kenta; Keyamura, Kenji; Kudo, Yuka; Katayama, Tsutomu

2008-12-26

ATP-DnaA is the initiator of chromosomal replication in Escherichia coli, and the activity of DnaA is regulated by the regulatory inactivation of the DnaA (RIDA) system. In this system, the Hda protein promotes DnaA-ATP hydrolysis to produce inactive ADP-DnaA in a mechanism that is mediated by the DNA-loaded form of the replicase sliding clamp. In this study, we first revealed that hda translation uses an unusual initiation codon, CUG, located downstream of the annotated initiation codon. The CUG initiation codon could be used for restricting the Hda level, as this initiation codon has a low translation efficiency, and the cellular Hda level is only approximately 100 molecules per cell. Hda translated using the correct reading frame was purified and found to have a high RIDA activity in vitro. Moreover, we found that Hda has a high affinity for ADP but not for other nucleotides, including ATP. ADP-Hda was active in the RIDA system in vitro and stable in a monomeric state, whereas apo-Hda formed inactive homomultimers. Both ADP-Hda and apo-Hda could form complexes with the DNA-loaded clamp; however, only ADP-Hda-DNA-clamp complexes were highly functional in the following interaction with DnaA. Formation of ADP-Hda was also observed in vivo, and mutant analysis suggested that ADP binding is crucial for cellular Hda activity. Thus, we propose that ADP is a crucial Hda ligand that promotes the activated conformation of the protein. ADP-dependent monomerization might enable the arginine finger of the Hda AAA+ domain to be accessible to ATP bound to the DnaA AAA+ domain.

DNA methylation analysis of phenotype specific stratified Indian population.

PubMed

Rotti, Harish; Mallya, Sandeep; Kabekkodu, Shama Prasada; Chakrabarty, Sanjiban; Bhale, Sameer; Bharadwaj, Ramachandra; Bhat, Balakrishna K; Dedge, Amrish P; Dhumal, Vikram Ram; Gangadharan, G G; Gopinath, Puthiya M; Govindaraj, Periyasamy; Joshi, Kalpana S; Kondaiah, Paturu; Nair, Sreekumaran; Nair, S N Venugopalan; Nayak, Jayakrishna; Prasanna, B V; Shintre, Pooja; Sule, Mayura; Thangaraj, Kumarasamy; Patwardhan, Bhushan; Valiathan, Marthanda Varma Sankaran; Satyamoorthy, Kapaettu

2015-05-08

DNA methylation and its perturbations are an established attribute to a wide spectrum of phenotypic variations and disease conditions. Indian traditional system practices personalized medicine through indigenous concept of distinctly descriptive physiological, psychological and anatomical features known as prakriti. Here we attempted to establish DNA methylation differences in these three prakriti phenotypes. Following structured and objective measurement of 3416 subjects, whole blood DNA of 147 healthy male individuals belonging to defined prakriti (Vata, Pitta and Kapha) between the age group of 20-30years were subjected to methylated DNA immunoprecipitation (MeDIP) and microarray analysis. After data analysis, prakriti specific signatures were validated through bisulfite DNA sequencing. Differentially methylated regions in CpG islands and shores were significantly enriched in promoters/UTRs and gene body regions. Phenotypes characterized by higher metabolism (Pitta prakriti) in individuals showed distinct promoter (34) and gene body methylation (204), followed by Vata prakriti which correlates to motion showed DNA methylation in 52 promoters and 139 CpG islands and finally individuals with structural attributes (Kapha prakriti) with 23 and 19 promoters and CpG islands respectively. Bisulfite DNA sequencing of prakriti specific multiple CpG sites in promoters and 5'-UTR such as; LHX1 (Vata prakriti), SOX11 (Pitta prakriti) and CDH22 (Kapha prakriti) were validated. Kapha prakriti specific CDH22 5'-UTR CpG methylation was also found to be associated with higher body mass index (BMI). Differential DNA methylation signatures in three distinct prakriti phenotypes demonstrate the epigenetic basis of Indian traditional human classification which may have relevance to personalized medicine.

Methylation-sensitive amplified polymorphism analysis of Verticillium wilt-stressed cotton (Gossypium).

PubMed

Wang, W; Zhang, M; Chen, H D; Cai, X X; Xu, M L; Lei, K Y; Niu, J H; Deng, L; Liu, J; Ge, Z J; Yu, S X; Wang, B H

2016-10-06

In this study, a methylation-sensitive amplification polymorphism analysis system was used to analyze DNA methylation level in three cotton accessions. Two disease-sensitive near-isogenic lines, PD94042 and IL41, and one disease-resistant Gossypium mustelinum accession were exposed to Verticillium wilt, to investigate molecular disease resistance mechanisms in cotton. We observed multiple different DNA methylation types across the three accessions following Verticillium wilt exposure. These included hypomethylation, hypermethylation, and other patterns. In general, the global DNA methylation level was significantly increased in the disease-resistant accession G. mustelinum following disease exposure. In contrast, there was no significant difference in the disease-sensitive accession PD94042, and a significant decrease was observed in IL41. Our results suggest that disease-resistant cotton might employ a mechanism to increase methylation level in response to disease stress. The differing methylation patterns, together with the increase in global DNA methylation level, might play important roles in tolerance to Verticillium wilt in cotton. Through cloning and analysis of differently methylated DNA sequences, we were also able to identify several genes that may contribute to disease resistance in cotton. Our results revealed the effect of DNA methylation on cotton disease resistance, and also identified genes that played important roles, which may shed light on the future cotton disease-resistant molecular breeding.

Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis.

PubMed

Maruyama, Kohei; Takeyama, Haruko; Nemoto, Etsuo; Tanaka, Tsuyoshi; Yoda, Kiyoshi; Matsunaga, Tadashi

2004-09-20

Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs. Copyright 2004 Wiley Periodicals, Inc.

DNA methylation in amphioxus: from ancestral functions to new roles in vertebrates.

PubMed

Albalat, Ricard; Martí-Solans, Josep; Cañestro, Cristian

2012-03-01

In vertebrates, DNA methylation is an epigenetic mechanism that modulates gene transcription, and plays crucial roles during development, cell fate maintenance, germ cell pluripotency and inheritable genome imprinting. DNA methylation might also play a role as a genome defense mechanism against the mutational activity derived from transposon mobility. In contrast to the heavily methylated genomes in vertebrates, most genomes in invertebrates are poorly or just moderately methylated, and the function of DNA methylation remains unclear. Here, we review the DNA methylation system in the cephalochordate amphioxus, which belongs to the most basally divergent group of our own phylum, the chordates. First, surveys of the amphioxus genome database reveal the presence of the DNA methylation machinery, DNA methyltransferases and methyl-CpG-binding domain proteins. Second, comparative genomics and analyses of conserved synteny between amphioxus and vertebrates provide robust evidence that the DNA methylation machinery of amphioxus represents the ancestral toolkit of chordates, and that its expansion in vertebrates was originated by the two rounds of whole-genome duplication that occurred in stem vertebrates. Third, in silico analysis of CpGo/e ratios throughout the amphioxus genome suggests a bimodal distribution of DNA methylation, consistent with a mosaic pattern comprising domains of methylated DNA interspersed with domains of unmethylated DNA, similar to the situation described in ascidians, but radically different to the globally methylated vertebrate genomes. Finally, we discuss potential roles of the DNA methylation system in amphioxus in the context of chordate genome evolution and the origin of vertebrates.

Significance of DNA bond strength in programmable nanoparticle thermodynamics and dynamics.

PubMed

Yu, Qiuyan; Hu, Jinglei; Hu, Yi; Wang, Rong

2018-04-04

Assembly of nanoparticles (NPs) coated with complementary DNA strands leads to novel crystals with nanosized basic units rather than classic atoms, ions or molecules. The assembly process is mediated by hybridization of DNA via specific base pairing interaction, and is kinetically linked to the disassociation of DNA duplexes. DNA-level physiochemical quantities, both thermodynamic and kinetic, are key to understanding this process and essential for the design of DNA-NP crystals. The melting transition properties are helpful to judge the thermostability and sensitivity of relative DNA probes or other applications. Three different cases are investigated by changing the linker length and the spacer length on which the melting properties depend using the molecular dynamics method. Melting temperature is determined by sigmoidal melting curves based on hybridization percentage versus temperature and the Lindemann melting rule simultaneously. We provide a computational strategy based on a coarse-grained model to estimate the hybridization enthalpy, entropy and free energy from percentages of hybridizations which are readily accessible in experiments. Importantly, the lifetime of DNA bond dehybridization based on temperature and the activation energy depending on DNA bond strength are also calculated. The simulation results are in good agreement with the theoretical analysis and the present experimental data. Our study provides a good strategy to predict the melting temperature which is important for the DNA-directed nanoparticle system, and bridges the dynamics and thermodynamics of DNA-directed nanoparticle systems by estimating the equilibrium constant from the hybridization of DNA bonds quantitatively.

Characterization of Aspergillus flavus strains from Brazilian Brazil nuts and cashew by RAPD and ribosomal DNA analysis.

PubMed

Midorikawa, G E O; Pinheiro, M R R; Vidigal, B S; Arruda, M C; Costa, F F; Pappas, G J; Ribeiro, S G; Freire, F; Miller, R N G

2008-07-01

The aim of this study was to determine the genetic variability in Aspergillus flavus populations from Brazil nut and cashew and develop a polymerase chain reaction (PCR) detection method. Chomatography analysis of 48 isolates identified 36 as aflatoxigenic (75%). One hundred and forty-one DNA bands were generated with 11 random amplified polymorphic DNA (RAPD) primers and analysed via unweighted pair group analysis, using arithmetic means (UPGMA). Isolates grouped according to host, with differentiation of those from A. occidentale also according to geographical origin. Aspergillus flavus-specific PCR primers ASPITSF2 and ASPITSR3 were designed from ribosomal DNA internal transcribed spacers (ITS 1 and 2), and an internal amplification control was developed, to prevent false negative results. Specificity to only A. flavus was confirmed against DNA from additional aspergilli and other fungi. RAPD-based characterization differentiated isolates according to plant host. The PCR primer pair developed showed specificity to A. flavus, with a detection limit of 10 fg. Genetic variability observed in A. flavus isolates from two Brazilian agroecosystems suggested reproductive isolation. The PCR detection method developed for A. flavus represents progress towards multiplex PCR detection of aflatoxigenic and nonaflatoxigenic strains in Hazard Analysis Critical Control Point systems.

Direct bisulfite sequencing for examination of DNA methylation with gene and nucleotide resolution from brain tissues.

PubMed

Parrish, R Ryley; Day, Jeremy J; Lubin, Farah D

2012-07-01

DNA methylation is an epigenetic modification that is essential for the development and mature function of the central nervous system. Due to the relevance of this modification to the transcriptional control of gene expression, it is often necessary to examine changes in DNA methylation patterns with both gene and single-nucleotide resolution. Here, we describe an in-depth basic protocol for direct bisulfite sequencing of DNA isolated from brain tissue, which will permit direct assessment of methylation status at individual genes as well as individual cytosine molecules/nucleotides within a genomic region. This method yields analysis of DNA methylation patterns that is robust, accurate, and reproducible, thereby allowing insights into the role of alterations in DNA methylation in brain tissue.

Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dong-Shan; Zhou, Guo-Hua; Luo, Ming; Ji, Xing-Hu; He, Zhi-Ke

2012-08-21

We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.

Use of wavelet-packet transforms to develop an engineering model for multifractal characterization of mutation dynamics in pathological and nonpathological gene sequences

NASA Astrophysics Data System (ADS)

Walker, David Lee

1999-12-01

This study uses dynamical analysis to examine in a quantitative fashion the information coding mechanism in DNA sequences. This exceeds the simple dichotomy of either modeling the mechanism by comparing DNA sequence walks as Fractal Brownian Motion (fbm) processes. The 2-D mappings of the DNA sequences for this research are from Iterated Function System (IFS) (Also known as the ``Chaos Game Representation'' (CGR)) mappings of the DNA sequences. This technique converts a 1-D sequence into a 2-D representation that preserves subsequence structure and provides a visual representation. The second step of this analysis involves the application of Wavelet Packet Transforms, a recently developed technique from the field of signal processing. A multi-fractal model is built by using wavelet transforms to estimate the Hurst exponent, H. The Hurst exponent is a non-parametric measurement of the dynamism of a system. This procedure is used to evaluate gene- coding events in the DNA sequence of cystic fibrosis mutations. The H exponent is calculated for various mutation sites in this gene. The results of this study indicate the presence of anti-persistent, random walks and persistent ``sub-periods'' in the sequence. This indicates the hypothesis of a multi-fractal model of DNA information encoding warrants further consideration. This work examines the model's behavior in both pathological (mutations) and non-pathological (healthy) base pair sequences of the cystic fibrosis gene. These mutations both natural and synthetic were introduced by computer manipulation of the original base pair text files. The results show that disease severity and system ``information dynamics'' correlate. These results have implications for genetic engineering as well as in mathematical biology. They suggest that there is scope for more multi-fractal models to be developed.

AQME: A forensic mitochondrial DNA analysis tool for next-generation sequencing data.

PubMed

Sturk-Andreaggi, Kimberly; Peck, Michelle A; Boysen, Cecilie; Dekker, Patrick; McMahon, Timothy P; Marshall, Charla K

2017-11-01

The feasibility of generating mitochondrial DNA (mtDNA) data has expanded considerably with the advent of next-generation sequencing (NGS), specifically in the generation of entire mtDNA genome (mitogenome) sequences. However, the analysis of these data has emerged as the greatest challenge to implementation in forensics. To address this need, a custom toolkit for use in the CLC Genomics Workbench (QIAGEN, Hilden, Germany) was developed through a collaborative effort between the Armed Forces Medical Examiner System - Armed Forces DNA Identification Laboratory (AFMES-AFDIL) and QIAGEN Bioinformatics. The AFDIL-QIAGEN mtDNA Expert, or AQME, generates an editable mtDNA profile that employs forensic conventions and includes the interpretation range required for mtDNA data reporting. AQME also integrates an mtDNA haplogroup estimate into the analysis workflow, which provides the analyst with phylogenetic nomenclature guidance and a profile quality check without the use of an external tool. Supplemental AQME outputs such as nucleotide-per-position metrics, configurable export files, and an audit trail are produced to assist the analyst during review. AQME is applied to standard CLC outputs and thus can be incorporated into any mtDNA bioinformatics pipeline within CLC regardless of sample type, library preparation or NGS platform. An evaluation of AQME was performed to demonstrate its functionality and reliability for the analysis of mitogenome NGS data. The study analyzed Illumina mitogenome data from 21 samples (including associated controls) of varying quality and sample preparations with the AQME toolkit. A total of 211 tool edits were automatically applied to 130 of the 698 total variants reported in an effort to adhere to forensic nomenclature. Although additional manual edits were required for three samples, supplemental tools such as mtDNA haplogroup estimation assisted in identifying and guiding these necessary modifications to the AQME-generated profile. Along with profile generation, AQME reported accurate haplogroups for 18 of the 19 samples analyzed. The single errant haplogroup assignment, although phylogenetically close, identified a bug that only affects partial mitogenome data. Future adjustments to AQME's haplogrouping tool will address this bug as well as enhance the overall scoring strategy to better refine and automate haplogroup assignments. As NGS enables broader use of the mtDNA locus in forensics, the availability of AQME and other forensic-focused mtDNA analysis tools will ease the transition and further support mitogenome analysis within routine casework. Toward this end, the AFMES-AFDIL has utilized the AQME toolbox in conjunction with the CLC Genomics Workbench to successfully validate and implement two NGS mitogenome methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Availability: A Metric for Nucleic Acid Strand Displacement Systems.

PubMed

Olson, Xiaoping; Kotani, Shohei; Padilla, Jennifer E; Hallstrom, Natalya; Goltry, Sara; Lee, Jeunghoon; Yurke, Bernard; Hughes, William L; Graugnard, Elton

2017-01-20

DNA strand displacement systems have transformative potential in synthetic biology. While powerful examples have been reported in DNA nanotechnology, such systems are plagued by leakage, which limits network stability, sensitivity, and scalability. An approach to mitigate leakage in DNA nanotechnology, which is applicable to synthetic biology, is to introduce mismatches to complementary fuel sequences at key locations. However, this method overlooks nuances in the secondary structure of the fuel and substrate that impact the leakage reaction kinetics in strand displacement systems. In an effort to quantify the impact of secondary structure on leakage, we introduce the concepts of availability and mutual availability and demonstrate their utility for network analysis. Our approach exposes vulnerable locations on the substrate and quantifies the secondary structure of fuel strands. Using these concepts, a 4-fold reduction in leakage has been achieved. The result is a rational design process that efficiently suppresses leakage and provides new insight into dynamic nucleic acid networks.

Evaluation of the branched-chain DNA assay for measurement of RNA in formalin-fixed tissues.

PubMed

Knudsen, Beatrice S; Allen, April N; McLerran, Dale F; Vessella, Robert L; Karademos, Jonathan; Davies, Joan E; Maqsodi, Botoul; McMaster, Gary K; Kristal, Alan R

2008-03-01

We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93-100%) than for qPCR (82.4-95%). Correlations between qPCR(FROZEN), the gold standard, and bDNA(FFPE) ranged from 0.60 to 0.94, similar to those from qPCR(FROZEN) and qPCR(FFPE). Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management.

Developmental validation of the HIrisPlex system: DNA-based eye and hair colour prediction for forensic and anthropological usage.

PubMed

Walsh, Susan; Chaitanya, Lakshmi; Clarisse, Lindy; Wirken, Laura; Draus-Barini, Jolanta; Kovatsi, Leda; Maeda, Hitoshi; Ishikawa, Takaki; Sijen, Titia; de Knijff, Peter; Branicki, Wojciech; Liu, Fan; Kayser, Manfred

2014-03-01

Forensic DNA Phenotyping or 'DNA intelligence' tools are expected to aid police investigations and find unknown individuals by providing information on externally visible characteristics of unknown suspects, perpetrators and missing persons from biological samples. This is especially useful in cases where conventional DNA profiling or other means remain non-informative. Recently, we introduced the HIrisPlex system, capable of predicting both eye and hair colour from DNA. In the present developmental validation study, we demonstrate that the HIrisPlex assay performs in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines providing an essential prerequisite for future HIrisPlex applications to forensic casework. The HIrisPlex assay produces complete profiles down to only 63 pg of DNA. Species testing revealed human specificity for a complete HIrisPlex profile, while only non-human primates showed the closest full profile at 20 out of the 24 DNA markers, in all animals tested. Rigorous testing of simulated forensic casework samples such as blood, semen, saliva stains, hairs with roots as well as extremely low quantity touch (trace) DNA samples, produced complete profiles in 88% of cases. Concordance testing performed between five independent forensic laboratories displayed consistent reproducible results on varying types of DNA samples. Due to its design, the assay caters for degraded samples, underlined here by results from artificially degraded DNA and from simulated casework samples of degraded DNA. This aspect was also demonstrated previously on DNA samples from human remains up to several hundreds of years old. With this paper, we also introduce enhanced eye and hair colour prediction models based on enlarged underlying databases of HIrisPlex genotypes and eye/hair colour phenotypes (eye colour: N = 9188 and hair colour: N = 1601). Furthermore, we present an online web-based system for individual eye and hair colour prediction from full and partial HIrisPlex DNA profiles. By demonstrating that the HIrisPlex assay is fully compatible with the SWGDAM guidelines, we provide the first forensically validated DNA test system for parallel eye and hair colour prediction now available to forensic laboratories for immediate casework application, including missing person cases. Given the robustness and sensitivity described here and in previous work, the HIrisPlex system is also suitable for analysing old and ancient DNA in anthropological and evolutionary studies. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Developmental validation of the IrisPlex system: determination of blue and brown iris colour for forensic intelligence.

PubMed

Walsh, Susan; Lindenbergh, Alexander; Zuniga, Sofia B; Sijen, Titia; de Knijff, Peter; Kayser, Manfred; Ballantyne, Kaye N

2011-11-01

The IrisPlex system consists of a highly sensitive multiplex genotyping assay together with a statistical prediction model, providing users with the ability to predict blue and brown human eye colour from DNA samples with over 90% precision. This 'DNA intelligence' system is expected to aid police investigations by providing phenotypic information on unknown individuals when conventional DNA profiling is not informative. Falling within the new area of forensic DNA phenotyping, this paper describes the developmental validation of the IrisPlex assay following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines for the application of DNA-based eye colour prediction to forensic casework. The IrisPlex assay produces complete SNP genotypes with only 31pg of DNA, approximately six human diploid cell equivalents, and is therefore more sensitive than commercial STR kits currently used in forensics. Species testing revealed human and primate specificity for a complete SNP profile. The assay is capable of producing accurate results from simulated casework samples such as blood, semen, saliva, hair, and trace DNA samples, including extremely low quantity samples. Due to its design, it can also produce full profiles with highly degraded samples often found in forensic casework. Concordance testing between three independent laboratories displayed reproducible results of consistent levels on varying types of simulated casework samples. With such high levels of sensitivity, specificity, consistency and reliability, this genotyping assay, as a core part of the IrisPlex system, operates in accordance with SWGDAM guidelines. Furthermore, as we demonstrated previously, the IrisPlex eye colour prediction system provides reliable results without the need for knowledge on the bio-geographic ancestry of the sample donor. Hence, the IrisPlex system, with its model-based prediction probability estimation of blue and brown human eye colour, represents a useful tool for immediate application in accredited forensic laboratories, to be used for forensic intelligence in tracing unknown individuals from crime scene samples. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Allosteric analysis of glucocorticoid receptor-DNA interface induced by cyclic Py-Im polyamide: a molecular dynamics simulation study.

PubMed

Wang, Yaru; Ma, Na; Wang, Yan; Chen, Guangju

2012-01-01

It has been extensively developed in recent years that cell-permeable small molecules, such as polyamide, can be programmed to disrupt transcription factor-DNA interfaces and can silence aberrant gene expression. For example, cyclic pyrrole-imidazole polyamide that competes with glucocorticoid receptor (GR) for binding to glucocorticoid response elements could be expected to affect the DNA dependent binding by interfering with the protein-DNA interface. However, how such small molecules affect the transcription factor-DNA interfaces and gene regulatory pathways through DNA structure distortion is not fully understood so far. In the present work, we have constructed some models, especially the ternary model of polyamides+DNA+GR DNA-binding domain (GRDBD) dimer, and carried out molecular dynamics simulations and free energy calculations for them to address how polyamide molecules disrupt the GRDBD and DNA interface when polyamide and protein bind at the same sites on opposite grooves of DNA. We found that the cyclic polyamide binding in minor groove of DNA can induce a large structural perturbation of DNA, i.e. a >4 Å widening of the DNA minor groove and a compression of the major groove by more than 4 Å as compared with the DNA molecule in the GRDBD dimer+DNA complex. Further investigations for the ternary system of polyamides+DNA+GRDBD dimer and the binary system of allosteric DNA+GRDBD dimer revealed that the compression of DNA major groove surface causes GRDBD to move away from the DNA major groove with the initial average distance of ∼4 Å to the final average distance of ∼10 Å during 40 ns simulation course. Therefore, this study straightforward explores how small molecule targeting specific sites in the DNA minor groove disrupts the transcription factor-DNA interface in DNA major groove, and consequently modulates gene expression.

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

Supercomputer analysis of purine and pyrimidine metabolism leading to DNA synthesis.

PubMed

Heinmets, F

1989-06-01

A model-system is established to analyze purine and pyrimidine metabolism leading to DNA synthesis. The principal aim is to explore the flow and regulation of terminal deoxynucleoside triophosphates (dNTPs) in various input and parametric conditions. A series of flow equations are established, which are subsequently converted to differential equations. These are programmed (Fortran) and analyzed on a Cray chi-MP/48 supercomputer. The pool concentrations are presented as a function of time in conditions in which various pertinent parameters of the system are modified. The system is formulated by 100 differential equations.

A strand graph semantics for DNA-based computation

PubMed Central

Petersen, Rasmus L.; Lakin, Matthew R.; Phillips, Andrew

2015-01-01

DNA nanotechnology is a promising approach for engineering computation at the nanoscale, with potential applications in biofabrication and intelligent nanomedicine. DNA strand displacement is a general strategy for implementing a broad range of nanoscale computations, including any computation that can be expressed as a chemical reaction network. Modelling and analysis of DNA strand displacement systems is an important part of the design process, prior to experimental realisation. As experimental techniques improve, it is important for modelling languages to keep pace with the complexity of structures that can be realised experimentally. In this paper we present a process calculus for modelling DNA strand displacement computations involving rich secondary structures, including DNA branches and loops. We prove that our calculus is also sufficiently expressive to model previous work on non-branching structures, and propose a mapping from our calculus to a canonical strand graph representation, in which vertices represent DNA strands, ordered sites represent domains, and edges between sites represent bonds between domains. We define interactions between strands by means of strand graph rewriting, and prove the correspondence between the process calculus and strand graph behaviours. Finally, we propose a mapping from strand graphs to an efficient implementation, which we use to perform modelling and simulation of DNA strand displacement systems with rich secondary structure. PMID:27293306

DNA-directed mutations. Leading and lagging strand specificity

NASA Technical Reports Server (NTRS)

Sinden, R. R.; Hashem, V. I.; Rosche, W. A.

1999-01-01

The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

Non-homologous end joining-mediated functional marker selection for DNA cloning in the yeast Kluyveromyces marxianus.

PubMed

Hoshida, Hisashi; Murakami, Nobutada; Suzuki, Ayako; Tamura, Ryoko; Asakawa, Jun; Abdel-Banat, Babiker M A; Nonklang, Sanom; Nakamura, Mikiko; Akada, Rinji

2014-01-01

The cloning of DNA fragments into vectors or host genomes has traditionally been performed using Escherichia coli with restriction enzymes and DNA ligase or homologous recombination-based reactions. We report here a novel DNA cloning method that does not require DNA end processing or homologous recombination, but that ensures highly accurate cloning. The method exploits the efficient non-homologous end-joining (NHEJ) activity of the yeast Kluyveromyces marxianus and consists of a novel functional marker selection system. First, to demonstrate the applicability of NHEJ to DNA cloning, a C-terminal-truncated non-functional ura3 selection marker and the truncated region were PCR-amplified separately, mixed and directly used for the transformation. URA3(+) transformants appeared on the selection plates, indicating that the two DNA fragments were correctly joined by NHEJ to generate a functional URA3 gene that had inserted into the yeast chromosome. To develop the cloning system, the shortest URA3 C-terminal encoding sequence that could restore the function of a truncated non-functional ura3 was determined by deletion analysis, and was included in the primers to amplify target DNAs for cloning. Transformation with PCR-amplified target DNAs and C-terminal truncated ura3 produced numerous transformant colonies, in which a functional URA3 gene was generated and was integrated into the chromosome with the target DNAs. Several K. marxianus circular plasmids with different selection markers were also developed for NHEJ-based cloning and recombinant DNA construction. The one-step DNA cloning method developed here is a relatively simple and reliable procedure among the DNA cloning systems developed to date. Copyright © 2013 John Wiley & Sons, Ltd.

The HIrisPlex-S system for eye, hair and skin colour prediction from DNA: Introduction and forensic developmental validation.

PubMed

Chaitanya, Lakshmi; Breslin, Krystal; Zuñiga, Sofia; Wirken, Laura; Pośpiech, Ewelina; Kukla-Bartoszek, Magdalena; Sijen, Titia; Knijff, Peter de; Liu, Fan; Branicki, Wojciech; Kayser, Manfred; Walsh, Susan

2018-07-01

Forensic DNA Phenotyping (FDP), i.e. the prediction of human externally visible traits from DNA, has become a fast growing subfield within forensic genetics due to the intelligence information it can provide from DNA traces. FDP outcomes can help focus police investigations in search of unknown perpetrators, who are generally unidentifiable with standard DNA profiling. Therefore, we previously developed and forensically validated the IrisPlex DNA test system for eye colour prediction and the HIrisPlex system for combined eye and hair colour prediction from DNA traces. Here we introduce and forensically validate the HIrisPlex-S DNA test system (S for skin) for the simultaneous prediction of eye, hair, and skin colour from trace DNA. This FDP system consists of two SNaPshot-based multiplex assays targeting a total of 41 SNPs via a novel multiplex assay for 17 skin colour predictive SNPs and the previous HIrisPlex assay for 24 eye and hair colour predictive SNPs, 19 of which also contribute to skin colour prediction. The HIrisPlex-S system further comprises three statistical prediction models, the previously developed IrisPlex model for eye colour prediction based on 6 SNPs, the previous HIrisPlex model for hair colour prediction based on 22 SNPs, and the recently introduced HIrisPlex-S model for skin colour prediction based on 36 SNPs. In the forensic developmental validation testing, the novel 17-plex assay performed in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, as previously shown for the 24-plex assay. Sensitivity testing of the 17-plex assay revealed complete SNP profiles from as little as 63 pg of input DNA, equalling the previously demonstrated sensitivity threshold of the 24-plex HIrisPlex assay. Testing of simulated forensic casework samples such as blood, semen, saliva stains, of inhibited DNA samples, of low quantity touch (trace) DNA samples, and of artificially degraded DNA samples as well as concordance testing, demonstrated the robustness, efficiency, and forensic suitability of the new 17-plex assay, as previously shown for the 24-plex assay. Finally, we provide an update to the publically available HIrisPlex website https://hirisplex.erasmusmc.nl/, now allowing the estimation of individual probabilities for 3 eye, 4 hair, and 5 skin colour categories from HIrisPlex-S input genotypes. The HIrisPlex-S DNA test represents the first forensically validated tool for skin colour prediction, and reflects the first forensically validated tool for simultaneous eye, hair and skin colour prediction from DNA. Copyright © 2018 Elsevier B.V. All rights reserved.

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

PubMed

Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

2011-01-01

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses.

PubMed

Villarreal, Jessica Varela; Jungfer, Christina; Obst, Ursula; Schwartz, Thomas

2013-09-01

Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems. © 2013 Elsevier B.V. All rights reserved.

The "Starch Wars" and the Early History of DNA Profiling.

PubMed

Aronson, J D

2006-01-01

Just as the movie Star Wars had a prequel, so did the "DNA Wars"-the series of legal, scientific, and personal battles that took place over the admissibility of forensic DNA evidence from 1989 to 1994. Between the late 1970s and the mid-1980s, another forensic identification technique became mired in controversy: electrophoresis-based blood protein analysis. Although the debates over blood analysis were every bit as rancorous and frustrating to almost everybody involved - so much so that they became known as the "Starch Wars" - their importance has not been adequately appreciated in the recent history of forensic science. After reviewing the early history of blood typing, I will describe the development of the Multi-System approach to blood protein analysis that took place in California from 1977 to 1978. I will then elucidate the history of the Starch Wars, and demonstrate the ways that they shaped subsequent disputes over DNA evidence, especially in California. I will show that: (a) many of the forensic scientists, law enforcement officials, and lawyers who became prominent players in the DNA Wars were deeply involved in the court cases involving protein electrophoresis; and (b) many of the issues that became controversial in the disputes over DNA evidence first emerged in the Starch Wars. In the conclusion, I will suggest various ways to improve the quality of forensic science based on my analysis of the Starch Wars. Copyright © 2006 Central Police University.

Sequence analysis of cultivated strawberry (Fragaria × ananassa Duch.) using microdissected single somatic chromosomes.

PubMed

Yanagi, Tomohiro; Shirasawa, Kenta; Terachi, Mayuko; Isobe, Sachiko

2017-01-01

Cultivated strawberry ( Fragaria  ×  ananassa Duch.) has homoeologous chromosomes because of allo-octoploidy. For example, two homoeologous chromosomes that belong to different sub-genome of allopolyploids have similar base sequences. Thus, when conducting de novo assembly of DNA sequences, it is difficult to determine whether these sequences are derived from the same chromosome. To avoid the difficulties associated with homoeologous chromosomes and demonstrate the possibility of sequencing allopolyploids using single chromosomes, we conducted sequence analysis using microdissected single somatic chromosomes of cultivated strawberry. Three hundred and ten somatic chromosomes of the Japanese octoploid strawberry 'Reiko' were individually selected under a light microscope using a microdissection system. DNA from 288 of the dissected chromosomes was successfully amplified using a DNA amplification kit. Using next-generation sequencing, we decoded the base sequences of the amplified DNA segments, and on the basis of mapping, we identified DNA sequences from 144 samples that were best matched to the reference genomes of the octoploid strawberry, F.  ×  ananassa , and the diploid strawberry, F. vesca . The 144 samples were classified into seven pseudo-molecules of F. vesca . The coverage rates of the DNA sequences from the single chromosome onto all pseudo-molecular sequences varied from 3 to 29.9%. We demonstrated an efficient method for sequence analysis of allopolyploid plants using microdissected single chromosomes. On the basis of our results, we believe that whole-genome analysis of allopolyploid plants can be enhanced using methodology that employs microdissected single chromosomes.

Authentication of forensic DNA samples.

PubMed

Frumkin, Dan; Wasserstrom, Adam; Davidson, Ariane; Grafit, Arnon

2010-02-01

Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system.

Luciferase assay to study the activity of a cloned promoter DNA fragment.

PubMed

Solberg, Nina; Krauss, Stefan

2013-01-01

Luciferase based assays have become an invaluable tool for the analysis of cloned promoter DNA fragments, both for verifying the ability of a potential promoter fragment to drive the expression of a luciferase reporter gene in various cellular contexts, and for dissecting binding elements in the promoter. Here, we describe the use of the Dual-Luciferase(®) Reporter Assay System created by Promega (Promega Corporation, Wisconsin, USA) to study the cloned 6.7 kilobases (kb) mouse (m) Tcf3 promoter DNA fragment in mouse embryonic derived neural stem cells (NSC). In this system, the expression of the firefly luciferase driven by the cloned mTcf3 promoter DNA fragment (including transcription initiation sites) is correlated with a co-transfected control reporter expressing Renilla luciferase from the herpes simplex virus (HSV) thymidine kinase promoter. Using an internal control reporter allows to normalize the activity of the experimental reporter to the internal control, which minimizes experimental variability.

Hereditary Disorders with Defective Repair of UV-Induced DNA Damage

PubMed Central

Moriwaki, Shinichi

2013-01-01

Nucleotide excision repair (NER) is an essential system for correcting ultraviolet (UV)—induced DNA damage. Lesions remaining in DNA due to reduced capacity of NER may result in cellular death, premature aging, mutagenesis and carcinogenesis of the skin. So, NER is an important protection against these changes. There are three representative genodermatoses resulting from genetic defects in NER: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). In Japan, CS is similarly rare but XP is more common and TTD is less common compared to Western countries. In 1998, we established the system for the diagnosis of these disorders and we have been performing DNA repair and genetic analysis for more than 400 samples since then. At present, there is no cure for any human genetic disorder. Early diagnosis and symptomatic treatment of neurological, ocular and dermatological abnormalities should contribute to prolonging life and elevating QOL in patients. PMID:23966815

Traumatic stress and accelerated DNA methylation age: A meta-analysis.

PubMed

Wolf, Erika J; Maniates, Hannah; Nugent, Nicole; Maihofer, Adam X; Armstrong, Don; Ratanatharathorn, Andrew; Ashley-Koch, Allison E; Garrett, Melanie; Kimbrel, Nathan A; Lori, Adriana; Va Mid-Atlantic Mirecc Workgroup; Aiello, Allison E; Baker, Dewleen G; Beckham, Jean C; Boks, Marco P; Galea, Sandro; Geuze, Elbert; Hauser, Michael A; Kessler, Ronald C; Koenen, Karestan C; Miller, Mark W; Ressler, Kerry J; Risbrough, Victoria; Rutten, Bart P F; Stein, Murray B; Ursano, Robert J; Vermetten, Eric; Vinkers, Christiaan H; Uddin, Monica; Smith, Alicia K; Nievergelt, Caroline M; Logue, Mark W

2018-06-01

Recent studies examining the association between posttraumatic stress disorder (PTSD) and accelerated aging, as defined by DNA methylation-based estimates of cellular age that exceed chronological age, have yielded mixed results. We conducted a meta-analysis of trauma exposure and PTSD diagnosis and symptom severity in association with accelerated DNA methylation age using data from 9 cohorts contributing to the Psychiatric Genomics Consortium PTSD Epigenetics Workgroup (combined N = 2186). Associations between demographic and cellular variables and accelerated DNA methylation age were also examined, as was the moderating influence of demographic variables. Meta-analysis of regression coefficients from contributing cohorts revealed that childhood trauma exposure (when measured with the Childhood Trauma Questionnaire) and lifetime PTSD severity evidenced significant, albeit small, meta-analytic associations with accelerated DNA methylation age (ps = 0.028 and 0.016, respectively). Sex, CD4T cell proportions, and natural killer cell proportions were also significantly associated with accelerated DNA methylation age (all ps < 0.02). PTSD diagnosis and lifetime trauma exposure were not associated with advanced DNA methylation age. There was no evidence of moderation of the trauma or PTSD variables by demographic factors. Results suggest that traumatic stress is associated with advanced epigenetic age and raise the possibility that cells integral to immune system maintenance and responsivity play a role in this. This study highlights the need for additional research into the biological mechanisms linking traumatic stress to accelerated DNA methylation age and the importance of furthering our understanding of the neurobiological and health consequences of PTSD. Published by Elsevier Ltd.

Circulating tumor DNA as a liquid biopsy for cancer.

PubMed

Heitzer, Ellen; Ulz, Peter; Geigl, Jochen B

2015-01-01

Targeted therapies have markedly changed the treatment of cancer over the past 10 years. However, almost all tumors acquire resistance to systemic treatment as a result of tumor heterogeneity, clonal evolution, and selection. Although genotyping is the most currently used method for categorizing tumors for clinical decisions, tumor tissues provide only a snapshot, or are often difficult to obtain. To overcome these issues, methods are needed for a rapid, cost-effective, and noninvasive identification of biomarkers at various time points during the course of disease. Because cell-free circulating tumor DNA (ctDNA) is a potential surrogate for the entire tumor genome, the use of ctDNA as a liquid biopsy may help to obtain the genetic follow-up data that are urgently needed. This review includes recent studies exploring the diagnostic, prognostic, and predictive potential of ctDNA as a liquid biopsy in cancer. In addition, it covers biological and technical aspects, including recent advances in the analytical sensitivity and accuracy of DNA analysis as well as hurdles that have to be overcome before implementation into clinical routine. Although the analysis of ctDNA is a promising area, and despite all efforts to develop suitable tools for a comprehensive analysis of tumor genomes from plasma DNA, the liquid biopsy is not yet routinely used as a clinical application. Harmonization of preanalytical and analytical procedures is needed to provide clinical standards to validate the liquid biopsy as a clinical biomarker in well-designed and sufficiently powered multicenter studies. © 2014 American Association for Clinical Chemistry.

Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts

PubMed Central

Thierry, Alain R.; Mouliere, Florent; Gongora, Celine; Ollier, Jeremy; Robert, Bruno; Ychou, Marc; Del Rio, Maguy; Molina, Franck

2010-01-01

Although circulating DNA (ctDNA) could be an attractive tool for early cancer detection, diagnosis, prognosis, monitoring or prediction of response to therapies, knowledge on its origin, form and rate of release is poor and often contradictory. Here, we describe an experimental system to systematically examine these aspects. Nude mice were xenografted with human HT29 or SW620 colorectal carcinoma (CRC) cells and ctDNA was analyzed by Q–PCR with highly specific and sensitive primer sets at different times post-graft. We could discriminate ctDNA from normal (murine) cells and from mutated and non-mutated tumor (human) cells by using species-specific KRAS or PSAT1 primers and by assessing the presence of the BRAF V600E mutation. The concentration of human (mutated and non-mutated) ctDNA increased significantly with tumor growth. Conversely, and differently from previous studies, low, constant level of mouse ctDNA was observed, thus facilitating the study of mutated and non-mutated tumor derived ctDNA. Finally, analysis of ctDNA fragmentation confirmed the predominance of low-size fragments among tumor ctDNA from mice with bigger tumors. Higher ctDNA fragmentation was also observed in plasma samples from three metastatic CRC patients in comparison to healthy individuals. Our data confirm the predominance of mononucleosome-derived fragments in plasma from xenografted animals and, as a consequence, of apoptosis as a source of ctDNA, in particular for tumor-derived ctDNA. Altogether, our results suggest that ctDNA features vary during CRC tumor development and our experimental system might be a useful tool to follow such variations. PMID:20494973

Stability analysis of an autocatalytic protein model

NASA Astrophysics Data System (ADS)

Lee, Julian

2016-05-01

A self-regulatory genetic circuit, where a protein acts as a positive regulator of its own production, is known to be the simplest biological network with a positive feedback loop. Although at least three components—DNA, RNA, and the protein—are required to form such a circuit, stability analysis of the fixed points of this self-regulatory circuit has been performed only after reducing the system to a two-component system, either by assuming a fast equilibration of the DNA component or by removing the RNA component. Here, stability of the fixed points of the three-component positive feedback loop is analyzed by obtaining eigenvalues of the full three-dimensional Hessian matrix. In addition to rigorously identifying the stable fixed points and saddle points, detailed information about the system can be obtained, such as the existence of complex eigenvalues near a fixed point.

DNA sequence-based comparative studies between non-extremophile and extremophile organisms with implications in exobiology

NASA Astrophysics Data System (ADS)

Holden, Todd; Marchese, P.; Tremberger, G., Jr.; Cheung, E.; Subramaniam, R.; Sullivan, R.; Schneider, P.; Flamholz, A.; Lieberman, D.; Cheung, T.

2008-08-01

We have characterized function related DNA sequences of various organisms using informatics techniques, including fractal dimension calculation, nucleotide and multi-nucleotide statistics, and sequence fluctuation analysis. Our analysis shows trends which differentiate extremophile from non-extremophile organisms, which could be reproduced in extraterrestrial life. Among the systems studied are radiation repair genes, genes involved in thermal shocks, and genes involved in drug resistance. We also evaluate sequence level changes that have occurred during short term evolution (several thousand generations) under extreme conditions.

High-throughput single-molecule telomere characterization.

PubMed

McCaffrey, Jennifer; Young, Eleanor; Lassahn, Katy; Sibert, Justin; Pastor, Steven; Riethman, Harold; Xiao, Ming

2017-11-01

We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG)n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7-bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG)n tract length at the end of each large telomere-terminal DNA segment. The methodology also permits subtelomere and haplotype-resolved analyses of SRE organization and variation, providing a window into the population dynamics and potential functions of these complex and structurally variant telomere-adjacent DNA regions. At its current stage of development, the assay can be used to identify and characterize telomere length distributions of 30-35 discrete telomeres simultaneously and accurately. The assay's utility is demonstrated using early versus late passage and senescent human diploid fibroblasts, documenting the anticipated telomere attrition on a global telomere-by-telomere basis as well as identifying subtelomere-specific biases for critically short telomeres. Similarly, we present the first global single-telomere-resolved analyses of two cancer cell lines. © 2017 McCaffrey et al.; Published by Cold Spring Harbor Laboratory Press.

A novel scoring system for gastric cancer risk assessment based on the expression of three CLIP4 DNA methylation-associated genes

PubMed Central

Hu, Chenggong; Zhou, Yongfang; Liu, Chang; Kang, Yan

2018-01-01

Gastric cancer (GC) is the fifth most common cancer and the third leading cause of cancer-associated mortality worldwide. In the current study, comprehensive bioinformatic analyses were performed to develop a novel scoring system for GC risk assessment based on CAP-Gly domain containing linker protein family member 4 (CLIP4) DNA methylation status. Two GC datasets with methylation sequencing information and mRNA expression profiling were downloaded from the The Cancer Genome Atlas and Gene Expression Omnibus databases. Differentially expressed genes (DEGs) between the CLIP4 hypermethylation and CLIP4 hypomethylation groups were screened using the limma package in R 3.3.1, and survival analysis of these DEGs was performed using the survival package. A risk scoring system was established via regression factor-weighted gene expression based on linear combination to screen the most important genes associated with CLIP4 methylation and prognosis. Genes associated with high/low-risk value were selected using the limma package. Functional enrichment analysis of the top 500 DEGs that positively and negatively associated with risk values was performed using DAVID 6.8 online and the gene set enrichment analysis (GSEA) software. In total, 35 genes were identified to be that significantly associated with prognosis and CLIP4 DNA methylation, and three prognostic signature genes, claudin-11 (CLDN11), apolipoprotein D (APOD), and chordin like 1 (CHRDL1), were used to establish a risk assessment system. The prognostic scoring system exhibited efficiency in classifying patients with different prognoses, where the low-risk groups had significantly longer overall survival times than those in the high-risk groups. CLDN11, APOD and CHRDL1 exhibited reduced expression in the hypermethylation and low-risk groups compare with the hypomethylation and high-risk groups, respectively. Multivariate Cox analysis indicated that risk value could be used as an independent prognostic factor. In functional analysis, six functional gene ontology terms and five GSEA pathways were associated with CLDN11, APOD and CHRDL1. The results established the credibility of the scoring system in this study. Additionally, these three genes, which were significantly associated with CLIP4 DNA methylation and GC risk assessment, were identified as potential prognostic biomarkers. PMID:29901187

The Helicobacter pylori HpyAXII restriction–modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

PubMed Central

Humbert, Olivier; Salama, Nina R.

2008-01-01

The naturally competent organism Helicobacter pylori encodes a large number of restriction–modification (R–M) systems that consist of a restriction endonuclease and a DNA methyltransferase. R–M systems are not only believed to limit DNA exchange among bacteria but may also have other cellular functions. We report a previously uncharacterized H. pylori type II R–M system, M.HpyAXII/R.HpyAXII. We show that this system targets GTAC sites, which are rare in the H. pylori chromosome but numerous in ribosomal RNA genes. As predicted, this type II R–M system showed attributes of a selfish element. Deletion of the methyltransferase M.HpyAXII is lethal when associated with an active endonuclease R.HpyAXII unless compensated by adaptive mutation or gene amplification. R.HpyAXII effectively restricted both unmethylated plasmid and chromosomal DNA during natural transformation and was predicted to belong to the novel ‘half pipe’ structural family of endonucleases. Analysis of a panel of clinical isolates revealed that R.HpyAXII was functional in a small number of H. pylori strains (18.9%, n = 37), whereas the activity of M.HpyAXII was highly conserved (92%, n = 50), suggesting that GTAC methylation confers a selective advantage to H. pylori. However, M.HpyAXII activity did not enhance H. pylori fitness during stomach colonization of a mouse infection model. PMID:18978016

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

PubMed Central

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

PubMed Central

Kavanagh, T A; Thanh, N D; Lao, N T; McGrath, N; Peter, S O; Horváth, E M; Dix, P J; Medgyesy, P

1999-01-01

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids. PMID:10388829

Heterogeneity, histological features and DNA ploidy in oral carcinoma by image-based analysis.

PubMed

Diwakar, N; Sperandio, M; Sherriff, M; Brown, A; Odell, E W

2005-04-01

Oral squamous carcinomas appear heterogeneous on DNA ploidy analysis. However, this may be partly a result of sample dilution or the detection limit of techniques. The aim of this study was to determine whether oral squamous carcinomas are heterogeneous for ploidy status using image-based ploidy analysis and to determine whether ploidy status correlates with histological parameters. Multiple samples from 42 oral squamous carcinomas were analysed for DNA ploidy using an image-based system and scored for histological parameters. 22 were uniformly aneuploid, 1 uniformly tetraploid and 3 uniformly diploid. 16 appeared heterogeneous but only 8 appeared to be genuinely heterogeneous when minor ploidy histogram peaks were taken into account. Ploidy was closely related to nuclear pleomorphism but not differentiation. Sample variation, detection limits and diagnostic criteria account for much of the ploidy heterogeneity observed. Confident diagnosis of diploid status in an oral squamous cell carcinoma requires a minimum of 5 samples.

Recruitment of DNA methyltransferase I to DNA repair sites.

PubMed

Mortusewicz, Oliver; Schermelleh, Lothar; Walter, Joachim; Cardoso, M Cristina; Leonhardt, Heinrich

2005-06-21

In mammalian cells, the replication of genetic and epigenetic information is directly coupled; however, little is known about the maintenance of epigenetic information in DNA repair. Using a laser microirradiation system to introduce DNA lesions at defined subnuclear sites, we tested whether the major DNA methyltransferase (Dnmt1) or one of the two de novo methyltransferases (Dnmt3a, Dnmt3b) are recruited to sites of DNA repair in vivo. Time lapse microscopy of microirradiated mammalian cells expressing GFP-tagged Dnmt1, Dnmt3a, or Dnmt3b1 together with red fluorescent protein-tagged proliferating cell nuclear antigen (PCNA) revealed that Dnmt1 and PCNA accumulate at DNA damage sites as early as 1 min after irradiation in S and non-S phase cells, whereas recruitment of Dnmt3a and Dnmt3b was not observed. Deletion analysis showed that Dnmt1 recruitment was mediated by the PCNA-binding domain. These data point to a direct role of Dnmt1 in the restoration of epigenetic information during DNA repair.

A novel mitochondrial DNA deletion in a patient with Pearson syndrome and neonatal diabetes mellitus provides insight into disease etiology, severity and progression.

PubMed

Chen, Xin-Yu; Zhao, Si-Yu; Wang, Yan; Wang, Dong; Dong, Chang-Hu; Yang, Ying; Wang, Zhi-Hua; Wu, Yuan-Ming

2016-07-01

Pearson syndrome (PS) is a rare, mitochondrial DNA (mtDNA) deletion disorder mainly affecting hematopoietic system and exocrine pancreas in early infancy, which is characterized by multi-organ involvement, variable manifestations and poor prognosis. Since the clinical complexity and uncertain outcome of PS, the ability to early diagnose and anticipate disease progression is of great clinical importance. We described a patient with severe anemia and hyperglycinemia at birth was diagnosed with neonatal diabetes mellitus, and later with PS. Genetic testing revealed that a novel mtDNA deletion existed in various non-invasive tissues from the patient. The disease course was monitored by mtDNA deletion heteroplasmy and mtDNA/nucleus DNA genome ratio in different tissues and at different time points, showing a potential genotype-phenotype correlation. Our findings suggest that for patient suspected for PS, it may be therapeutically important to first perform detailed mtDNA analysis on non-invasive tissues at the initial diagnosis and during disease progression.

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.

Role of DNA-DNA Interactions on the Structure and Thermodynamics of Bacteriophages Lambda and P4

PubMed Central

Petrov, Anton S.; Harvey, Stephen C.

2010-01-01

Electrostatic interactions play an important role in both packaging of DNA inside bacteriophages and its release into bacterial cells. While at physiological conditions DNA strands repel each other, the presence of polyvalent cations such as spermine and spermidine in solutions leads to the formation of DNA condensates. In this study, we discuss packaging of DNA into bacteriophages P4 and Lambda under repulsive and attractive conditions using a coarse-grained model of DNA and capsids. Packaging under repulsive conditions leads to the appearance of the coaxial spooling conformations; DNA occupies all available space inside the capsid. Under the attractive potential both packed systems reveal toroidal conformations, leaving the central part of the capsids empty. We also present a detailed thermodynamic analysis of packaging and show that the forces required to pack the genomes in the presence of polyamines are significantly lower than those observed under repulsive conditions. The analysis reveals that in both the repulsive and attractive regimes the entropic penalty of DNA confinement has a significant non-negligible contribution into the total energy of packaging. Additionally we report the results of simulations of DNA condensation inside partially packed Lambda. We found that at low densities DNA behaves as free unconfined polymer and condenses into the toroidal structures; at higher densities rearrangement of the genome into toroids becomes hindered, and condensation results in the formation of non-equilibrium structures. In all cases packaging in a specific conformation occurs as a result of interplay between bending stresses experienced by the confined polymer and interactions between the strands. PMID:21074621

The Ins and Outs of DNA Fingerprinting the Infectious Fungi

PubMed Central

Soll, David R.

2000-01-01

DNA fingerprinting methods have evolved as major tools in fungal epidemiology. However, no single method has emerged as the method of choice, and some methods perform better than others at different levels of resolution. In this review, requirements for an effective DNA fingerprinting method are proposed and procedures are described for testing the efficacy of a method. In light of the proposed requirements, the most common methods now being used to DNA fingerprint the infectious fungi are described and assessed. These methods include restriction fragment length polymorphisms (RFLP), RFLP with hybridization probes, randomly amplified polymorphic DNA and other PCR-based methods, electrophoretic karyotyping, and sequencing-based methods. Procedures for computing similarity coefficients, generating phylogenetic trees, and testing the stability of clusters are then described. To facilitate the analysis of DNA fingerprinting data, computer-assisted methods are described. Finally, the problems inherent in the collection of test and control isolates are considered, and DNA fingerprinting studies of strain maintenance during persistent or recurrent infections, microevolution in infecting strains, and the origin of nosocomial infections are assessed in light of the preceding discussion of the ins and outs of DNA fingerprinting. The intent of this review is to generate an awareness of the need to verify the efficacy of each DNA fingerprinting method for the level of genetic relatedness necessary to answer the epidemiological question posed, to use quantitative methods to analyze DNA fingerprint data, to use computer-assisted DNA fingerprint analysis systems to analyze data, and to file data in a form that can be used in the future for retrospective and comparative studies. PMID:10756003

Real-time PCR Machine System Modeling and a Systematic Approach for the Robust Design of a Real-time PCR-on-a-Chip System

PubMed Central

Lee, Da-Sheng

2010-01-01

Chip-based DNA quantification systems are widespread, and used in many point-of-care applications. However, instruments for such applications may not be maintained or calibrated regularly. Since machine reliability is a key issue for normal operation, this study presents a system model of the real-time Polymerase Chain Reaction (PCR) machine to analyze the instrument design through numerical experiments. Based on model analysis, a systematic approach was developed to lower the variation of DNA quantification and achieve a robust design for a real-time PCR-on-a-chip system. Accelerated lift testing was adopted to evaluate the reliability of the chip prototype. According to the life test plan, this proposed real-time PCR-on-a-chip system was simulated to work continuously for over three years with similar reproducibility in DNA quantification. This not only shows the robustness of the lab-on-a-chip system, but also verifies the effectiveness of our systematic method for achieving a robust design. PMID:22315563

Infrared fluorescent automated detection of thirteen short tandem repeat polymorphisms and one gender-determining system of the CODIS core system.

PubMed

Ricci, U; Sani, I; Guarducci, S; Biondi, C; Pelagatti, S; Lazzerini, V; Brusaferri, A; Lapini, M; Andreucci, E; Giunti, L; Giovannucci Uzielli, M L

2000-11-01

We used an infrared (IR) automated fluorescence monolaser sequencer for the analysis of 13 autosomal short tandem repeat (STR) systems (TPOX, D3S1358, FGA, CSF1PO, D5S818, D7S820, D8S1179, TH01, vWA, D13S317, D16S359, D18S51, D21S11) and the X-Y homologous gene amelogenin system. These two systems represent the core of the combined DNA index systems (CODIS). Four independent multiplex reactions, based on the polymerase chain reaction (PCR) technique and on the direct labeling of the forward primer of every primer pair, with a new molecule (IRDye800), were set up, permitting the exact characterization of the alleles by comparison with ladders of specific sequenced alleles. This is the first report of the whole analysis of the STRs of the CODIS core using an IR automated DNA sequencer. The protocol was used to solve paternity/maternity tests and for population studies. The electrophoretic system also proved useful for the correct typing of those loci differing in size by only 2 bp. A sensibility study demonstrated that the test can detect an average of 10 pg of undegraded human DNA. We also performed a preliminary study analyzing some forensic samples and mixed stains, which suggested the usefulness of using this analytical system for human identification as well as for forensic purposes.

Ribosomal DNA transcription in the dorsal raphe nucleus is increased in residual but not in paranoid schizophrenia.

PubMed

Krzyżanowska, Marta; Steiner, Johann; Brisch, Ralf; Mawrin, Christian; Busse, Stefan; Braun, Katharina; Jankowski, Zbigniew; Bernstein, Hans-Gert; Bogerts, Bernhard; Gos, Tomasz

2015-03-01

The central serotonergic system is implicated in the pathogenesis of schizophrenia, where the imbalance between dopamine, serotonin and glutamate plays a key pathophysiological role. The dorsal raphe nucleus (DRN) is the main source of serotonergic innervation of forebrain limbic structures disturbed in schizophrenia patients. The study was carried out on paraffin-embedded brains from 17 (8 paranoid and 9 residual) schizophrenia patients and 28 matched controls without mental disorders. The transcriptional activity of ribosomal DNA (rDNA) in DRN neurons was evaluated by the AgNOR silver-staining method. An increased rDNA transcriptional activity was found in schizophrenia patients in the cumulative analysis of all DRN subnuclei (t test, P = 0.02). Further subgroup analysis revealed that it was an effect specific for residual schizophrenia versus paranoid schizophrenia or control groups (ANOVA, P = 0.002). This effect was confounded neither by suicide nor by antipsychotic medication. Our findings suggest that increased activity of rDNA in DRN neurons is a distinct phenomenon in schizophrenia, particularly in residual patients. An activation of the rDNA transcription in DRN neurons may represent a compensatory mechanism to overcome the previously described prefrontal serotonergic hypofunction in this diagnostic subgroup.

Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

PubMed

Singh, Sheetal; Shih, Shyh-Jen; Vaughan, Andrew T M

2014-01-01

Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter- and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location.

Environmental Barcoding: A Next-Generation Sequencing Approach for Biomonitoring Applications Using River Benthos

PubMed Central

Hajibabaei, Mehrdad; Shokralla, Shadi; Zhou, Xin; Singer, Gregory A. C.; Baird, Donald J.

2011-01-01

Timely and accurate biodiversity analysis poses an ongoing challenge for the success of biomonitoring programs. Morphology-based identification of bioindicator taxa is time consuming, and rarely supports species-level resolution especially for immature life stages. Much work has been done in the past decade to develop alternative approaches for biodiversity analysis using DNA sequence-based approaches such as molecular phylogenetics and DNA barcoding. On-going assembly of DNA barcode reference libraries will provide the basis for a DNA-based identification system. The use of recently introduced next-generation sequencing (NGS) approaches in biodiversity science has the potential to further extend the application of DNA information for routine biomonitoring applications to an unprecedented scale. Here we demonstrate the feasibility of using 454 massively parallel pyrosequencing for species-level analysis of freshwater benthic macroinvertebrate taxa commonly used for biomonitoring. We designed our experiments in order to directly compare morphology-based, Sanger sequencing DNA barcoding, and next-generation environmental barcoding approaches. Our results show the ability of 454 pyrosequencing of mini-barcodes to accurately identify all species with more than 1% abundance in the pooled mixture. Although the approach failed to identify 6 rare species in the mixture, the presence of sequences from 9 species that were not represented by individuals in the mixture provides evidence that DNA based analysis may yet provide a valuable approach in finding rare species in bulk environmental samples. We further demonstrate the application of the environmental barcoding approach by comparing benthic macroinvertebrates from an urban region to those obtained from a conservation area. Although considerable effort will be required to robustly optimize NGS tools to identify species from bulk environmental samples, our results indicate the potential of an environmental barcoding approach for biomonitoring programs. PMID:21533287

[Metabolic changes in cells under electromagnetic radiation of mobile communication systems].

PubMed

Iakimenko, I L; Sidorik, E P; Tsybulin, A S

2011-01-01

Review is devoted to the analysis of biological effects of microwaves. The results of last years' researches indicated the potential risks of long-term low-level microwaves exposure for human health. The analysis of metabolic changes in living cells under the exposure of microwaves from mobile communication systems indicates that this factor is stressful for cells. Among the reproducible effects of low-level microwave radiation are overexpression of heat shock proteins, an increase of reactive oxygen species level, an increase of intracellular Ca2+, damage of DNA, inhibition of DNA reparation, and induction of apoptosis. Extracellular-signal-regulated kinases ERK and stress-related kinases p38MAPK are involved in metabolic changes. Analysis of current data suggests that the concept of exceptionally thermal mechanism of biological effects of microwaves is not correct. In turn, this raises the question of the need to revaluation of modern electromagnetic standards based on thermal effects of non-ionizing radiation on biological systems.

Fully Integrated Microfluidic Device for Direct Sample-to-Answer Genetic Analysis

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Grodzinski, Piotr

Integration of microfluidics technology with DNA microarrays enables building complete sample-to-answer systems that are useful in many applications such as clinic diagnostics. In this chapter, a fully integrated microfluidic device [1] that consists of microfluidic mixers, valves, pumps, channels, chambers, heaters, and a DNA microarray sensor to perform DNA analysis of complex biological sample solutions is present. This device can perform on-chip sample preparation (including magnetic bead-based cell capture, cell preconcentration and purification, and cell lysis) of complex biological sample solutions (such as whole blood), polymerase chain reaction, DNA hybridization, and electrochemical detection. A few novel microfluidic techniques were developed and employed. A micromix-ing technique based on a cavitation microstreaming principle was implemented to enhance target cell capture from whole blood samples using immunomagnetic beads. This technique was also employed to accelerate DNA hybridization reaction. Thermally actuated paraffin-based microvalves were developed to regulate flows. Electrochemical pumps and thermopneumatic pumps were integrated on the chip to provide pumping of liquid solutions. The device is completely self-contained: no external pressure sources, fluid storage, mechanical pumps, or valves are necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Pathogenic bacteria detection from ~mL whole blood samples and single-nucleotide polymorphism analysis directly from diluted blood were demonstrated. The device provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus has a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Spatial Representativeness of Environmental DNA Metabarcoding Signal for Fish Biodiversity Assessment in a Natural Freshwater System.

PubMed

Civade, Raphaël; Dejean, Tony; Valentini, Alice; Roset, Nicolas; Raymond, Jean-Claude; Bonin, Aurélie; Taberlet, Pierre; Pont, Didier

2016-01-01

In the last few years, the study of environmental DNA (eDNA) has drawn attention for many reasons, including its advantages for monitoring and conservation purposes. So far, in aquatic environments, most of eDNA research has focused on the detection of single species using species-specific markers. Recently, species inventories based on the analysis of a single generalist marker targeting a larger taxonomic group (eDNA metabarcoding) have proven useful for bony fish and amphibian biodiversity surveys. This approach involves in situ filtering of large volumes of water followed by amplification and sequencing of a short discriminative fragment from the 12S rDNA mitochondrial gene. In this study, we went one step further by investigating the spatial representativeness (i.e. ecological reliability and signal variability in space) of eDNA metabarcoding for large-scale fish biodiversity assessment in a freshwater system including lentic and lotic environments. We tested the ability of this approach to characterize large-scale organization of fish communities along a longitudinal gradient, from a lake to the outflowing river. First, our results confirm that eDNA metabarcoding is more efficient than a single traditional sampling campaign to detect species presence, especially in rivers. Second, the species list obtained using this approach is comparable to the one obtained when cumulating all traditional sampling sessions since 1995 and 1988 for the lake and the river, respectively. In conclusion, eDNA metabarcoding gives a faithful description of local fish biodiversity in the study system, more specifically within a range of a few kilometers along the river in our study conditions, i.e. longer than a traditional fish sampling site.

Evaluation of the Branched-Chain DNA Assay for Measurement of RNA in Formalin-Fixed Tissues

PubMed Central

Knudsen, Beatrice S.; Allen, April N.; McLerran, Dale F.; Vessella, Robert L.; Karademos, Jonathan; Davies, Joan E.; Maqsodi, Botoul; McMaster, Gary K.; Kristal, Alan R.

2008-01-01

We evaluated the branched-chain DNA (bDNA) assay QuantiGene Reagent System to measure RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. The QuantiGene Reagent System does not require RNA isolation, avoids enzymatic preamplification, and has a simple workflow. Five selected genes were measured by bDNA assay; quantitative polymerase chain reaction (qPCR) was used as a reference method. Mixed-effect statistical models were used to partition the overall variance into components attributable to xenograft, sample, and assay. For FFPE tissues, the coefficients of reliability were significantly higher for the bDNA assay (93–100%) than for qPCR (82.4–95%). Correlations between qPCRFROZEN, the gold standard, and bDNAFFPE ranged from 0.60 to 0.94, similar to those from qPCRFROZEN and qPCRFFPE. Additionally, the sensitivity of the bDNA assay in tissue homogenates was 10-fold higher than in purified RNA. In 9- to 13-year-old blocks with poor RNA quality, the bDNA assay allowed the correct identification of the overexpression of known cancer genes. In conclusion, the QuantiGene Reagent System is considerably more reliable, reproducible, and sensitive than qPCR, providing an alternative method for the measurement of gene expression in FFPE tissues. It also appears to be well suited for the clinical analysis of FFPE tissues with diagnostic or prognostic gene expression biomarker panels for use in patient treatment and management. PMID:18276773

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.

Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons.

PubMed

Kim, Eun Hye; Lee, Hwan Young; Yang, In Seok; Jung, Sang-Eun; Yang, Woo Ick; Shin, Kyoung-Jin

2016-05-01

The next-generation sequencing (NGS) method has been utilized to analyze short tandem repeat (STR) markers, which are routinely used for human identification purposes in the forensic field. Some researchers have demonstrated the successful application of the NGS system to STR typing, suggesting that NGS technology may be an alternative or additional method to overcome limitations of capillary electrophoresis (CE)-based STR profiling. However, there has been no available multiplex PCR system that is optimized for NGS analysis of forensic STR markers. Thus, we constructed a multiplex PCR system for the NGS analysis of 18 markers (13CODIS STRs, D2S1338, D19S433, Penta D, Penta E and amelogenin) by designing amplicons in the size range of 77-210 base pairs. Then, PCR products were generated from two single-sources, mixed samples and artificially degraded DNA samples using a multiplex PCR system, and were prepared for sequencing on the MiSeq system through construction of a subsequent barcoded library. By performing NGS and analyzing the data, we confirmed that the resultant STR genotypes were consistent with those of CE-based typing. Moreover, sequence variations were detected in targeted STR regions. Through the use of small-sized amplicons, the developed multiplex PCR system enables researchers to obtain successful STR profiles even from artificially degraded DNA as well as STR loci which are analyzed with large-sized amplicons in the CE-based commercial kits. In addition, successful profiles can be obtained from mixtures up to a 1:19 ratio. Consequently, the developed multiplex PCR system, which produces small size amplicons, can be successfully applied to STR NGS analysis of forensic casework samples such as mixtures and degraded DNA samples. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Detection of early changes in lung cell cytology by flow-systems analysis techniques, July 1--December 31, 1975

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

Steinkamp, J.A.; Ingram, M.; Hansen, K.M.

1976-03-01

This report summarizes results of preliminary experiments to demonstrate the feasibility of using automated flow-systems analysis in detecting early changes of respiratory epithelium exposed to physical and chemical agents associated with the by-products of nonnuclear energy production. The Syrian hamster was selected as the experimental test animal to begin investigation of the effects of toxic agents to cells of the respiratory tract. Since initiation of the program approximately six months ago, the goals have been acquisition of adequate numbers of exfoliated cells from the lung; adaptation of cytological techniques developed on human exfoliated gynecological samples to hamster lung epithelium formore » obtaining single-cell suspensions; utilization of existing cell staining methods to measure DNA content in lung cells; and analysis of DNA content and cell size. As the flow-system cell analysis technology is adapted to the measurement of exfoliated lung cells, rapid and quantitative determination of early changes in the physical and biochemical cellular properties will be attempted as a function of exposure to the toxic agents. (auth)« less

Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

PubMed

Kogoma, T

1997-06-01

Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.

PubMed

González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred; Llosa, Matxalen

2017-06-15

Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination. Copyright © 2017 American Society for Microbiology.

The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome

PubMed Central

González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred

2017-01-01

ABSTRACT Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery. IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site-specific integrase activity in bacteria, as an integrase in human cells. Although it is not efficient as a site-specific integrase, we found that TrwC is active in human cells and promotes random integration of the transferred DNA in the human genome, probably acting as a DNA chaperone until it is integrated by host mechanisms. TrwC-DNA complexes can be delivered to human cells through a type IV secretion system involved in pathogenesis. Thus, TrwC could be used in vivo to transfer the DNA of interest into the appropriate cell and promote its integration. If used in combination with a site-specific nuclease, it could lead to site-specific integration of the incoming DNA by homologous recombination. PMID:28411218

The T4 Phage DNA Mimic Protein Arn Inhibits the DNA Binding Activity of the Bacterial Histone-like Protein H-NS*

PubMed Central

Ho, Chun-Han; Wang, Hao-Ching; Ko, Tzu-Ping; Chang, Yuan-Chih; Wang, Andrew H.-J.

2014-01-01

The T4 phage protein Arn (Anti restriction nuclease) was identified as an inhibitor of the restriction enzyme McrBC. However, until now its molecular mechanism remained unclear. In the present study we used structural approaches to investigate biological properties of Arn. A structural analysis of Arn revealed that its shape and negative charge distribution are similar to dsDNA, suggesting that this protein could act as a DNA mimic. In a subsequent proteomic analysis, we found that the bacterial histone-like protein H-NS interacts with Arn, implying a new function. An electrophoretic mobility shift assay showed that Arn prevents H-NS from binding to the Escherichia coli hns and T4 p8.1 promoters. In vitro gene expression and electron microscopy analyses also indicated that Arn counteracts the gene-silencing effect of H-NS on a reporter gene. Because McrBC and H-NS both participate in the host defense system, our findings suggest that T4 Arn might knock down these mechanisms using its DNA mimicking properties. PMID:25118281

Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting - strengths and weaknesses.

PubMed

Hormann, Wymke; Hahn, Melanie; Gerlach, Stefan; Hochstrate, Nicola; Affeldt, Kai; Giesen, Joyce; Fechner, Kai; Damoiseaux, Jan G M C

2017-11-27

Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis. We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months. Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation. Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

Nutriomes and personalised nutrition for DNA damage prevention, telomere integrity maintenance and cancer growth control.

PubMed

Fenech, Michael F

2014-01-01

DNA damage at the base sequence and chromosome level is a fundamental cause of developmental and degenerative diseases. Multiple micronutrients and their interactions with the inherited and/or acquired genome determine DNA damage and genomic instability rates. The challenge is to identify for each individual the combination of micronutrients and their doses (i.e. the nutriome) that optimises genome stability, including telomere integrity and functionality and DNA repair. Using nutrient array systems with high-content analysis diagnostics of DNA damage, cell death and cell growth, it is possible to define, on an individual basis, the optimal nutriome for DNA damage prevention and cancer growth control. This knowledge can also be used to improve culture systems for cells used in therapeutics such as stem cells to ensure that they are not genetically aberrant when returned to the body. Furthermore, this information could be used to design dietary patterns that deliver the micronutrient combinations and concentrations required for preventing DNA damage by micronutrient deficiency or excess. Using this approach, new knowledge could be obtained to identify the dietary restrictions and/or supplementations required to control specific cancers, which is particularly important given that reliable validated advice is not yet available for those diagnosed with cancer.

Immobilization of methotrexate anticancer drug onto the graphene surface and interaction with calf thymus DNA and 4T1 cancer cells.

PubMed

Karimi Shervedani, Reza; Mirhosseini, Hadiseh; Samiei Foroushani, Marzieh; Torabi, Mostafa; Rahsepar, Fatemeh Rahnemaye; Norouzi-Barough, Leila

2018-02-01

Immobilization of methotrexate (MTX) anticancer drug onto the graphene surface is reported through three methods, including either covalent linkage via (a) EDC/NHS organic activators and (b) electrografting of MTX diazonium salt, or (c) noncovalent bonding, resulting in three different systems. To evaluate the interaction ability of the immobilized MTX with biological species, calf thymus DNA (ctDNA), mouse 4T1 breast tumor, and Human foreskin fibroblast (hFF) cells as models of the primary intracellular target of anticancer drugs, cancer and normal cells, respectively, are examined. The features of the constructed systems and their interactions with ctDNA are followed by surface analysis techniques and electrochemical methods. The results indicate that (i) the amount of the immobilized MTX on the graphene surface is affected by type of the immobilization method; and a maximum value of (Γ=9.3±0.9pmolcm -2 ) is found via electrografting method, (ii) graphene-modified-MTX has high affinity for ctDNA in a wide dynamic range of concentrations, and (iii) the nature of the interaction is of electrostatic and/or hydrogen bonding type, formed most probably between OH, NH and CO groups of MTX and different DNA functions. Finally, electrochemical impedance spectroscopy results approved the high affinity of the systems for 4T1 cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

The TubR-centromere complex adopts a double-ring segrosome structure in Type III partition systems.

PubMed

Martín-García, Bárbara; Martín-González, Alejandro; Carrasco, Carolina; Hernández-Arriaga, Ana M; Ruíz-Quero, Rubén; Díaz-Orejas, Ramón; Aicart-Ramos, Clara; Moreno-Herrero, Fernando; Oliva, María A

2018-05-14

In prokaryotes, the centromere is a specialized segment of DNA that promotes the assembly of the segrosome upon binding of the Centromere Binding Protein (CBP). The segrosome structure exposes a specific surface for the interaction of the CBP with the motor protein that mediates DNA movement during cell division. Additionally, the CBP usually controls the transcriptional regulation of the segregation system as a cell cycle checkpoint. Correct segrosome functioning is therefore indispensable for accurate DNA segregation. Here, we combine biochemical reconstruction and structural and biophysical analysis to bring light to the architecture of the segrosome complex in Type III partition systems. We present the particular features of the centromere site, tubC, of the model system encoded in Clostridium botulinum prophage c-st. We find that the split centromere site contains two different iterons involved in the binding and spreading of the CBP, TubR. The resulting nucleoprotein complex consists of a novel double-ring structure that covers part of the predicted promoter. Single molecule data provides a mechanism for the formation of the segrosome structure based on DNA bending and unwinding upon TubR binding.

Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ® swabs.

PubMed

Ambers, Angie; Wiley, Rachel; Novroski, Nicole; Budowle, Bruce

2018-01-01

Previous studies have shown that nylon flocked swabs outperform traditional fiber swabs in DNA recovery due to their innovative design and lack of internal absorbent core to entrap cellular materials. The microFLOQ ® Direct swab, a miniaturized version of the 4N6 FLOQSwab ® , has a small swab head that is treated with a lysing agent which allows for direct amplification and DNA profiling from sample collection to final result in less than two hours. Additionally, the microFLOQ ® system subsamples only a minute portion of a stain and preserves the vast majority of the sample for subsequent testing or re-analysis, if desired. The efficacy of direct amplification of DNA from dilute bloodstains, saliva stains, and touch samples was evaluated using microFLOQ ® Direct swabs and the GlobalFiler™ Express system. Comparisons were made to traditional methods to assess the robustness of this alternate workflow. Controlled studies with 1:19 and 1:99 dilutions of bloodstains and saliva stains consistently yielded higher STR peak heights than standard methods with 1ng input DNA from the same samples. Touch samples from common items yielded single source and mixed profiles that were consistent with primary users of the objects. With this novel methodology/workflow, no sample loss occurs and therefore more template DNA is available during amplification. This approach may have important implications for analysis of low quantity and/or degraded samples that plague forensic casework. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluation of partial 16S ribosomal DNA sequencing for identification of nocardia species by using the MicroSeq 500 system with an expanded database.

PubMed

Cloud, Joann L; Conville, Patricia S; Croft, Ann; Harmsen, Dag; Witebsky, Frank G; Carroll, Karen C

2004-02-01

Identification of clinically significant nocardiae to the species level is important in patient diagnosis and treatment. A study was performed to evaluate Nocardia species identification obtained by partial 16S ribosomal DNA (rDNA) sequencing by the MicroSeq 500 system with an expanded database. The expanded portion of the database was developed from partial 5' 16S rDNA sequences derived from 28 reference strains (from the American Type Culture Collection and the Japanese Collection of Microorganisms). The expanded MicroSeq 500 system was compared to (i). conventional identification obtained from a combination of growth characteristics with biochemical and drug susceptibility tests; (ii). molecular techniques involving restriction enzyme analysis (REA) of portions of the 16S rRNA and 65-kDa heat shock protein genes; and (iii). when necessary, sequencing of a 999-bp fragment of the 16S rRNA gene. An unknown isolate was identified as a particular species if the sequence obtained by partial 16S rDNA sequencing by the expanded MicroSeq 500 system was 99.0% similar to that of the reference strain. Ninety-four nocardiae representing 10 separate species were isolated from patient specimens and examined by using the three different methods. Sequencing of partial 16S rDNA by the expanded MicroSeq 500 system resulted in only 72% agreement with conventional methods for species identification and 90% agreement with the alternative molecular methods. Molecular methods for identification of Nocardia species provide more accurate and rapid results than the conventional methods using biochemical and susceptibility testing. With an expanded database, the MicroSeq 500 system for partial 16S rDNA was able to correctly identify the human pathogens N. brasiliensis, N. cyriacigeorgica, N. farcinica, N. nova, N. otitidiscaviarum, and N. veterana.

Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases

PubMed Central

Feschenko, Vladimir V.; Rajman, Luis A.; Lovett, Susan T.

2003-01-01

Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability. Such rearrangements underlie several human genetic diseases. In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats. We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR. For perfect repeats of ≈100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion. Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases. Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases. For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system. By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins. Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment. Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA. PMID:12538867

Analysis of DNA Sequences by an Optical Time-Integrating Correlator: Proof-of-Concept Experiments.

DTIC Science & Technology

1992-05-01

DNA ANALYSIS STRATEGY 4 2.1 Representation of DNA Bases 4 2.2 DNA Analysis Strategy 6 3.0 CUSTOM GENERATORS FOR DNA SEQUENCES 10 3.1 Hardware Design 10...of the DNA bases where each base is represented by a 7-bits long pseudorandom sequence. 5 Figure 4: Coarse analysis of a DNA sequence. 7 Figure 5: Fine...a 20-bases long database. 32 xiii LIST OF TABLES PAGE Table 1: Short representations of the DNA bases where each base is represented by 7-bits long

Induction of mucosal IgA by a novel jet delivery technique for HIV-1 DNA.

PubMed

Lundholm, P; Asakura, Y; Hinkula, J; Lucht, E; Wahren, B

1999-04-09

Novel ways of delivering plasmid DNA to elicit humoral IgA, IgG and cell-mediated immune responses in mice were investigated. Intraoral administration of DNA in the cheek, using a jet immunization technique, elicited the highest IgA mucosal responses. Intranasal immunization gave strong mucosal IgA responses and persistent systemic IgG. Immunoglobulin isotype analysis revealed an IgG1 profile for intramuscular tongue and gene gun immunizations and an IgG2a profile following oral jet injection and intranasal application. The route of delivery was of importance for the characteristics and quality of the mucosal immune response following DNA immunization. For DNA vaccine delivery, the intraoral jet injection technique has the advantages of being a simple and rapid way of administering the DNA in solution and of provoking specific mucosal IgA when administered in the mucosal associated lymphoid tissue.

Successful amplification of DNA aboard the International Space Station.

PubMed

Boguraev, Anna-Sophia; Christensen, Holly C; Bonneau, Ashley R; Pezza, John A; Nichols, Nicole M; Giraldez, Antonio J; Gray, Michelle M; Wagner, Brandon M; Aken, Jordan T; Foley, Kevin D; Copeland, D Scott; Kraves, Sebastian; Alvarez Saavedra, Ezequiel

2017-01-01

As the range and duration of human ventures into space increase, it becomes imperative that we understand the effects of the cosmic environment on astronaut health. Molecular technologies now widely used in research and medicine will need to become available in space to ensure appropriate care of astronauts. The polymerase chain reaction (PCR) is the gold standard for DNA analysis, yet its potential for use on-orbit remains under-explored. We describe DNA amplification aboard the International Space Station (ISS) through the use of a miniaturized miniPCR system. Target sequences in plasmid, zebrafish genomic DNA, and bisulfite-treated DNA were successfully amplified under a variety of conditions. Methylation-specific primers differentially amplified bisulfite-treated samples as would be expected under standard laboratory conditions. Our findings establish proof of concept for targeted detection of DNA sequences during spaceflight and lay a foundation for future uses ranging from environmental monitoring to on-orbit diagnostics.

Automated one-step DNA sequencing based on nanoliter reaction volumes and capillary electrophoresis.

PubMed

Pang, H M; Yeung, E S

2000-08-01

An integrated system with a nano-reactor for cycle-sequencing reaction coupled to on-line purification and capillary gel electrophoresis has been demonstrated. Fifty nanoliters of reagent solution, which includes dye-labeled terminators, polymerase, BSA and template, was aspirated and mixed with the template inside the nano-reactor followed by cycle-sequencing reaction. The reaction products were then purified by a size-exclusion chromatographic column operated at 50 degrees C followed by room temperature on-line injection of the DNA fragments into a capillary for gel electrophoresis. Over 450 bases of DNA can be separated and identified. As little as 25 nl reagent solution can be used for the cycle-sequencing reaction with a slightly shorter read length. Significant savings on reagent cost is achieved because the remaining stock solution can be reused without contamination. The steps of cycle sequencing, on-line purification, injection, DNA separation, capillary regeneration, gel-filling and fluidic manipulation were performed with complete automation. This system can be readily multiplexed for high-throughput DNA sequencing or PCR analysis directly from templates or even biological materials.

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

PubMed Central

Allison, J; Hall, L; MacIntyre, I; Craig, R K

1981-01-01

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein. Images Fig. 1. Fig. 2. Fig. 3. PMID:6896146

Ammonia- and nitrite-oxidizing bacterial communities in a pilot-scale chloraminated drinking water distribution system.

PubMed

Regan, John M; Harrington, Gregory W; Noguera, Daniel R

2002-01-01

Nitrification in drinking water distribution systems is a common operational problem for many utilities that use chloramines for secondary disinfection. The diversity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the distribution systems of a pilot-scale chloraminated drinking water treatment system was characterized using terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene (ribosomal DNA [rDNA]) cloning and sequencing. For ammonia oxidizers, 16S rDNA-targeted T-RFLP indicated the presence of Nitrosomonas in each of the distribution systems, with a considerably smaller peak attributable to Nitrosospira-like AOB. Sequences of AOB amplification products aligned within the Nitrosomonas oligotropha cluster and were closely related to N. oligotropha and Nitrosomonas ureae. The nitrite-oxidizing communities were comprised primarily of Nitrospira, although Nitrobacter was detected in some samples. These results suggest a possible selection of AOB related to N. oligotropha and N. ureae in chloraminated systems and demonstrate the presence of NOB, indicating a biological mechanism for nitrite loss that contributes to a reduction in nitrite-associated chloramine decay.

Ammonia- and Nitrite-Oxidizing Bacterial Communities in a Pilot-Scale Chloraminated Drinking Water Distribution System

PubMed Central

Regan, John M.; Harrington, Gregory W.; Noguera, Daniel R.

2002-01-01

Nitrification in drinking water distribution systems is a common operational problem for many utilities that use chloramines for secondary disinfection. The diversity of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the distribution systems of a pilot-scale chloraminated drinking water treatment system was characterized using terminal restriction fragment length polymorphism (T-RFLP) analysis and 16S rRNA gene (ribosomal DNA [rDNA]) cloning and sequencing. For ammonia oxidizers, 16S rDNA-targeted T-RFLP indicated the presence of Nitrosomonas in each of the distribution systems, with a considerably smaller peak attributable to Nitrosospira-like AOB. Sequences of AOB amplification products aligned within the Nitrosomonas oligotropha cluster and were closely related to N. oligotropha and Nitrosomonas ureae. The nitrite-oxidizing communities were comprised primarily of Nitrospira, although Nitrobacter was detected in some samples. These results suggest a possible selection of AOB related to N. oligotropha and N. ureae in chloraminated systems and demonstrate the presence of NOB, indicating a biological mechanism for nitrite loss that contributes to a reduction in nitrite-associated chloramine decay. PMID:11772611

The phase behavior of cationic lipid-DNA complexes.

PubMed Central

May, S; Harries, D; Ben-Shaul, A

2000-01-01

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram. PMID:10733951

Optimized pH method for DNA elution from buccal cells collected in Whatman FTA cards.

PubMed

Lema, Carolina; Kohl-White, Kendra; Lewis, Laurie R; Dao, Dat D

2006-01-01

DNA is the most accessible biologic material for obtaining information from the human genome because of its molecular stability and its presence in every nucleated cell. Currently, single nucleotide polymorphism genotyping and DNA methylation are the main DNA-based approaches to deriving genomic and epigenomic disease biomarkers. Upon the discontinuation of the Schleicher & Schuell IsoCode product (Dassel, Germany), which was a treated paper system to elute DNA from several biologic sources for polymerase chain reaction (PCR) analysis, a high-yielding DNA elution method was imperative. We describe here an improved procedure of the not fully validated Whatman pH-based elution protocol. Our DNA elution procedure from buccal cells collected in Whatman FTA cards (Whatman Inc., Florham Park, NJ) yielded approximately 4 microg of DNA from a 6-mm FTA card punch and was successfully applied for HLA-DQB1 genotyping. The genotypes showed complete concordance with data obtained from blood of the same subjects. The achieved high DNA yield from buccal cells suggests a potential cost-effective tool for genomic and epigenomic disease biomarkers development.

Moving environmental DNA methods from concept to practice for monitoring aquatic macroorganisms

USGS Publications Warehouse

Goldberg, Caren S.; Strickler, Katherine M.; Pilliod, David S.

2015-01-01

The discovery that macroorganisms can be detected from their environmental DNA (eDNA) in aquatic systems has immense potential for the conservation of biological diversity. This special issue contains 11 papers that review and advance the field of eDNA detection of vertebrates and other macroorganisms, including studies of eDNA production, transport, and degradation; sample collection and processing to maximize detection rates; and applications of eDNA for conservation using citizen scientists. This body of work is an important contribution to the ongoing efforts to take eDNA detection of macroorganisms from technical breakthrough to established, reliable method that can be used in survey, monitoring, and research applications worldwide. While the rapid advances in this field are remarkable, important challenges remain, including consensus on best practices for collection and analysis, understanding of eDNA diffusion and transport, and avoidance of inhibition in sample collection and processing. Nonetheless, as demonstrated in this special issue, eDNA techniques for research and monitoring are beginning to realize their potential for contributing to the conservation of biodiversity globally.

A common anchor facilitated GO-DNA nano-system for multiplex microRNA analysis in live cells.

PubMed

Yu, Jiantao; He, Sihui; Shao, Chen; Zhao, Haoran; Li, Jing; Tian, Leilei

2018-04-19

The design of a nano-system for the detection of intracellular microRNAs is challenging as it must fulfill complex requirements, i.e., it must have a high sensitivity to determine the dynamic expression level, a good reliability for multiplex and simultaneous detection, and a satisfactory biostability to work in biological environments. Instead of employing a commonly used physisorption or a full-conjugation strategy, here, a GO-DNA nano-system was developed under graft/base-pairing construction. The common anchor sequence was chemically grafted to GO to base-pair with various microRNA probes; and the hybridization with miRNAs drives the dyes on the probes to leave away from GO, resulting in "turned-on" fluorescence. This strategy not only simplifies the synthesis but also efficiently balances the loading yields of different probes. Moreover, the conjugation yield of GO with a base-paired hybrid has been improved by more than two-fold compared to that of the conjugation with a single strand. We demonstrated that base-paired DNA probes could be efficiently delivered into cells along with GO and are properly stabilized by the conjugated anchor sequence. The resultant GO-DNA nano-system exhibited high stability in a complex biological environment and good resistance to nucleases, and was able to accurately discriminate various miRNAs without cross-reaction. With all of these positive features, the GO-DNA nano-system can simultaneously detect three miRNAs and monitor their dynamic expression levels.

CCR6+ Th cell distribution differentiates systemic lupus erythematosus patients based on anti-dsDNA antibody status.

PubMed

Zhong, Wei; Jiang, Zhenyu; Wu, Jiang; Jiang, Yanfang; Zhao, Ling

2018-01-01

Systemic lupus erythematosus (SLE) disease has been shown to be associated with the generation of multiple auto-antibodies. Among these, anti-dsDNA antibodies (anti-DNAs) are specific and play a pathogenic role in SLE. Indeed, anti-DNA + SLE patients display a worse disease course. The generation of these pathogenic anti-DNAs has been attributed to the interaction between aberrant T helper (Th) cells and autoimmune B cells. Thus, in this study we have investigated whether CCR6 + Th cells have the ability to differentiate SLE patients based on anti-DNA status, and if their distribution has any correlation with disease activity. We recruited 25 anti-DNA + and 25 anti-DNA - treatment-naive onset SLE patients, matched for various clinical characteristics in our nested matched case-control study. CCR6 + Th cells and their additional subsets were analyzed in each patient by flow cytometry. Anti-DNA + SLE patients specifically had a higher percentage of Th cells expressing CCR6 and CXCR3. Further analysis of CCR6 + Th cell subsets showed that anti-DNA + SLE patients had elevated proportions of Th9, Th17, Th17.1 and CCR4/CXCR3 double-negative (DN) cells. However, the proportions of CCR6 - Th subsets, including Th1 and Th2 cells, did not show any association with anti-DNA status. Finally, we identified a correlation between CCR6 + Th subsets and clinical indicators, specifically in anti-DNA + SLE patients. Our data indicated that CCR6 + Th cells and their subsets were elevated and correlated with disease activity in anti-DNA + SLE patients. We speculated that CCR6 + Th cells may contribute to distinct disease severity in anti-DNA + SLE patients.

Evaluating Protocols for Porcine Faecal Microbiome Recollection, Storage and DNA Extraction: from the Farm to the Lab.

PubMed

Muiños-Bühl, Anixa; González-Recio, Oscar; Muñoz, María; Óvilo, Cristina; García-Casco, Juan; Fernández, Ana I

2018-06-01

There is a growing interest in understanding the role of the gut microbiome on productive and meat quality-related traits in livestock species in order to develop new useful tools for improving pig production systems and industry. Faecal samples are analysed as a proxy of gut microbiota and here the selection of suitable protocols for faecal sampling and DNA isolation is a critical first step in order to obtain reliable results, even more to compare results obtained from different studies. The aim of the current study was to establish in a cost-effective way, using automated ribosomal intergenic spacer analysis technique, a protocol for porcine faecal sampling and storage at farm and slaughterhouse and to determine the most efficient microbiota DNA isolation kit among those most widely used. Operational Taxonomic Unit profiles were compared from Iberian pig faecal samples collected from rectum or ground, stored with liquid N 2 , room temperature or RNAlater, and processed with QIAamp DNA Stool (Qiagen), PowerFecal DNA Isolation (Mobio) or SpeedTools Tissue DNA extraction (Biotools) commercial kits. The results, focused on prokaryote sampling, based on DNA yield and quality, OTU number and Sørensen similarity Indexes, indicate that the recommended protocol for porcine faecal microbiome sampling at farm should include: the collection from porcine rectum to avoid contamination; the storage in liquid N 2 or even at room temperature, but not in RNAlater; and the isolation of microbiota DNA using PowerFecal DNA Isolation kit. These conditions provide more reliable DNA samples for further microbiome analysis.

Geometry-dependent DNA-TiO2 immobilization mechanism: A spectroscopic approach

NASA Astrophysics Data System (ADS)

Silva-Moraes, M. O.; Garcia-Basabe, Y.; de Souza, R. F. B.; Mota, A. J.; Passos, R. R.; Galante, D.; Fonseca Filho, H. D.; Romaguera-Barcelay, Y.; Rocco, M. L. M.; Brito, W. R.

2018-06-01

DNA nucleotides are used as a molecular recognition system on electrodes modified to be applied in the detection of various diseases, but immobilization mechanisms, as well as, charge transfers are not satisfactorily described in the literature. An electrochemical and spectroscopic study was carried out to characterize the molecular groups involved in the direct immobilization of DNA structures on the surface of nanostructured TiO2 with the aim of evaluating the influence of the geometrical aspects. X-ray photoelectron spectroscopy at O1s and P2p core levels indicate that immobilization of DNA samples occurs through covalent (Psbnd Osbnd Ti) bonds. X-ray absorption spectra at the Ti2p edge reinforce this conclusion. A new species at 138.5 eV was reported from P2p XPS spectra analysis which plays an important role in DNA-TiO2 immobilization. The Psbnd Osbnd Ti/Osbnd Ti ratio showed that quantitatively the DNA immobilization mechanism is dependent on their geometry, becoming more efficient for plasmid ds-DNA structures than for PCR ds-DNA structures. The analysis of photoabsorption spectra at C1s edge revealed that the molecular groups that participate in the C1s → LUMO electronic transitions have different pathways in the charge transfer processes at the DNA-TiO2 interface. Our results may contribute to additional studies of immobilization mechanisms understanding the influence of the geometry of different DNA molecules on nanostructured semiconductor and possible impact to the charge transfer processes with application in biosensors or aptamers.

Availability: A Metric for Nucleic Acid Strand Displacement Systems

PubMed Central

2016-01-01

DNA strand displacement systems have transformative potential in synthetic biology. While powerful examples have been reported in DNA nanotechnology, such systems are plagued by leakage, which limits network stability, sensitivity, and scalability. An approach to mitigate leakage in DNA nanotechnology, which is applicable to synthetic biology, is to introduce mismatches to complementary fuel sequences at key locations. However, this method overlooks nuances in the secondary structure of the fuel and substrate that impact the leakage reaction kinetics in strand displacement systems. In an effort to quantify the impact of secondary structure on leakage, we introduce the concepts of availability and mutual availability and demonstrate their utility for network analysis. Our approach exposes vulnerable locations on the substrate and quantifies the secondary structure of fuel strands. Using these concepts, a 4-fold reduction in leakage has been achieved. The result is a rational design process that efficiently suppresses leakage and provides new insight into dynamic nucleic acid networks. PMID:26875531

Microbial Toluene Removal in Hypoxic Model Constructed Wetlands Occurs Predominantly via the Ring Monooxygenation Pathway.

PubMed

Martínez-Lavanchy, P M; Chen, Z; Lünsmann, V; Marin-Cevada, V; Vilchez-Vargas, R; Pieper, D H; Reiche, N; Kappelmeyer, U; Imparato, V; Junca, H; Nijenhuis, I; Müller, J A; Kuschk, P; Heipieper, H J

2015-09-01

In the present study, microbial toluene degradation in controlled constructed wetland model systems, planted fixed-bed reactors (PFRs), was queried with DNA-based methods in combination with stable isotope fractionation analysis and characterization of toluene-degrading microbial isolates. Two PFR replicates were operated with toluene as the sole external carbon and electron source for 2 years. The bulk redox conditions in these systems were hypoxic to anoxic. The autochthonous bacterial communities, as analyzed by Illumina sequencing of 16S rRNA gene amplicons, were mainly comprised of the families Xanthomonadaceae, Comamonadaceae, and Burkholderiaceae, plus Rhodospirillaceae in one of the PFR replicates. DNA microarray analyses of the catabolic potentials for aromatic compound degradation suggested the presence of the ring monooxygenation pathway in both systems, as well as the anaerobic toluene pathway in the PFR replicate with a high abundance of Rhodospirillaceae. The presence of catabolic genes encoding the ring monooxygenation pathway was verified by quantitative PCR analysis, utilizing the obtained toluene-degrading isolates as references. Stable isotope fractionation analysis showed low-level of carbon fractionation and only minimal hydrogen fractionation in both PFRs, which matches the fractionation signatures of monooxygenation and dioxygenation. In combination with the results of the DNA-based analyses, this suggests that toluene degradation occurs predominantly via ring monooxygenation in the PFRs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

DNA Binding Peptide Directed Synthesis of Continuous DNA Nanowires for Analysis of Large DNA Molecules by Scanning Electron Microscope.

PubMed

Kim, Kyung-Il; Lee, Seonghyun; Jin, Xuelin; Kim, Su Ji; Jo, Kyubong; Lee, Jung Heon

2017-01-01

Synthesis of smooth and continuous DNA nanowires, preserving the original structure of native DNA, and allowing its analysis by scanning electron microscope (SEM), is demonstrated. Gold nanoparticles densely assembled on the DNA backbone via thiol-tagged DNA binding peptides work as seeds for metallization of DNA. This method allows whole analysis of DNA molecules with entangled 3D features. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In silico studies to explore the mutagenic ability of 5-halo/oxy/li-oxy-uracil bases with guanine of DNA base pairs.

PubMed

Jana, Kalyanashis; Ganguly, Bishwajit

2014-10-16

DNA nucleobases are reactive in nature and undergo modifications by deamination, oxidation, alkylation, or hydrolysis processes. Many such modified bases are susceptible to mutagenesis when formed in cellular DNA. The mutagenesis can occur by mispairing with DNA nucleobases by a DNA polymerase during replication. We have performed a study of mispairing of DNA bases with unnatural bases computationally. 5-Halo uracils have been studied as mispairs in mutagenesis; however, the reports on their different forms are scarce in the literature. The stability of mispairs with keto form, enol form, and ionized form of 5-halo-uracil has been computed with the M06-2X/6-31+G** level of theory. The enol form of 5-halo-uracil showed remarkable stability toward DNA mispair compared to the corresponding keto and ionized forms. (F)U-G mispair showed the highest stability in the series and (Halo)(U(enol/ionized)-G mispair interactions energies are more stable than the natural G-C basepair of DNA. To enhance the stability of DNA mispairs, we have introduced the hydroxyl group in the place of halogen atoms, which provides additional hydrogen-bonding interactions in the system while forming the 5-membered ring. The study has been further extended with lithiated 5-hydroxymethyl-uracil to stabilize the DNA mispair. (CH2OLi)U(ionized)-G mispair has shown the highest stability (ΔG = -32.4 kcal/mol) with multi O-Li interactions. AIM (atoms in molecules) and EDA (energy decomposition analysis) analysis has been performed to examine the nature of noncovalent interactions in such mispairs. EDA analysis has shown that electrostatic energy mainly contributes toward the interaction energy of mispairs. The higher stability achieved in these studied mispairs can play a pivotal role in the mutagenesis and can help to attain the mutation for many desired biological processes.

Fluorescence enhancement of quercetin complexes by silver nanoparticles and its analytical application

NASA Astrophysics Data System (ADS)

Liu, Ping; Zhao, Liangliang; Wu, Xia; Huang, Fei; Wang, Minqin; Liu, Xiaodan

2014-03-01

It is found that the plasmon effect of silver nanoparticles (AgNPs) helps to enhance the fluorescence intensity of the quercetin (Qu) and nucleic acids system. Qu exhibited strong fluorescence enhancement when it bound to nucleic acids in the presence of AgNPs. Based on this, a sensitive method for the determination of nucleic acids was developed. The detection limits for the nucleic acids (S/N = 3) were reduced to the ng mL-1 level. The interaction mechanism of the AgNPs-fish sperm DNA (fsDNA)-Qu system was also investigated in this paper. This complex system of Qu and AgNPs was also successfully used for the detection of nucleic acids in agarose gel electrophoresis analysis. Preliminary results indicated that AgNPs also helped to improve sensitivity in the fluorescence image analysis of Qu combined with cellular contents in Arabidopsis thaliana protoplasts.

A highly efficient transposon mutagenesis system for the tomato pathogen Clavibacter michiganensis subsp. michiganensis.

PubMed

Kirchner, O; Gartemann, K H; Zellermann, E M; Eichenlaub, R; Burger, A

2001-11-01

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 x 10(6) transformants per microg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 x 10(3) transposon mutants per pg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

Belimumab in the treatment of systemic lupus erythematosus: high disease activity predictors of response.

PubMed

van Vollenhoven, Ronald F; Petri, Michelle A; Cervera, Ricard; Roth, David A; Ji, Beulah N; Kleoudis, Christi S; Zhong, Z John; Freimuth, William

2012-08-01

To identify factors that predict response to belimumab treatment in the phase 3 BLISS trials of autoantibody-positive systemic lupus erythematosus (SLE) and further analyse clinical efficacy in various patient subsets. The BLISS trials compared belimumab 1 and 10 mg/kg versus placebo, all plus standard SLE therapy, over 52 or 76 weeks. Pooled subgroup analyses of week 52 SLE responder index rates (the primary endpoint in both trials) were performed based on demographic characteristics and baseline disease activity indicators. Pooled multivariate analysis was performed to determine predictors of response and treatment effect. Pooled univariate and multivariate analyses (N=1684) identified baseline factors associated with an increased benefit of belimumab versus placebo. These factors included the Safety Of Estrogens In Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENA-SLEDAI) ≥10, low complement, anti-dsDNA positivity and corticosteroid use. Efficacy outcomes were assessed in the low complement/anti-dsDNA-positive and SELENA-SLEDAI ≥10 subgroups. Week 52 SLE Responder Index rates in the low complement/anti-dsDNA-positive subgroup were 31.7%, 41.5% (p=0.002) and 51.5% (p<0.001) with placebo and belimumab 1 mg/kg and 10 mg/kg, respectively; corresponding rates in the SELENA-SLEDAI ≥10 subgroup were 44.3%, 58.0% (p<0.001) and 63.2% (p<0.001). Further analysis of secondary endpoints in the low complement/anti-dsDNA-positive subgroup showed that compared with placebo, belimumab produced greater benefits regarding severe flares, corticosteroid use and health-related quality of life. These findings suggest that belimumab has greater therapeutic benefit than standard therapy alone in patients with higher disease activity, anti-dsDNA positivity, low complement or corticosteroid treatment at baseline. CLINICALTRIALS.GOV: identifiers NCT00424476 and NCT00410384.

Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis.

PubMed

De Santis, Riccardo; Ciammaruconi, Andrea; Faggioni, Giovanni; D'Amelio, Raffaele; Marianelli, Cinzia; Lista, Florigio

2009-04-07

Brucellosis is an important zoonosis caused by the genus Brucella. In addition Brucella represents potential biological warfare agents due to the high contagious rates for humans and animals. Therefore, the strain typing epidemiological tool may be crucial for tracing back source of infection in outbreaks and discriminating naturally occurring outbreaks versus bioterroristic event. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 15 polymorphic markers was previously described. The obtained MLVA band profiles may be resolved by techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing. In this paper a rapid, accurate and reproducible system, based on the Lab on a chip technology was set up for Brucella spp. genotyping. Seventeen DNA samples of Brucella strains isolated in Sicily, previously genotyped, and twelve DNA samples, provided by MLVA Brucella VNTR ring trial, were analyzed by MLVA-15 on Agilent 2100. The DNA fragment sizes produced by Agilent, compared with those expected, showed discrepancies; therefore, in order to assign the correct alleles to the Agilent DNA fragment sizes, a conversion table was produced. In order to validate the system twelve unknown DNA samples were analyzed by this method obtaining a full concordance with the VNTR ring trial results. In this paper we described a rapid and specific detection method for the characterization of Brucella isolates. The comparison of the MLVA typing data produced by Agilent system with the data obtained by standard sequencing or ethidium bromide slab gel electrophoresis showed a general concordance of the results. Therefore this platform represents a fair compromise among costs, speed and specificity compared to any conventional molecular typing technique.

Systems-Level Analysis of EGFR Inhibition-DNA Damage Combination Treatment in Breast Cancer

DTIC Science & Technology

2011-09-01

optimal when erlotinib was given 24 hours before doxorubicin. Conversely, erlotinib given after doxorubicin slightly desensitized the cells to...status of BT-20, and potentially other triple negative breast cancer cells. Although the driving oncogenes for this subtype are not currently known...agents. In fact (for reasons that are currently unclear), these cells were typically desensitized to DNA damage when pre-treated with erlotinib

Nucleotide sequence and regulatory studies of VGF, a nervous system-specific mRNA that is rapidly and relatively selectively induced by nerve growth factor.

PubMed

Salton, S R

1991-09-01

A nervous system-specific mRNA that is rapidly induced in PC12 cells to a greater extent by nerve growth factor (NGF) than by epidermal growth factor treatment has been cloned. The polypeptide deduced from the nucleic acid sequence of the NGF33.1 cDNA clone contains regions of amino acid sequence identity with that predicted by the cDNA clone VGF, and further analysis suggests that both NGF33.1 and VGF cDNA clones very likely correspond to the same mRNA (VGF). In this report both the nucleic acid sequence that corresponds to VGF mRNA and the polypeptide predicted by the NGF33.1 cDNA clone are presented. Genomic Southern analysis and database comparison did not detect additional sequences with high homology to the VGF gene. Induction of VGF mRNA by depolarization and phorbol 12-myristate 13-acetate treatment was greater than by serum stimulation or protein kinase A pathway activation. These studies suggest that VGF mRNA is induced to the greatest extent by NGF treatment and that VGF is one of the most rapidly regulated neuronal mRNAs identified in PC12 cells.

DNAtraffic--a new database for systems biology of DNA dynamics during the cell life.

PubMed

Kuchta, Krzysztof; Barszcz, Daniela; Grzesiuk, Elzbieta; Pomorski, Pawel; Krwawicz, Joanna

2012-01-01

DNAtraffic (http://dnatraffic.ibb.waw.pl/) is dedicated to be a unique comprehensive and richly annotated database of genome dynamics during the cell life. It contains extensive data on the nomenclature, ontology, structure and function of proteins related to the DNA integrity mechanisms such as chromatin remodeling, histone modifications, DNA repair and damage response from eight organisms: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli and Arabidopsis thaliana. DNAtraffic contains comprehensive information on the diseases related to the assembled human proteins. DNAtraffic is richly annotated in the systemic information on the nomenclature, chemistry and structure of DNA damage and their sources, including environmental agents or commonly used drugs targeting nucleic acids and/or proteins involved in the maintenance of genome stability. One of the DNAtraffic database aim is to create the first platform of the combinatorial complexity of DNA network analysis. Database includes illustrations of pathways, damage, proteins and drugs. Since DNAtraffic is designed to cover a broad spectrum of scientific disciplines, it has to be extensively linked to numerous external data sources. Our database represents the result of the manual annotation work aimed at making the DNAtraffic much more useful for a wide range of systems biology applications.

DNAtraffic—a new database for systems biology of DNA dynamics during the cell life

PubMed Central

Kuchta, Krzysztof; Barszcz, Daniela; Grzesiuk, Elzbieta; Pomorski, Pawel; Krwawicz, Joanna

2012-01-01

DNAtraffic (http://dnatraffic.ibb.waw.pl/) is dedicated to be a unique comprehensive and richly annotated database of genome dynamics during the cell life. It contains extensive data on the nomenclature, ontology, structure and function of proteins related to the DNA integrity mechanisms such as chromatin remodeling, histone modifications, DNA repair and damage response from eight organisms: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Escherichia coli and Arabidopsis thaliana. DNAtraffic contains comprehensive information on the diseases related to the assembled human proteins. DNAtraffic is richly annotated in the systemic information on the nomenclature, chemistry and structure of DNA damage and their sources, including environmental agents or commonly used drugs targeting nucleic acids and/or proteins involved in the maintenance of genome stability. One of the DNAtraffic database aim is to create the first platform of the combinatorial complexity of DNA network analysis. Database includes illustrations of pathways, damage, proteins and drugs. Since DNAtraffic is designed to cover a broad spectrum of scientific disciplines, it has to be extensively linked to numerous external data sources. Our database represents the result of the manual annotation work aimed at making the DNAtraffic much more useful for a wide range of systems biology applications. PMID:22110027

Maternal smoking does not affect the amount of cell-free fetal DNA in maternal plasma during the 1st trimester of pregnancy.

PubMed

Tarquini, F; Picchiassi, E; Centra, M; Pennacchi, L; Galeone, F; Bini, V; Di Renzo, G C; Coata, G

2015-01-01

CffDNA, from 344 non-smoking, 38 smoking and 33 ex-smoking pregnant women at 11 (+0)-13 (+6) gestational weeks, was extracted and quantified by the multicopy DYS14, as the fetal DNA marker and using the quantitative real-time PCR 7300 detection system. The smoking habit was based on maternal self-report, confirmed by cotinine levels and male fetuses were verified by phenotype at birth. The genders of newborns were compared with DYS14-cffDNA analysis, achieving a 100% diagnostic accuracy of the test. A total of 177 non-smokers, 18 smokers and 22 ex-smoker pregnancies with male fetuses were identified by the cffDNA concentration. Results showed that smoking status was not associated with different amounts of DYS14-cffDNA (p = 0.159), suggesting the possibility of offering cffDNA testing to all pregnant women, even if they are active smokers or ex-smokers, and the test can be unadjusted for smoking status.

DNA-magnetic bead detection using disposable cards and the anisotropic magnetoresistive sensor

NASA Astrophysics Data System (ADS)

Hien, L. T.; Quynh, L. K.; Huyen, V. T.; Tu, B. D.; Hien, N. T.; Phuong, D. M.; Nhung, P. H.; Giang, D. T. H.; Duc, N. H.

2016-12-01

A disposable card incorporating specific DNA probes targeting the 16 S rRNA gene of Streptococcus suis was developed for magnetically labeled target DNA detection. A single-stranded target DNA was hybridized with the DNA probe on the SPA/APTES/PDMS/Si as-prepared card, which was subsequently magnetically labeled with superparamagnetic beads for detection using an anisotropic magnetoresistive (AMR) sensor. An almost linear response between the output signal of the AMR sensor and amount of single-stranded target DNA varied from 4.5 to 18 pmol was identified. From the sensor output signal response towards the mass of magnetic beads which were directly immobilized on the disposable card surface, the limit of detection was estimated about 312 ng ferrites, which corresponds to 3.8 μemu. In comparison with DNA detection by conventional biosensor based on magnetic bead labeling, disposable cards are featured with higher efficiency and performances, ease of use and less running cost with respects to consumables for biosensor in biomedical analysis systems operating with immobilized bioreceptor.

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

PubMed

Pineda, Gina M; Montgomery, Anne H; Thompson, Robyn; Indest, Brooke; Carroll, Marion; Sinha, Sudhir K

2014-11-01

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample. In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Analysis of ploidy and the patterns of amplified fragment length polymorphism and methylation sensitive amplified polymorphism in strawberry plants recovered from cryopreservation.

PubMed

Hao, Yu-Jin; You, Chun-Xiang; Deng, Xui-Xin

2002-01-01

Shoot-tips of 10 strawberry genotypes were successfully cryopreserved using a modified encapsulation-dehydration method. All genotypes survived cryopreservation with high survival and regeneration rates. Eight Joho single-bud sibling lines were established as a model system for genetic analysis. Although cytological examination found chromosomal variation in both non-cryopreserved and cryopreserved samples, the ploidy constitution remained relatively stable after cryopreservation. DNA samples digested with MseI and PstI were used for amplified fragmentation length polymorphism (AFLP) assay. In 16 primer combinations, only one, namely, PCCA-MCAG, detected one site where band pattern changed after cryopreservation, which might be contributed to the change in DNA methylation status at PstI recognition site. Methylation sensitive amplified polymorphism (MSAP) assay was carried out for further investigation on the influence of cryopreservation on DNA methylation status. It was found that cryopreservation induced a significant change in DNA methylation status.

A comprehensive complex systems approach to the study and analysis of mammalian cell cycle control system in the presence of DNA damage stress.

PubMed

Abroudi, Ali; Samarasinghe, Sandhya; Kulasiri, Don

2017-09-21

Not many models of mammalian cell cycle system exist due to its complexity. Some models are too complex and hard to understand, while some others are too simple and not comprehensive enough. Moreover, some essential aspects, such as the response of G1-S and G2-M checkpoints to DNA damage as well as the growth factor signalling, have not been investigated from a systems point of view in current mammalian cell cycle models. To address these issues, we bring a holistic perspective to cell cycle by mathematically modelling it as a complex system consisting of important sub-systems that interact with each other. This retains the functionality of the system and provides a clearer interpretation to the processes within it while reducing the complexity in comprehending these processes. To achieve this, we first update a published ODE mathematical model of cell cycle with current knowledge. Then the part of the mathematical model relevant to each sub-system is shown separately in conjunction with a diagram of the sub-system as part of this representation. The model sub-systems are Growth Factor, DNA damage, G1-S, and G2-M checkpoint signalling. To further simplify the model and better explore the function of sub-systems, they are further divided into modules. Here we also add important new modules of: chk-related rapid cell cycle arrest, p53 modules expanded to seamlessly integrate with the rapid arrest module, Tyrosine phosphatase modules that activate Cyc_Cdk complexes and play a crucial role in rapid and delay arrest at both G1-S and G2-M, Tyrosine Kinase module that is important for inactivating nuclear transport of CycB_cdk1 through Wee1 to resist M phase entry, Plk1-Related module that is crucial in activating Tyrosine phosphatases and inactivating Tyrosine kinase, and APC-Related module to show steps in CycB degradation. This multi-level systems approach incorporating all known aspects of cell cycle allowed us to (i) study, through dynamic simulation of an ODE model, comprehensive details of cell cycle dynamics under normal and DNA damage conditions revealing the role and value of the added new modules and elements, (ii) assess, through a global sensitivity analysis, the most influential sub-systems, modules and parameters on system response, such as G1-S and G2-M transitions, and (iii) probe deeply into the relationship between DNA damage and cell cycle progression and test the biological evidence that G1-S is relatively inefficient in arresting damaged cells compared to G2-M checkpoint. To perform sensitivity analysis, Self-Organizing Map with Correlation Coefficient Analysis (SOMCCA) is developed which shows that Growth Factor and G1-S Checkpoint sub-systems and 13 parameters in the modules within them are crucial for G1-S and G2-M transitions. To study the relative efficiency of DNA damage checkpoints, a Checkpoint Efficiency Evaluator (CEE) is developed based on perturbation studies and statistical Type II error. Accordingly, cell cycle is about 96% efficient in arresting damaged cells with G2-M checkpoint being more efficient than G1-S. Further, both checkpoint systems are near perfect (98.6%) in passing healthy cells. Thus this study has shown the efficacy of the proposed systems approach to gain a better understanding of different aspects of mammalian cell cycle system separately and as an integrated system that will also be useful in investigating targeted therapy in future cancer treatments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Electromagnetic Fields Exposure Limits

DTIC Science & Technology

2018-01-01

analysis, synthesis, integration and validation of knowledge derived through the scientific method. In NATO, S&T is addressed using different...Panel • NMSG NATO Modelling and Simulation Group • SAS System Analysis and Studies Panel • SCI Systems Concepts and Integration Panel • SET... integrity or morphology. They later also failed to find a lack of direct DNA damage in human blood (strand breaks, alkali-labile sites, and incomplete

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

PubMed

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 10 2 -10 5 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets

PubMed Central

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing. PMID:28740546

An Improved Multiplex Real-Time SYBR Green PCR Assay for Analysis of 24 Target Genes from 16 Bacterial Species in Fecal DNA Samples from Patients with Foodborne Illnesses.

PubMed

Kawase, Jun; Etoh, Yoshiki; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

2016-05-20

Here, we developed a new version of our original screening system (Rapid Foodborne Bacterial Screening 24; RFBS24), which can simultaneously detect 24 genes of foodborne pathogens in fecal DNA samples. This new version (RFBS24 ver. 5) detected all known stx2 subtypes, enterotoxigenic Escherichia coli (STh genotype), and Vibrio parahaemolyticus (trh2), which were not detected by the original RFBS24 assay. The detection limits of RFBS24 ver. 5 were approximately 5.6 × 10(-2)-5.6 × 10(-5) (ng DNA)/reaction, significantly lower (10- to 100-fold) than those of the original RFBS24 for the 22 target genes analyzed here. We also tested the new assay on fecal DNA samples from patients infected with Salmonella, Campylobacter, or enterohemorrhagic E. coli. The number of bacterial target genes detected by RFBS24 ver. 5 was greater than that detected by RFBS24. RFBS24 ver. 5 combined with an Ultra Clean Fecal DNA Isolation Kit showed adequate performance (sensitivity and specificity 89% and 100%, respectively, for Salmonella spp. and 100% and 83%, respectively, for Campylobacter jejuni) in terms of rapid detection of a causative pathogen during foodborne-illness outbreaks. Thus, RFBS24 ver. 5 is more useful than the previous assay system for detection of foodborne pathogens and offers quick simultaneous analysis of many targets and thus facilitates rapid dissemination of information to public health officials.

Nucleic acid extraction techniques and application to the microchip.

PubMed

Price, Carol W; Leslie, Daniel C; Landers, James P

2009-09-07

As recently as the early 1990s, DNA purification was time-consuming, requiring the use of toxic, hazardous reagents. The advent of solid phase extraction techniques and the availability of commercial kits for quick and reliable DNA extraction has relegated those early techniques largely to the history books. High quality DNA can now be extracted from whole blood, serum, saliva, urine, stool, cerebral spinal fluid, tissues, and cells in less time without sacrificing recovery. Having achieved such a radical change in the methodology of DNA extraction, focus has shifted to adapting these methods to a miniaturized system, or "lab-on-a-chip" (A. Manz, N. Graber and H. M. Widmer, Sens. Actuators, B, 1990, 1, 244-248). Manz et al.'s concept of a "miniaturized total chemical analysis system" (microTAS) involved a silicon chip that incorporated sample pretreatment, separation and detection. This review will focus on the first of these steps, sample pretreatment in the form of DNA purification. The intention of this review is to provide an overview of the fundamentals of nucleic acid purification and solid phase extraction (SPE) and to discuss specific microchip DNA extraction successes and challenges. In order to fully appreciate the advances in DNA purification, a brief review of the history of DNA extraction is provided so that the reader has an understanding of the impact that the development of SPE techniques have had. This review will highlight the different methods of nucleic acid extraction (Table 1), including relevant citations, but without an exhaustive summary of the literature. A recent review by Wen et al. (J. Wen, L. A. Legendre, J. M. Bienvenue and J. P. Landers, Anal. Chem., 2008, 80, 6472-6479) covers solid phase extraction methods with a greater focus on their incorporation into integrated microfluidic systems.

PHYLOGENETIC DIVERSITY IN DRINKING WATER BACTERIA IN A DISTRIBUTION SYSTEM SIMULATOR

EPA Science Inventory

This work was carried out to characterize the composition of microbial populations in a distribution system simulator (DSS) by direct sequence analysis of 16S rDNA clone libraries. Bacterial populations were examined in chlorinated distribution water and chloraminated DSS feed an...

Efficient generation of transgenic cattle using the DNA transposon and their analysis by next-generation sequencing

PubMed Central

Yum, Soo-Young; Lee, Song-Jeon; Kim, Hyun-Min; Choi, Woo-Jae; Park, Ji-Hyun; Lee, Won-Wu; Kim, Hee-Soo; Kim, Hyeong-Jong; Bae, Seong-Hun; Lee, Je-Hyeong; Moon, Joo-Yeong; Lee, Ji-Hyun; Lee, Choong-Il; Son, Bong-Jun; Song, Sang-Hoon; Ji, Su-Min; Kim, Seong-Jin; Jang, Goo

2016-01-01

Here, we efficiently generated transgenic cattle using two transposon systems (Sleeping Beauty and Piggybac) and their genomes were analyzed by next-generation sequencing (NGS). Blastocysts derived from microinjection of DNA transposons were selected and transferred into recipient cows. Nine transgenic cattle have been generated and grown-up to date without any health issues except two. Some of them expressed strong fluorescence and the transgene in the oocytes from a superovulating one were detected by PCR and sequencing. To investigate genomic variants by the transgene transposition, whole genomic DNA were analyzed by NGS. We found that preferred transposable integration (TA or TTAA) was identified in their genome. Even though multi-copies (i.e. fifteen) were confirmed, there was no significant difference in genome instabilities. In conclusion, we demonstrated that transgenic cattle using the DNA transposon system could be efficiently generated, and all those animals could be a valuable resource for agriculture and veterinary science. PMID:27324781

Role of Bacillus subtilis Error Prevention Oxidized Guanine System in Counteracting Hexavalent Chromium-Promoted Oxidative DNA Damage

PubMed Central

Santos-Escobar, Fernando; Gutiérrez-Corona, J. Félix

2014-01-01

Chromium pollution is potentially detrimental to bacterial soil communities, compromising carbon and nitrogen cycles that are essential for life on earth. It has been proposed that intracellular reduction of hexavalent chromium [Cr(VI)] to trivalent chromium [Cr(III)] may cause bacterial death by a mechanism that involves reactive oxygen species (ROS)-induced DNA damage; the molecular basis of the phenomenon was investigated in this work. Here, we report that Bacillus subtilis cells lacking a functional error prevention oxidized guanine (GO) system were significantly more sensitive to Cr(VI) treatment than cells of the wild-type (WT) strain, suggesting that oxidative damage to DNA is involved in the deleterious effects of the oxyanion. In agreement with this suggestion, Cr(VI) dramatically increased the ROS concentration and induced mutagenesis in a GO-deficient B. subtilis strain. Alkaline gel electrophoresis (AGE) analysis of chromosomal DNA of WT and ΔGO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ΔGO strain was more susceptible to DNA glycosylase Fpg attack, suggesting that chromium genotoxicity is associated with 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G) lesions. In support of this notion, specific monoclonal antibodies detected the accumulation of 8-oxo-G lesions in the chromosomes of B. subtilis cells subjected to Cr(VI) treatment. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves radical oxygen attack of DNA, generating 8-oxo-G, and that such effects are counteracted by the prevention and repair GO system. PMID:24973075

Highly sensitive and selective microRNA detection based on DNA-bio-bar-code and enzyme-assisted strand cycle exponential signal amplification.

PubMed

Dong, Haifeng; Meng, Xiangdan; Dai, Wenhao; Cao, Yu; Lu, Huiting; Zhou, Shufeng; Zhang, Xueji

2015-04-21

Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNP-DNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Longiaru, Mathew

2009-05-01

DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

DOE-FG02-00ER62797 Final Report

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

Sweedler, J.V.

2004-12-01

Specific Aims The overall goal of this proposal has been to develop and interface a new technology, molecular gates, with microfabricated systems to add an important capability to microfabricated DNA measurement systems. This project specifically focused on demonstrating how molecular gates could be used to capture a single analyte band, among a stream of bands from a separation or a flow injection analysis experiment, and release it for later measurement, thus allowing further manipulations on the selected analyte. Since the original proposal, the molecular gate concept has been greatly expanded to allow the gates to be used as externally controllablemore » intelligent interconnects in multilayer microfluidic networks. We have demonstrated: (1) the ability of the molecular gates to work with a much wider range of biological molecules including DNA, proteins and small metabolites; and (2) the capability of performing an electrophoretic separation and sequestering individual picoliter volume components (or even classes of components) into separate channels for further analysis. Both capabilities will enable characterization of small mass amounts of complex mixtures of DNA, proteins and even small molecules--allowing them to be further separated and chemically characterized.« less

MethylViewer: computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects.

PubMed

Pardo, Carolina E; Carr, Ian M; Hoffman, Christopher J; Darst, Russell P; Markham, Alexander F; Bonthron, David T; Kladde, Michael P

2011-01-01

Bisulfite sequencing is a widely-used technique for examining cytosine DNA methylation at nucleotide resolution along single DNA strands. Probing with cytosine DNA methyltransferases followed by bisulfite sequencing (MAPit) is an effective technique for mapping protein-DNA interactions. Here, MAPit methylation footprinting with M.CviPI, a GC methyltransferase we previously cloned and characterized, was used to probe hMLH1 chromatin in HCT116 and RKO colorectal cancer cells. Because M.CviPI-probed samples contain both CG and GC methylation, we developed a versatile, visually-intuitive program, called MethylViewer, for evaluating the bisulfite sequencing results. Uniquely, MethylViewer can simultaneously query cytosine methylation status in bisulfite-converted sequences at as many as four different user-defined motifs, e.g. CG, GC, etc., including motifs with degenerate bases. Data can also be exported for statistical analysis and as publication-quality images. Analysis of hMLH1 MAPit data with MethylViewer showed that endogenous CG methylation and accessible GC sites were both mapped on single molecules at high resolution. Disruption of positioned nucleosomes on single molecules of the PHO5 promoter was detected in budding yeast using M.CviPII, increasing the number of enzymes available for probing protein-DNA interactions. MethylViewer provides an integrated solution for primer design and rapid, accurate and detailed analysis of bisulfite sequencing or MAPit datasets from virtually any biological or biochemical system.

S - and N-alkylating agents diminish the fluorescence of fluorescent dye-stained DNA.

PubMed

Giesche, Robert; John, Harald; Kehe, Kai; Schmidt, Annette; Popp, Tanja; Balzuweit, Frank; Thiermann, Horst; Gudermann, Thomas; Steinritz, Dirk

2017-01-25

Sulfur mustard (SM), a chemical warfare agent, causes DNA alkylation, which is believed to be the main cause of its toxicity. SM DNA adducts are commonly used to verify exposure to this vesicant. However, the required analytical state-of-the-art mass-spectrometry methods are complex, use delicate instruments, are not mobile, and require laboratory infrastructure that is most likely not available in conflict zones. Attempts have thus been made to develop rapid detection methods that can be used in the field. The analysis of SM DNA adducts (HETE-G) by immunodetection is a convenient and suitable method. For a diagnostic assessment, HETE-G levels must be determined in relation to the total DNA in the sample. Total DNA can be easily visualized by the use of fluorescent DNA dyes. This study examines whether SM and related compounds affect total DNA staining, an issue that has not been investigated before. After pure DNA was extracted from human keratinocytes (HaCaT cells), DNA was exposed to different S- and N-alkylating agents. Our experiments revealed a significant, dose-dependent decrease in the fluorescence signal of fluorescent dye-stained DNA after exposure to alkylating agents. After mass spectrometry and additional fluorescence measurements ruled out covalent modifications of ethidium bromide (EthBr) by SM, we assumed that DNA crosslinks caused DNA condensation and thereby impaired access of the fluorescent dyes to the DNA. DNA digestion by restriction enzymes restored fluorescence, a fact that strengthened our hypothesis. However, monofunctional agents, which are unable to crosslink DNA, also decreased the fluorescence signal. In subsequent experiments, we demonstrated that protons produced during DNA alkylation caused a pH decrease that was found responsible for the reduction in fluorescence. The use of an appropriate buffer system eliminated the adverse effect of alkylating agents on DNA staining with fluorescent dyes. An appropriate buffer system is thus crucial for DNA quantification with fluorescent dyes in the presence of alkylating compounds. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Deinococcus geothermalis: The Pool of Extreme Radiation Resistance Genes Shrinks

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

Makarova, Kira S.; Omelchenko, Marina V.; Gaidamakova, Elena K.

Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation (IR), ultraviolet light (UV) and desiccation. The mesophile Deinococcus radiodurans was the first member of this group whose genome was completely sequenced. Analysis of the genome sequence of D. radiodurans, however, failed to identify unique DNA repair systems. To further delineate the genes underlying the resistance phenotypes, we report the whole-genome sequence of a second Deinococcus species, the thermophile Deinococcus geothermalis, which at itsoptimal growth temperature is as resistant to IR, UV and desiccation as D. radiodurans, and a comparative analysis of the two Deinococcus genomes. Many D. radioduransmore » genes previously implicated in resistance, but for which no sensitive phenotype was observed upon disruption, are absent in D. geothermalis. In contrast, most D. radiodurans genes whose mutants displayed a radiation-sensitive phenotype in D. radiodurans are conserved in D. geothermalis. Supporting the existence of a Deinococcus radiation response regulon, a common palindromic DNA motif was identified in a conserved set of genes associated with resistance, and a dedicated transcriptional regulator was predicted. We present the case that these two species evolved essentially the same diverse set of gene families, and that the extreme stress-resistance phenotypes of the Deinococcus lineage emerged progressively by amassing cell-cleaning systems from different sources, but not by acquisition of novel DNA repair systems. Our reconstruction of the genomic evolution of the Deinococcus-Thermus phylum indicates that the corresponding set of enzymes proliferated mainly in the common ancestor of Deinococcus. Results of the comparative analysis weaken the arguments for a role of higher-order chromosome alignment structures in resistance; more clearly define and substantially revise downward the number of uncharacterized genes that might participate in DNA repair and contribute to resistance; and strengthen the case for a role in survival of systems involved in manganese and iron homeostasis.« less

A nonenzymatic DNA nanomachine for biomolecular detection by target recycling of hairpin DNA cascade amplification.

PubMed

Zheng, Jiao; Li, Ningxing; Li, Chunrong; Wang, Xinxin; Liu, Yucheng; Mao, Guobin; Ji, Xinghu; He, Zhike

2018-06-01

Synthetic enzyme-free DNA nanomachine performs quasi-mechanical movements in response to external intervention, suggesting the promise of constructing sensitive and specific biosensors. Herein, a smart DNA nanomachine biosensor for biomolecule (such as nucleic acid, thrombin and adenosine) detection is developed by target-assisted enzyme-free hairpin DNA cascade amplifier. The whole DNA nanomachine system is constructed on gold nanoparticle which decorated with hundreds of locked hairpin substrate strands serving as DNA tracks, and the DNA nanomachine could be activated by target molecule toehold-mediated exchange on gold nanoparticle surface, resulted in the fluorescence recovery of fluorophore. The process is repeated so that each copy of the target can open multiplex fluorophore-labeled hairpin substrate strands, resulted in amplification of the fluorescence signal. Compared with the conventional biosensors of catalytic hairpin assembly (CHA) without substrate in solution, the DNA nanomachine could generate 2-3 orders of magnitude higher fluorescence signal. Furthermore, the DNA nanomachine could be used for nucleic acid, thrombin and adenosine highly sensitive specific detection based on isothermal, and homogeneous hairpin DNA cascade signal amplification in both buffer and a complicated biomatrix, and this kind of DNA nanomachine could be efficiently applied in the field of biomedical analysis. Copyright © 2018 Elsevier B.V. All rights reserved.

DNA polymeric films as a support for cell growth as a new material for regenerative medicine: Compatibility and applicability.

PubMed

Jayme, Cristiano Ceron; de Paula, Leonardo Barcelos; Rezende, Nayara; Calori, Italo Rodrigo; Franchi, Leonardo Pereira; Tedesco, Antonio Claudio

2017-11-15

DNA polymeric films (DNA-PFs) are a promising drug delivery system (DDS) in modern medicine. In this study, we evaluated the growth behavior of oral squamous cell carcinoma (OSCC) cells on DNA-PFs. The morphological, biochemical, and cytometric features of OSCC cell adhesion on DNA-PFs were also assessed. An initial, temporary alteration in cell morphology was observed at early time points owing to the inhibition of cell attachment to the film, which then returned to a normal morphological state at later time points. MTT and resazurin assays showed a moderate reduction in cell viability related to increased DNA concentration in the DNA-PFs. Flow cytometry studies showed low cytotoxicity of DNA-PFs, with cell viabilities higher than 90% in all the DNA-PFs tested. Flow cytometric cell cycle analysis also showed average cell cycle phase distributions at later time points, indicating that OSCC cell growth is maintained in the presence of DNA-PFs. These results show high biocompatibility of DNA-PFs and suggest their use in designing "dressing material," where the DNA film acts as a support for cell growth, or with incorporation of active or photoactive compounds, which can induce tissue regeneration and are useful to treat many diseases, especially oral cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA-mediated inhibition of peroxidase-like activities on platinum nanoparticles for simple and rapid colorimetric detection of nucleic acids.

PubMed

Chen, Weiwei; Fang, Xueen; Li, Hua; Cao, Hongmei; Kong, Jilie

2017-08-15

In this research, we found that the peroxidase-like activities of noncovalent DNA-Pt hybrid nanoparticles could be obviously blocked, when Pt nanoparticles (PtNPs) were synthesized in situ using DNA as a template. Moreover, this self-assembled synthetic process was very convenient and rapid (within few mintues), and the inhibition mediated by DNA was also very effective. First, by the paper-based analytical device (PAD) we found the catalytic activities of DNA-Pt hybrid nanoparticles exhibited a linear response to the concentration of DNA in the range from 0.0075 to 0.25µM. Then, with the magnetic bead isolated system and target DNA-induced hybridization chain reaction (HCR), we realized the specific target DNA analysis with a low detection of 0.228nM, and demonstrated its effectivity in distinguishing the target DNA from other interferences. To our knowledge, this is the first report that used the nanoassembly between DNA and PtNPs for colorimetric detection of nucleic acids, which was based on DNA-mediated inhibition of catalytic activities of platinum nanoparticles. The results may be useful for understanding the interactions between DNA and metal nanoparticles, and for development of other convenient and effective analytical strategies. Copyright © 2017. Published by Elsevier B.V.

Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes.

PubMed

Chen, Chao; Wang, Lianrong; Chen, Si; Wu, Xiaolin; Gu, Meijia; Chen, Xi; Jiang, Susu; Wang, Yunfu; Deng, Zixin; Dedon, Peter C; Chen, Shi

2017-04-25

Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction-modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems, 6m A methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA from Escherichia coli B7A and Salmonella enterica serovar Cerro 87, strains possessing PT-based R-M genes, revealed d(G PS 6m A) dinucleotides in the G PS 6m AAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(G PS A) motifs per genome, with 6m A arising from a yet-to-be-identified methyltransferase. To further explore PT and 6m A in another consensus sequence, G PS 6m ATC, we engineered a strain of E. coli HST04 to express Dnd genes from Hahella chejuensis KCTC2396 (PT in G PS ATC) and Dam methyltransferase from E. coli DH10B ( 6m A in G 6m ATC). Based on this model, in vitro studies revealed reduced Dam activity in G PS ATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed 6m A in all 2,058 G PS ATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that 6m A can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.

DNA Damage Repair System in Plants: A Worldwide Research Update.

PubMed

Gimenez, Estela; Manzano-Agugliaro, Francisco

2017-10-30

Living organisms are usually exposed to various DNA damaging agents so the mechanisms to detect and repair diverse DNA lesions have developed in all organisms with the result of maintaining genome integrity. Defects in DNA repair machinery contribute to cancer, certain diseases, and aging. Therefore, conserving the genomic sequence in organisms is key for the perpetuation of life. The machinery of DNA damage repair (DDR) in prokaryotes and eukaryotes is similar. Plants also share mechanisms for DNA repair with animals, although they differ in other important details. Plants have, surprisingly, been less investigated than other living organisms in this context, despite the fact that numerous lethal mutations in animals are viable in plants. In this manuscript, a worldwide bibliometric analysis of DDR systems and DDR research in plants was made. A comparison between both subjects was accomplished. The bibliometric analyses prove that the first study about DDR systems in plants (1987) was published thirteen years later than that for other living organisms (1975). Despite the increase in the number of papers about DDR mechanisms in plants in recent decades, nowadays the number of articles published each year about DDR systems in plants only represents 10% of the total number of articles about DDR. The DDR research field was done by 74 countries while the number of countries involved in the DDR & Plant field is 44. This indicates the great influence that DDR research in the plant field currently has, worldwide. As expected, the percentage of studies published about DDR systems in plants has increased in the subject area of agricultural and biological sciences and has diminished in medicine with respect to DDR studies in other living organisms. In short, bibliometric results highlight the current interest in DDR research in plants among DDR studies and can open new perspectives in the research field of DNA damage repair.

Internal validation of STRmix™ for the interpretation of single source and mixed DNA profiles.

PubMed

Moretti, Tamyra R; Just, Rebecca S; Kehl, Susannah C; Willis, Leah E; Buckleton, John S; Bright, Jo-Anne; Taylor, Duncan A; Onorato, Anthony J

2017-07-01

The interpretation of DNA evidence can entail analysis of challenging STR typing results. Genotypes inferred from low quality or quantity specimens, or mixed DNA samples originating from multiple contributors, can result in weak or inconclusive match probabilities when a binary interpretation method and necessary thresholds (such as a stochastic threshold) are employed. Probabilistic genotyping approaches, such as fully continuous methods that incorporate empirically determined biological parameter models, enable usage of more of the profile information and reduce subjectivity in interpretation. As a result, software-based probabilistic analyses tend to produce more consistent and more informative results regarding potential contributors to DNA evidence. Studies to assess and internally validate the probabilistic genotyping software STRmix™ for casework usage at the Federal Bureau of Investigation Laboratory were conducted using lab-specific parameters and more than 300 single-source and mixed contributor profiles. Simulated forensic specimens, including constructed mixtures that included DNA from two to five donors across a broad range of template amounts and contributor proportions, were used to examine the sensitivity and specificity of the system via more than 60,000 tests comparing hundreds of known contributors and non-contributors to the specimens. Conditioned analyses, concurrent interpretation of amplification replicates, and application of an incorrect contributor number were also performed to further investigate software performance and probe the limitations of the system. In addition, the results from manual and probabilistic interpretation of both prepared and evidentiary mixtures were compared. The findings support that STRmix™ is sufficiently robust for implementation in forensic laboratories, offering numerous advantages over historical methods of DNA profile analysis and greater statistical power for the estimation of evidentiary weight, and can be used reliably in human identification testing. With few exceptions, likelihood ratio results reflected intuitively correct estimates of the weight of the genotype possibilities and known contributor genotypes. This comprehensive evaluation provides a model in accordance with SWGDAM recommendations for internal validation of a probabilistic genotyping system for DNA evidence interpretation. Copyright © 2017. Published by Elsevier B.V.

Drastic stabilization of parallel DNA hybridizations by a polylysine comb-type copolymer with hydrophilic graft chain.

PubMed

Miyoshi, Daisuke; Ueda, Yu-Mi; Shimada, Naohiko; Nakano, Shu-Ichi; Sugimoto, Naoki; Maruyama, Atsushi

2014-09-01

Electrostatic interactions play a major role in protein-DNA interactions. As a model system of a cationic protein, herein we focused on a comb-type copolymer of a polycation backbone and dextran side chains, poly(L-lysine)-graft-dextran (PLL-g-Dex), which has been reported to form soluble interpolyelectrolyte complexes with DNA strands. We investigated the effects of PLL-g-Dex on the conformation and thermodynamics of DNA oligonucleotides forming various secondary structures. Thermodynamic analysis of the DNA structures showed that the parallel conformations involved in both DNA duplexes and triplexes were significantly and specifically stabilized by PLL-g-Dex. On the basis of thermodynamic parameters, it was further possible to design DNA switches that undergo structural transition responding to PLL-g-Dex from an antiparallel duplex to a parallel triplex even with mismatches in the third strand hybridization. These results suggest that polycationic molecules are able to induce structural polymorphism of DNA oligonucleotides, because of the conformation-selective stabilization effects. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Design and analysis of DNA strand displacement devices using probabilistic model checking

PubMed Central

Lakin, Matthew R.; Parker, David; Cardelli, Luca; Kwiatkowska, Marta; Phillips, Andrew

2012-01-01

Designing correct, robust DNA devices is difficult because of the many possibilities for unwanted interference between molecules in the system. DNA strand displacement has been proposed as a design paradigm for DNA devices, and the DNA strand displacement (DSD) programming language has been developed as a means of formally programming and analysing these devices to check for unwanted interference. We demonstrate, for the first time, the use of probabilistic verification techniques to analyse the correctness, reliability and performance of DNA devices during the design phase. We use the probabilistic model checker prism, in combination with the DSD language, to design and debug DNA strand displacement components and to investigate their kinetics. We show how our techniques can be used to identify design flaws and to evaluate the merits of contrasting design decisions, even on devices comprising relatively few inputs. We then demonstrate the use of these components to construct a DNA strand displacement device for approximate majority voting. Finally, we discuss some of the challenges and possible directions for applying these methods to more complex designs. PMID:22219398

Anti-DNA Ig peptides promote Treg cell activity in systemic lupus erythematosus patients.

PubMed

Hahn, Bevra H; Anderson, Marissa; Le, Elizabeth; La Cava, Antonio

2008-08-01

Treg cells oppose autoreactive responses in several autoimmune diseases, and their frequency is reduced in systemic lupus erythematosus (SLE). In murine lupus models, treatment with anti-DNA Ig-based peptides can expand the number of Treg cells in vivo. This study was undertaken to test the possibility that functional human Treg cells can be induced by exposure to anti-DNA Ig-based peptides. Peripheral blood mononuclear cells were isolated from 36 lupus patients and 32 healthy individuals matched for ethnicity, sex, and age. Short-term culture experiments in the presence of several independent stimuli including anti-DNA Ig peptides were followed by flow cytometric analysis for identification of CD4+,CD25(high) T cells, cell sorting for in vitro suppression assays, and analysis of correlations between the expression of forkhead box P3 (FoxP3) and serologic and clinical characteristics of the SLE patients. The number of in vitro CD4+,CD25(high) T cells increased after culture with anti-DNA Ig peptides in the SLE patients, but not in the controls. The expanded CD4+,CD25(high) T cells required FoxP3 for cell contact-mediated suppression of proliferation and interferon-gamma production in target CD4+,CD25- T cells. The induction of FoxP3 in SLE Treg cells occurred only in seropositive patients, and was correlated with anti-DNA and IgG serum titers. These results suggest a new modality to reverse the functional deficit of Treg cells in SLE patients with positive autoimmune serology, and identify a new strategy to enhance immunoregulatory T cell activity in human SLE.

Ultrasensitive determination of DNA sequences by flow injection chemiluminescence using silver ions as labels.

PubMed

Zheng, Lichun; Liu, Xiuhui; Zhou, Min; Ma, Yongjun; Wu, Guofan; Lu, Xiaoquan

2014-10-27

We presented a new strategy for ultrasensitive detection of DNA sequences based on the novel detection probe which was labeled with Ag(+) using metallothionein (MT) as a bridge. The assay relied on a sandwich-type DNA hybridization in which the DNA targets were first hybridized to the captured oligonucleotide probes immobilized on Fe3O4@Au composite magnetic nanoparticles (MNPs), and then the Ag(+)-modified detection probes were used to monitor the presence of the specific DNA targets. After being anchored on the hybrids, Ag(+) was released down through acidic treatment and sensitively determined by a coupling flow injection-chemiluminescent reaction system (Ag(+)-Mn(2+)-K2S2O8-H3PO4-luminol) (FI-CL). The experiment results showed that the CL intensities increased linearly with the concentrations of DNA targets in the range from 10 to 500 pmol L(-1) with a detection limit of 3.3 pmol L(-1). The high sensitivity in this work may be ascribed to the high molar ratio of Ag(+)-MT, the sensitive determination of Ag(+) by the coupling FI-CL reaction system and the perfect magnetic separation based on Fe3O4@Au composite MNPs. Moreover, the proposed strategy exhibited excellent selectivity against the mismatched DNA sequences and could be applied to real samples analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.

PubMed

Demeke, Tigst; Ratnayaka, Indira; Phan, Anh

2009-01-01

The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

Interactions of the SAP Domain of Human Ku70 with DNA Substrate: A Molecular Dynamics Study

NASA Technical Reports Server (NTRS)

Hu, Shaowen; Carra, Claudio; Huff, Janice; Pluth, Janice M.; Cucinotta, Francis A.

2007-01-01

NASA is developing a systems biology approach to improve the assessment of health risks associated with space radiation. The primary toxic and mutagenic lesion following radiation exposure is the DNA double strand break (DSB), thus a model incorporating proteins and pathways important in response and repair of this lesion is critical. One key protein heterodimer for systems models of radiation effects is the Ku70/80 complex. The Ku70/80 complex is important in the initial binding of DSB ends following DNA damage, and is a component of nonhomologous end joining repair, the primary pathway for DSB repair in mammalian cells. The SAP domain of Ku70 (residues 556-609), contains an a helix-extended strand-helix motif and similar motifs have been found in other nucleic acid-binding proteins critical for DNA repair. However, the exact mechanism of damage recognition and substrate specificity for the Ku heterodimer remains unclear in part due to the absence of a high-resolution structure of the SAP/DNA complex. We performed a series of molecular dynamics (MD) simulations on a system with the SAP domain of Ku70 and a 10 base pairs DNA duplex. Large-scale conformational changes were observed and some putative binding modes were suggested based on energetic analysis. These modes are consistent with previous experimental investigations. In addition, the results indicate that cooperation of SAP with other domains of Ku70/80 is necessary to explain the high affinity of binding as observed in experiments.

One simple DNA extraction device and its combination with modified visual loop-mediated isothermal amplification for rapid on-field detection of genetically modified organisms.

PubMed

Zhang, Miao; Liu, Yinan; Chen, Lili; Quan, Sheng; Jiang, Shimeng; Zhang, Dabing; Yang, Litao

2013-01-02

Quickness, simplicity, and effectiveness are the three major criteria for establishing a good molecular diagnosis method in many fields. Herein we report a novel detection system for genetically modified organisms (GMOs), which can be utilized to perform both on-field quick screening and routine laboratory diagnosis. In this system, a newly designed inexpensive DNA extraction device was used in combination with a modified visual loop-mediated isothermal amplification (vLAMP) assay. The main parts of the DNA extraction device included a silica gel membrane filtration column and a modified syringe. The DNA extraction device could be easily operated without using other laboratory instruments, making it applicable to an on-field GMO test. High-quality genomic DNA (gDNA) suitable for polymerase chain reaction (PCR) and isothermal amplification could be quickly isolated from plant tissues using this device within 15 min. In the modified vLAMP assay, a microcrystalline wax encapsulated detection bead containing SYBR green fluorescent dye was introduced to avoid dye inhibition and cross-contaminations from post-LAMP operation. The system was successfully applied and validated in screening and identification of GM rice, soybean, and maize samples collected from both field testing and the Grain Inspection, Packers, and Stockyards Administration (GIPSA) proficiency test program, which demonstrated that it was well-adapted to both on-field testing and/or routine laboratory analysis of GMOs.

Homogeneous electrochemical immunoassay of aflatoxin B1 in foodstuff using proximity-hybridization-induced omega-like DNA junctions and exonuclease III-triggered isothermal cycling signal amplification.

PubMed

Tang, Juan; Huang, Yapei; Liu, Huiqiong; Zhang, Cengceng; Tang, Dianping

2016-12-01

A new homogeneous electrochemical immunosensing platform was designed for sensitive detection of aflatoxin B 1 (AFB 1 ) in foodstuff. The system consisted of anti-AFB 1 antibody labeled DNA 1 (Ab-DNA 1 ), AFB 1 -bovine serum albumin (BSA)-conjugated DNA 2 (AFB 1 -DNA 2 ), and methylene blue functionalized hairpin DNA. Owing to a specific antigen-antibody reaction between anti-AFB 1 and AFB 1 -BSA, the immunocomplex formed assisted the proximity hybridization of DNA 1 with DNA 2 , thus resulting in the formation of an omega-like DNA junction. Thereafter, the junction opened the hairpin DNA to construct a new double-stranded DNA, which could be readily cleaved by exonuclease III to release the omega-like DNA junction and methylene blue. The dissociated DNA junction could repeatedly hybridize with residual hairpin DNA molecules with exonuclease III-based isothermal cycling amplification, thereby releasing numerous free methylene blue molecules into the detection solution. The as-produced free methylene blue molecules could be captured by a negatively charged indium tin oxide electrode, each of which could produce an electronic signal within the applied potentials. On introduction of target AFB 1 , the analyte competed with AFB 1 -DNA 2 for the conjugated anti-AFB 1 on the Ab-DNA 1 , subsequently decreasing the amount of omega-like DNA junctions formed, hence causing methylene blue labeled hairpin DNA to move far away from the electrode surface. Under optimal conditions the detectable electrochemical signal decreased with increasing amount of target AFB 1 in a dynamic working range of 0.01-30 ng mL -1 with a detection limit of 4.8 pg mL -1 . In addition, the precision and reproducibility of this system were acceptable. Finally, the method was further evaluated for analysis of naturally contaminated or AFB 1 -spiked peanut samples, giving results that matched well with those obtained with a commercial AFB 1 ELISA kit.

Analysis of autoimmune bone marrow by antibody-phage display: somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes.

PubMed

Seal, S N; Hoet, R M; Raats, J M; Radic, M Z

2000-09-01

To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.

Mammalian Homologs of Yeast Checkpoint Genes

DTIC Science & Technology

2001-07-01

previous cycle we developed systems and reagents for expression and analysis of all of the pertinent proteins, and are made headway on association of Chk2...function, with emphasis on p53 regulation, cell cycle regulation, and complementation of ATM defects. Saccharomyces Schizosaceharomy Homo sapiens...RAD53, two essential genes, play a central role in DNA damage checkpoints at all cell cycle stages. Our lab showed that Rad9 is a regulator coupling DNA

DNA Damage and Oxidative Stress in Dyskeratosis Congenita: Analysis of Pathways and Therapeutic Stategies Using CPISPR and iPSC Model Systems

DTIC Science & Technology

2016-06-01

telomeres and characterized by a classical clinical triad of leukoplakia, skin dyspigmentation and nail dystrophy with concomitant marrow failure...DC symptomology, to a degree, corresponds to critically shortened telomeres that limits cellular replicative potential and thus prematurely exhausts...stem cell pools. Our previous findings support a hypothesis whereby shortened telomeres increase DNA damage responses within the cell leading to

Global DNA hypomethylation in peripheral blood leukocytes as a biomarker for cancer risk: a meta-analysis.

PubMed

Woo, Hae Dong; Kim, Jeongseon

2012-01-01

Good biomarkers for early detection of cancer lead to better prognosis. However, harvesting tumor tissue is invasive and cannot be routinely performed. Global DNA methylation of peripheral blood leukocyte DNA was evaluated as a biomarker for cancer risk. We performed a meta-analysis to estimate overall cancer risk according to global DNA hypomethylation levels among studies with various cancer types and analytical methods used to measure DNA methylation. Studies were systemically searched via PubMed with no language limitation up to July 2011. Summary estimates were calculated using a fixed effects model. The subgroup analyses by experimental methods to determine DNA methylation level were performed due to heterogeneity within the selected studies (p<0.001, I(2): 80%). Heterogeneity was not found in the subgroup of %5-mC (p = 0.393, I(2): 0%) and LINE-1 used same target sequence (p = 0.097, I(2): 49%), whereas considerable variance remained in LINE-1 (p<0.001, I(2): 80%) and bladder cancer studies (p = 0.016, I(2): 76%). These results suggest that experimental methods used to quantify global DNA methylation levels are important factors in the association study between hypomethylation levels and cancer risk. Overall, cancer risks of the group with the lowest DNA methylation levels were significantly higher compared to the group with the highest methylation levels [OR (95% CI): 1.48 (1.28-1.70)]. Global DNA hypomethylation in peripheral blood leukocytes may be a suitable biomarker for cancer risk. However, the association between global DNA methylation and cancer risk may be different based on experimental methods, and region of DNA targeted for measuring global hypomethylation levels as well as the cancer type. Therefore, it is important to select a precise and accurate surrogate marker for global DNA methylation levels in the association studies between global DNA methylation levels in peripheral leukocyte and cancer risk.

Benefits and Limitations of DNA Barcoding and Metabarcoding in Herbal Product Authentication.

PubMed

Raclariu, Ancuta Cristina; Heinrich, Michael; Ichim, Mihael Cristin; de Boer, Hugo

2018-03-01

Herbal medicines play an important role globally in the health care sector and in industrialised countries they are often considered as an alternative to mono-substance medicines. Current quality and authentication assessment methods rely mainly on morphology and analytical phytochemistry-based methods detailed in pharmacopoeias. Herbal products however are often highly processed with numerous ingredients, and even if these analytical methods are accurate for quality control of specific lead or marker compounds, they are of limited suitability for the authentication of biological ingredients. To review the benefits and limitations of DNA barcoding and metabarcoding in complementing current herbal product authentication. Recent literature relating to DNA based authentication of medicinal plants, herbal medicines and products are summarised to provide a basic understanding of how DNA barcoding and metabarcoding can be applied to this field. Different methods of quality control and authentication have varying resolution and usefulness along the value chain of these products. DNA barcoding can be used for authenticating products based on single herbal ingredients and DNA metabarcoding for assessment of species diversity in processed products, and both methods should be used in combination with appropriate hyphenated chemical methods for quality control. DNA barcoding and metabarcoding have potential in the context of quality control of both well and poorly regulated supply systems. Standardisation of protocols for DNA barcoding and DNA sequence-based identification are necessary before DNA-based biological methods can be implemented as routine analytical approaches and approved by the competent authorities for use in regulated procedures. © 2017 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd. © 2017 The Authors. Phytochemical Analysis Published by John Wiley & Sons Ltd.

Influence of PNA containing 8-aza-7-deazaadenine on structure stability and binding affinity of PNA·DNA duplex: insights from thermodynamics, counter ion, hydration and molecular dynamics analysis.

PubMed

Gupta, Sharad K; Sur, Souvik; Prasad Ojha, Rajendra; Tandon, Vibha

2013-07-01

This paper describes the synthesis of a novel 8-aza-7-deazapurin-2,6-diamine (DPP)-containing peptide nucleic acid (PNA) monomer and Boc protecting group-based oligomerization of PNA, replacing adenine (A) with DPP monomers in the PNA strand. The PNA oligomers were synthesized against the biologically relevant SV40 promoter region (2494-AATTTTTTTTATTTA-2508) of pEGFP-N3 plasmid. The DPP-PNA·DNA duplex showed enhanced stability as compared to normal duplex (A-PNA·DNA). The electronic distribution of DPP monomer suggested that DPP had better electron donor properties over 2,6-diamino purine. UV melting and thermodynamic analysis revealed that the PNA oligomer containing a diaminopyrazolo(3,4-d)pyrimidine moiety (DPP) stabilized the PNA·DNA hybrids compared to A-PNA·DNA. DPP-PNA·DNA duplex showed higher water activity (Δnw = 38.5) in comparison to A-PNA·DNA duplex (Δnw = 14.5). The 50 ns molecular dynamics simulations of PNA·DNA duplex containing DPP or unmodified nucleobase-A showed average H-bond distances in the DPP-dT base pair of 2.90 Å (OH-N bond) and 2.91 Å (NH-N bond), which were comparably shorter than in the A-dT base pair, in which the average distances were 3.18 Å (OH-N bond) and 2.97 Å (NH-N bond), and there was one additional H-bond in the DPP-dT base pair of around 2.98 Å (O2H-N2 bond), supporting the higher stability of DPP-PNA·DNA. The analysis of molecular dynamics simulation data showed that the system binding free energy increased at a rate of approximately -4.5 kcal mol(-1) per DPP base of the PNA·DNA duplex. In summary, increased thermal stability, stronger hydrogen bonding and more stable conformation in the DPP-PNA·DNA duplex make it a better candidate as antisense/antigene therapeutic agents.

Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction.

PubMed

Janku, F; Huang, H J; Fujii, T; Shelton, D N; Madwani, K; Fu, S; Tsimberidou, A M; Piha-Paul, S A; Wheler, J J; Zinner, R G; Naing, A; Hong, D S; Karp, D D; Cabrilo, G; Kopetz, E S; Subbiah, V; Luthra, R; Kee, B K; Eng, C; Morris, V K; Karlin-Neumann, G A; Meric-Bernstam, F

2017-03-01

Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy. Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care. Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001). Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Hierarchy of Certain Types of DNA Splicing Systems

NASA Astrophysics Data System (ADS)

Yusof, Yuhani; Sarmin, Nor Haniza; Goode, T. Elizabeth; Mahmud, Mazri; Heng, Fong Wan

A Head splicing system (H-system)consists of a finite set of strings (words) written over a finite alphabet, along with a finite set of rules that acts on the strings by iterated cutting and pasting to create a splicing language. Any interpretation that is aligned with Tom Head's original idea is one in which the strings represent double-stranded deoxyribonucleic acid (dsDNA) and the rules represent the cutting and pasting action of restriction enzymes and ligase, respectively. A new way of writing the rule sets is adopted so as to make the biological interpretation transparent. This approach is used in a formal language- theoretic analysis of the hierarchy of certain classes of splicing systems, namely simple, semi-simple and semi-null splicing systems. The relations between such systems and their associated languages are given as theorems, corollaries and counterexamples.

Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles.

PubMed

Tu, Thomas; Budzinska, Magdalena A; Vondran, Florian W R; Shackel, Nicholas A; Urban, Stephan

2018-02-07

Chronic infection by the Hepatitis B Virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (cccDNA), integration of HBV DNA into the host cell genome is regularly observed in the liver of infected patients. While reported as a pro-oncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well-understood, chiefly due to the lack of in vitro infection models that have detectable integration events. Here, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10000 cells, with the most consistent detection in Huh7-NTCP cells. Integration rate remained stable between 3 and 9 days post-infection. HBV DNA integration was efficiently blocked by treatment with 200nM of the HBV entry inhibitor Myrcludex B, but not with 10μM Tenofovir, 100U Interferon alpha, or 1μM of the capsid assembly inhibitor GLS4. This suggests integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration. Importance Hepatitis B Virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the hepadnaviridae family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer formation and persistence of virus infection. However, when and the mechanism(s) by which HBV DNA integration occurs is not clear. Here, we have developed and characterized an in vitro system to reliably detect HBV DNA integrations that result from a true HBV infection event and that closely resemble those found in patient tissues. Using this model, we show that integration already occurs when the infection is first established. Importantly, we provide here a system to analyze molecular factors involved in HBV integration, which can be used to develop strategies to halt its formation. Copyright © 2018 American Society for Microbiology.

Highly-sensitive microRNA detection based on bio-bar-code assay and catalytic hairpin assembly two-stage amplification.

PubMed

Tang, Songsong; Gu, Yuan; Lu, Huiting; Dong, Haifeng; Zhang, Kai; Dai, Wenhao; Meng, Xiangdan; Yang, Fan; Zhang, Xueji

2018-04-03

Herein, a highly-sensitive microRNA (miRNA) detection strategy was developed by combining bio-bar-code assay (BBA) with catalytic hairpin assembly (CHA). In the proposed system, two nanoprobes of magnetic nanoparticles functionalized with DNA probes (MNPs-DNA) and gold nanoparticles with numerous barcode DNA (AuNPs-DNA) were designed. In the presence of target miRNA, the MNP-DNA and AuNP-DNA hybridized with target miRNA to form a "sandwich" structure. After "sandwich" structures were separated from the solution by the magnetic field and dehybridized by high temperature, the barcode DNA sequences were released by dissolving AuNPs. The released barcode DNA sequences triggered the toehold strand displacement assembly of two hairpin probes, leading to recycle of barcode DNA sequences and producing numerous fluorescent CHA products for miRNA detection. Under the optimal experimental conditions, the proposed two-stage amplification system could sensitively detect target miRNA ranging from 10 pM to 10 aM with a limit of detection (LOD) down to 97.9 zM. It displayed good capability to discriminate single base and three bases mismatch due to the unique sandwich structure. Notably, it presented good feasibility for selective multiplexed detection of various combinations of synthetic miRNA sequences and miRNAs extracted from different cell lysates, which were in agreement with the traditional polymerase chain reaction analysis. The two-stage amplification strategy may be significant implication in the biological detection and clinical diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system.

PubMed

Mu, Di; Yan, Liang; Tang, Hui; Liao, Yong

2015-10-01

To develop a sensitive and accurate assay system for the quantification of covalently closed circular HBV DNA (cccDNA) for future clinical monitoring of cccDNA fluctuation during antiviral therapy in the liver of infected patients. A droplet digital PCR (ddPCR)-based assay system detected template DNA input at the single copy level (or ~10(-5) pg of plasmid HBV DNA) by using serially diluted plasmid HBV DNA samples. Compared with the conventional quantitative PCR assay in the detection of cccDNA, which required at least 50 ng of template DNA input, a parallel experiment applying a ddPCR system demonstrates that the lowest detection limit of cccDNA from HepG2.215 cellular DNA samples is around 1 ng, which is equivalent to 0.54 ± 0.94 copies of cccDNA. In addition, we demonstrated that the addition of cccDNA-safe exonuclease and utilization of cccDNA-specific primers in the ddPCR assay system significantly improved the detection accuracy of HBV cccDNA from HepG2.215 cellular DNA samples. The ddPCR-based cccDNA detection system is a sensitive and accurate assay for the quantification of cccDNA in HBV-transfected HepG2.215 cellular DNA samples and may represent an important method for future application in monitoring cccDNA fluctuation during antiviral therapy.

Inferring Phylogenetic Relationships of Indian Citron (Citrus medica L.) based on rbcL and matK Sequences of Chloroplast DNA.

PubMed

Uchoi, Ajit; Malik, Surendra Kumar; Choudhary, Ravish; Kumar, Susheel; Rohini, M R; Pal, Digvender; Ercisli, Sezai; Chaudhury, Rekha

2016-06-01

Phylogenetic relationships of Indian Citron (Citrus medica L.) with other important Citrus species have been inferred through sequence analyses of rbcL and matK gene region of chloroplast DNA. The study was based on 23 accessions of Citrus genotypes representing 15 taxa of Indian Citrus, collected from wild, semi-wild, and domesticated stocks. The phylogeny was inferred using the maximum parsimony (MP) and neighbor-joining (NJ) methods. Both MP and NJ trees separated all the 23 accessions of Citrus into five distinct clusters. The chloroplast DNA (cpDNA) analysis based on rbcL and matK sequence data carried out in Indian taxa of Citrus was useful in differentiating all the true species and species/varieties of probable hybrid origin in distinct clusters or groups. Sequence analysis based on rbcL and matK gene provided unambiguous identification and disposition of true species like C. maxima, C. medica, C. reticulata, and related hybrids/cultivars. The separation of C. maxima, C. medica, and C. reticulata in distinct clusters or sub-clusters supports their distinctiveness as the basic species of edible Citrus. However, the cpDNA sequence analysis of rbcL and matK gene could not find any clear cut differentiation between subgenera Citrus and Papeda as proposed in Swingle's system of classification.

TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

NASA Astrophysics Data System (ADS)

Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

2016-02-01

Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle.

TPhP exposure disturbs carbohydrate metabolism, lipid metabolism, and the DNA damage repair system in zebrafish liver

PubMed Central

Du, Zhongkun; Zhang, Yan; Wang, Guowei; Peng, Jianbiao; Wang, Zunyao; Gao, Shixiang

2016-01-01

Triphenyl phosphate is a high production volume organophosphate flame retardant that has been detected in multiple environmental media at increasing concentrations. The environmental and health risks of triphenyl phosphate have drawn attention because of the multiplex toxicity of this chemical compound. However, few studies have paid close attention to the impacts of triphenyl phosphate on liver metabolism. We investigated hepatic histopathological, metabolomic and transcriptomic responses of zebrafish after exposure to 0.050 mg/L and 0.300 mg/L triphenyl phosphate for 7 days. Metabolomic analysis revealed significant changes in the contents of glucose, UDP-glucose, lactate, succinate, fumarate, choline, acetylcarnitine, and several fatty acids. Transcriptomic analysis revealed that related pathways, such as the glycosphingolipid biosynthesis, PPAR signaling pathway and fatty acid elongation, were significantly affected. These results suggest that triphenyl phosphate exposure markedly disturbs hepatic carbohydrate and lipid metabolism in zebrafish. Moreover, DNA replication, the cell cycle, and non-homologous end-joining and base excision repair were strongly affected, thus indicating that triphenyl phosphate hinders the DNA damage repair system in zebrafish liver cells. The present study provides a systematic analysis of the triphenyl phosphate-induced toxic effects in zebrafish liver and demonstrates that low concentrations of triphenyl phosphate affect normal metabolism and cell cycle. PMID:26898711

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification.

PubMed

Zeng, Yan; Wan, Yi; Zhang, Dun; Qi, Peng

2015-01-01

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3'-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14 pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria. Copyright © 2014 Elsevier B.V. All rights reserved.

Analysis of DNA Sequences by An Optical Time-Integrating Correlator: Proof-Of-Concept Experiments.

DTIC Science & Technology

1992-05-01

TABLES xv LIST OF ABBREVIATIONS xvii 1.0 INTRODUCTION 1 2.0 DNA ANALYSIS STRATEGY 4 2.1 Representation of DNA Bases 4 2.2 DNA Analysis Strategy 6 3.0...Zehnder architecture. 3 Figure 3: Short representations of the DNA bases where each base is represented by a 7-bits long pseudorandom sequence. 5... DNA bases where each base is represented by 7-bits long pseudorandom sequences. 4 Table 2: Long representations of the DNA bases with 255-bits maximum

Decoding DNA labels by melting curve analysis using real-time PCR.

PubMed

Balog, József A; Fehér, Liliána Z; Puskás, László G

2017-12-01

Synthetic DNA has been used as an authentication code for a diverse number of applications. However, existing decoding approaches are based on either DNA sequencing or the determination of DNA length variations. Here, we present a simple alternative protocol for labeling different objects using a small number of short DNA sequences that differ in their melting points. Code amplification and decoding can be done in two steps using quantitative PCR (qPCR). To obtain a DNA barcode with high complexity, we defined 8 template groups, each having 4 different DNA templates, yielding 158 (>2.5 billion) combinations of different individual melting temperature (Tm) values and corresponding ID codes. The reproducibility and specificity of the decoding was confirmed by using the most complex template mixture, which had 32 different products in 8 groups with different Tm values. The industrial applicability of our protocol was also demonstrated by labeling a drone with an oil-based paint containing a predefined DNA code, which was then successfully decoded. The method presented here consists of a simple code system based on a small number of synthetic DNA sequences and a cost-effective, rapid decoding protocol using a few qPCR reactions, enabling a wide range of authentication applications.

Development and analysis of a tick-borne encephalitis virus infectious clone using a novel and rapid strategy.

PubMed

Gritsun, T S; Gould, E A

1998-12-01

In less than 1 month we have constructed an infectious clone of attenuated tick-borne encephalitis virus (strain Vasilchenko) from 100 microl of unpurified virus suspension using long high fidelity PCR and a modified bacterial cloning system. Optimization of the 3' antisense primer concentration was essential to achieve PCR synthesis of an 11 kb cDNA copy of RNA from infectious virus. A novel system utilising two antisense primers, a 14-mer for reverse transcription and a 35-mer for long PCR, produced high yields of genomic length cDNA. Use of low copy number Able K cells and an incubation temperature of 28 degrees C increased the genetic stability of cloned cDNA. Clones containing 11 kb cDNA inserts produced colonies of reduced size, thus providing a positive selection system for full length clones. Sequencing of the infectious clone emphasised the improved fidelity of the method compared with conventional PCR and cloning methods. A simple and rapid strategy for genetic manipulation of the infectious clone is also described. These developments represent a significant advance in recombinant technology and should be applicable to positive stranded RNA viruses which cannot easily be purified or genetically manipulated.

One-milliliter wet-digestion for inductively coupled plasma mass spectrometry (ICP-MS): determination of platinum-DNA adducts in cells treated with platinum(II) complexes.

PubMed

Yamada, Kanae; Kato, Naoyuki; Takagi, Akimitsu; Koi, Minoru; Hemmi, Hiromichi

2005-08-01

Platinum (Pt)-DNA adducts formed by the anti-tumor agent cisplatin are recognized by the DNA mismatch repair (MMR) system. To investigate the involvement of MMR proteins including hMLH1 in the removal of these adducts, we developed a mL-scale wet-digestion method for inductively coupled plasma mass spectrometry (ICP-MS). The detection limit was 0.01 ng mL(-1) Pt, which corresponded to 2 pg Pt/microg DNA when 10 microg of DNA was used. The mean relative errors were 5.4% or better for a dynamic range of 0.01-10 ng mL(-1) Pt. DNA (approximately 500 microg) had no matrix effect. To improve the accuracy, DNA preparations were treated with ribonuclease and the apparent reduction in the concentration of Pt was corrected using cellular DNA levels, which were determined with Hoechst 33258. No significant differences were observed, in terms of the formation of Pt-DNA adducts or their removal over 6 h, between hMLH1-deficient HCT116 cells, a human colorectal cancer cell line, and hMLH1-complemented HCT116+ch3 cells (n=5; P>0.05), indicating that the hMLH1-dependent DNA repair systems contribute to neither the formation nor the removal of the adducts at detectable levels. In addition, approximately 19% of the adducts were removed within 6 h in both cell lines. A time course analysis (~24 h) suggested that the removal of cisplatin-generated Pt-DNA adducts follows first-order kinetics (t(1/2)=32 h). The amount of Pt-DNA adduct formed by oxaliplatin in 1 h was 56% (ratio of means) of that generated by an equimolar concentration of cisplatin in HCT116. The proposed procedure could be useful for determining Pt-DNA adducts formed by Pt(II) complexes.

Developmental validation of the PowerPlex(®) ESI 16/17 Fast and PowerPlex(®) ESX 16/17 Fast Systems.

PubMed

McLaren, Robert S; Bourdeau-Heller, Jeanne; Patel, Jaynish; Thompson, Jonelle M; Pagram, Jenny; Loake, Thomas; Beesley, David; Pirttimaa, Markus; Hill, Carolyn R; Duewer, David L; Kline, Margaret C; Butler, John M; Storts, Douglas R

2014-11-01

The PowerPlex(®) ESI 16 Fast, ESI 17 Fast, ESX 16 Fast, and ESX 17 Fast Systems represent faster cycling versions (50min or less) of the PowerPlex(®) ESI and ESX Systems released by Promega in 2009 to accommodate the ENFSI and EDNAP groups' call for new STR multiplexes for Europe. In addition to amplification of purified DNA samples, these new faster cycling systems allow for direct amplification from single-source blood and buccal samples deposited on FTA(®) and nonFTA paper as well as from SwabSolution™ extracts of buccal swabs without the need for purification and quantitation. There are no changes to the autosomal primer pair sequences in the PowerPlex(®) ESI Fast and ESX Fast Systems compared to the original multiplexes, and full concordance at all autosomal loci and amelogenin was observed with data generated previously with the original PowerPlex(®) ESI and ESX Systems. This paper describes the developmental validation study performed on these new fast systems following guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and those of the DNA Advisory Board (DAB). Validation data demonstrate that these systems are sensitive for detecting low levels of DNA while also being capable of generating robust profiles from the high amount of input DNA present in direct-amplification samples. These systems are also tolerant to both high concentrations of PCR inhibitors as well as to slight variations in the final concentration of master mix and primer pair present in the amplification reaction that might be encountered due to pipetting error. The results of this validation study demonstrate that these systems may be used on multiple thermal cyclers and capillary electrophoresis platforms. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Optimal dose selection of N-methyl-N-nitrosourea for the rat comet assay to evaluate DNA damage in organs with different susceptibility to cytotoxicity.

PubMed

Kitamoto, Sachiko; Matsuyama, Ryoko; Uematsu, Yasuaki; Ogata, Keiko; Ota, Mika; Yamada, Toru; Miyata, Kaori; Funabashi, Hitoshi; Saito, Koichi

2015-07-01

The in vivo rodent alkaline comet assay (comet assay) is a promising technique to evaluate DNA damage in vivo. However, there is no agreement on a method to evaluate DNA damage in organs where cytotoxicity is observed. As a part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the comet assay, we examined DNA damage in the liver, stomach, and bone marrow of rats given three oral doses of N-methyl-N-nitrosourea (MNU) up to the maximum tolerated dose based on systemic toxicity. MNU significantly increased the % tail DNA in all the organs. Histopathological analysis showed no cytotoxic effect on the liver, indicating clearly that MNU has a genotoxic potential in the liver. In the stomach, however, the cytotoxic effects were very severe at systemically non-toxic doses. Low-dose MNU significantly increased the % tail DNA even at a non-cytotoxic dose, indicating that MNU has a genotoxic potential also in the stomach. Part of the DNA damage at cytotoxic doses was considered to be a secondary effect of severe cell damage. In the bone marrow, both the % tail DNA and incidence of micronucleated polychromatic erythrocytes significantly increased at non-hematotoxic doses, which were different from the non-cytotoxic doses for liver and stomach. These findings indicate that an optimal dose for detecting DNA damage may vary among organs and that careful attention is required to select an optimum dose for the comet assay based on systemic toxicity such as mortality and clinical observations. The present study shows that when serious cytotoxicity is suggested by increased % hedgehogs in the comet assay, histopathological examination should be included for the evaluation of a positive response. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of stress on endocrine and metabolic processes and redirection: Crosstalk between subcellular compartments

USDA-ARS?s Scientific Manuscript database

Recent advances in genome analysis and biochemical pathway mapping have advanced our understanding of how biological systems have evolved over time. Protein and DNA marker comparisons suggest that several of these systems are both ancient in origin but highly conserved into today’s evolved species. ...

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

PubMed Central

Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

2014-01-01

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

Assessing Date Palm Genetic Diversity Using Different Molecular Markers.

PubMed

Atia, Mohamed A M; Sakr, Mahmoud M; Adawy, Sami S

2017-01-01

Molecular marker technologies which rely on DNA analysis provide powerful tools to assess biodiversity at different levels, i.e., among and within species. A range of different molecular marker techniques have been developed and extensively applied for detecting variability in date palm at the DNA level. Recently, the employment of gene-targeting molecular marker approaches to study biodiversity and genetic variations in many plant species has increased the attention of researchers interested in date palm to carry out phylogenetic studies using these novel marker systems. Molecular markers are good indicators of genetic distances among accessions, because DNA-based markers are neutral in the face of selection. Here we describe the employment of multidisciplinary molecular marker approaches: amplified fragment length polymorphism (AFLP), start codon targeted (SCoT) polymorphism, conserved DNA-derived polymorphism (CDDP), intron-targeted amplified polymorphism (ITAP), simple sequence repeats (SSR), and random amplified polymorphic DNA (RAPD) to assess genetic diversity in date palm.

Application of DNA-based methods in forensic entomology.

PubMed

Wells, Jeffrey D; Stevens, Jamie R

2008-01-01

A forensic entomological investigation can benefit from a variety of widely practiced molecular genotyping methods. The most commonly used is DNA-based specimen identification. Other applications include the identification of insect gut contents and the characterization of the population genetic structure of a forensically important insect species. The proper application of these procedures demands that the analyst be technically expert. However, one must also be aware of the extensive list of standards and expectations that many legal systems have developed for forensic DNA analysis. We summarize the DNA techniques that are currently used in, or have been proposed for, forensic entomology and review established genetic analyses from other scientific fields that address questions similar to those in forensic entomology. We describe how accepted standards for forensic DNA practice and method validation are likely to apply to insect evidence used in a death or other forensic entomological investigation.

Antimalarial, antimicrobial, cytotoxic, DNA interaction and SOD like activities of tetrahedral copper(II) complexes

NASA Astrophysics Data System (ADS)

Mehta, Jugal V.; Gajera, Sanjay B.; Patel, Mohan N.

2015-02-01

The mononuclear copper(II) complexes with P, O-donor ligand and different fluoroquinolones have been synthesized and characterized by elemental analysis, electronic spectra, TGA, EPR, FT-IR and LC-MS spectroscopy. An antimicrobial efficiency of the complexes has been tested against five different microorganisms in terms of minimum inhibitory concentration (MIC) and displays very good antimicrobial activity. The binding strength and binding mode of the complexes with Herring Sperm DNA (HS DNA) have been investigated by absorption titration and viscosity measurement studies. The studies suggest the classical intercalative mode of DNA binding. Gel electrophoresis assay determines the ability of the complexes to cleave the supercoiled form of pUC19 DNA. Synthesized complexes have been tested for their SOD mimic activity using nonenzymatic NBT/NADH/PMS system and found to have good antioxidant activity. All the complexes show good cytotoxic and in vitro antimalarial activities.

Petri-net-based 2D design of DNA walker circuits.

PubMed

Gilbert, David; Heiner, Monika; Rohr, Christian

2018-01-01

We consider localised DNA computation, where a DNA strand walks along a binary decision graph to compute a binary function. One of the challenges for the design of reliable walker circuits consists in leakage transitions, which occur when a walker jumps into another branch of the decision graph. We automatically identify leakage transitions, which allows for a detailed qualitative and quantitative assessment of circuit designs, design comparison, and design optimisation. The ability to identify leakage transitions is an important step in the process of optimising DNA circuit layouts where the aim is to minimise the computational error inherent in a circuit while minimising the area of the circuit. Our 2D modelling approach of DNA walker circuits relies on coloured stochastic Petri nets which enable functionality, topology and dimensionality all to be integrated in one two-dimensional model. Our modelling and analysis approach can be easily extended to 3-dimensional walker systems.

Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein–DNA complexes

PubMed Central

Ebenstein, Yuval; Gassman, Natalie; Kim, Soohong; Weiss, Shimon

2011-01-01

Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA. PMID:19452448

CMG-biotools, a free workbench for basic comparative microbial genomics.

PubMed

Vesth, Tammi; Lagesen, Karin; Acar, Öncel; Ussery, David

2013-01-01

Today, there are more than a hundred times as many sequenced prokaryotic genomes than were present in the year 2000. The economical sequencing of genomic DNA has facilitated a whole new approach to microbial genomics. The real power of genomics is manifested through comparative genomics that can reveal strain specific characteristics, diversity within species and many other aspects. However, comparative genomics is a field not easily entered into by scientists with few computational skills. The CMG-biotools package is designed for microbiologists with limited knowledge of computational analysis and can be used to perform a number of analyses and comparisons of genomic data. The CMG-biotools system presents a stand-alone interface for comparative microbial genomics. The package is a customized operating system, based on Xubuntu 10.10, available through the open source Ubuntu project. The system can be installed on a virtual computer, allowing the user to run the system alongside any other operating system. Source codes for all programs are provided under GNU license, which makes it possible to transfer the programs to other systems if so desired. We here demonstrate the package by comparing and analyzing the diversity within the class Negativicutes, represented by 31 genomes including 10 genera. The analyses include 16S rRNA phylogeny, basic DNA and codon statistics, proteome comparisons using BLAST and graphical analyses of DNA structures. This paper shows the strength and diverse use of the CMG-biotools system. The system can be installed on a vide range of host operating systems and utilizes as much of the host computer as desired. It allows the user to compare multiple genomes, from various sources using standardized data formats and intuitive visualizations of results. The examples presented here clearly shows that users with limited computational experience can perform complicated analysis without much training.

Detection of the Odor Signature of Ovarian Cancer using DNA-Decorated Carbon Nanotube Field Effect Transistor Arrays

NASA Astrophysics Data System (ADS)

Kehayias, Christopher; Kybert, Nicholas; Yodh, Jeremy; Johnson, A. T. Charlie

Carbon nanotubes are low-dimensional materials that exhibit remarkable chemical and bio-sensing properties and have excellent compatibility with electronic systems. Here, we present a study that uses an electronic olfaction system based on a large array of DNA-carbon nanotube field effect transistors vapor sensors to analyze the VOCs of blood plasma samples collected from patients with malignant ovarian cancer, patients with benign ovarian lesions, and age-matched healthy subjects. Initial investigations involved coating each CNT sensor with single-stranded DNA of a particular base sequence. 10 distinct DNA oligomers were used to functionalize the carbon nanotube field effect transistors, providing a 10-dimensional sensor array output response. Upon performing a statistical analysis of the 10-dimensional sensor array responses, we showed that blood samples from patients with malignant cancer can be reliably differentiated from those of healthy control subjects with a p-value of 3 x 10-5. The results provide preliminary evidence that the blood of ovarian cancer patients contains a discernable volatile chemical signature that can be detected using DNA-CNT nanoelectronic vapor sensors, a first step towards a minimally invasive electronic diagnostic technology for ovarian cancer.

Identification of cytomegalovirus and human herpesvirus-6 DNA in a patient with corneal endotheliitis.

PubMed

Yokogawa, Hideaki; Kobayashi, Akira; Yamazaki, Natsuko; Sugiyama, Kazuhisa

2013-03-01

To report the case of a patient with unilateral corneal endotheliitis in which both cytomegalovirus (CMV) and human herpesvirus-6 (HHV6) DNA was identified in the aqueous humor. A 67-year-old man with corneal endotheliitis OD was referred to us for decreased visual acuity. Local corneal stromal edema, pigmented keratic precipitates, a coin-shaped lesion and minimal anterior chamber reaction were observed by slit-lamp biomicroscopy. Cells with owl's eye appearance in the endothelial cell layer were observed by in vivo laser confocal microscopy. The patient had rheumatoid arthritis, which was treated by oral prednisolone and intravenous abatacept. Polymerase chain reaction analysis of aqueous humor samples detected both CMV and HHV6 DNA, but not other HHVs. Treatment with topical ganciclovir and systemic valganciclovir resulted in a clear cornea. A patient with corneal endotheliitis had both CMV and HHV6 DNA identified in the aqueous humor. Although both viruses were identified in this case, clinical manifestations resembled CMV corneal endotheliitis, and it was unclear whether HHV6 could affect the clinical course. Systemic abatacept and corticosteroid therapy might play a positive role in cases with both CMV and HHV6 DNA in this corneal endotheliitis.

Molecular thermal transistor: Dimension analysis and mechanism

NASA Astrophysics Data System (ADS)

Behnia, S.; Panahinia, R.

2018-04-01

Recently, large challenge has been spent to realize high efficient thermal transistors. Outstanding properties of DNA make it as an excellent nano material in future technologies. In this paper, we introduced a high efficient DNA based thermal transistor. The thermal transistor operates when the system shows an increase in the thermal flux despite of decreasing temperature gradient. This is what called as negative differential thermal resistance (NDTR). Based on multifractal analysis, we could distinguish regions with NDTR state from non-NDTR state. Moreover, Based on dimension spectrum of the system, it is detected that NDTR state is accompanied by ballistic transport regime. The generalized correlation sum (analogous to specific heat) shows that an irregular decrease in the specific heat induces an increase in the mean free path (mfp) of phonons. This leads to the occurrence of NDTR.

A convenient and adaptable package of DNA sequence analysis programs for microcomputers.

PubMed Central

Pustell, J; Kafatos, F C

1982-01-01

We describe a package of DNA data handling and analysis programs designed for microcomputers. The package is convenient for immediate use by persons with little or no computer experience, and has been optimized by trial in our group for a year. By typing a single command, the user enters a system which asks questions or gives instructions in English. The system will enter, alter, and manage sequence files or a restriction enzyme library. It generates the reverse complement, translates, calculates codon usage, finds restriction sites, finds homologies with various degrees of mismatch, and graphs amino acid composition or base frequencies. A number of options for data handling and printing can be used to produce figures for publication. The package will be available in ANSI Standard FORTRAN for use with virtually any FORTRAN compiler. PMID:6278412

Plasma ctDNA RAS mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients

PubMed Central

Vidal, J; Muinelo, L; Dalmases, A; Jones, F; Edelstein, D; Iglesias, M; Orrillo, M; Abalo, A; Rodríguez, C; Brozos, E; Vidal, Y; Candamio, S; Vázquez, F; Ruiz, J; Guix, M; Visa, L; Sikri, V; Albanell, J; Bellosillo, B; López, R; Montagut, C

2017-01-01

Abstract Background RAS assessment is mandatory for therapy decision in metastatic colorectal cancer (mCRC) patients. This determination is based on tumor tissue, however, genotyping of circulating tumor (ct)DNA offers clear advantages as a minimally invasive method that represents tumor heterogeneity. Our study aims to evaluate the use of ctDNA as an alternative for determining baseline RAS status and subsequent monitoring of RAS mutations during therapy as a component of routine clinical practice. Patients and methods RAS mutational status in plasma was evaluated in mCRC patients by OncoBEAM™ RAS CRC assay. Concordance of results in plasma and tissue was retrospectively evaluated. RAS mutations were also prospectively monitored in longitudinal plasma samples from selected patients. Results Analysis of RAS in tissue and plasma samples from 115 mCRC patients showed a 93% overall agreement. Plasma/tissue RAS discrepancies were mainly explained by spatial and temporal tumor heterogeneity. Analysis of clinico-pathological features showed that the site of metastasis (i.e. peritoneal, lung), the histology of the tumor (i.e. mucinous) and administration of treatment previous to blood collection negatively impacted the detection of RAS in ctDNA. In patients with baseline mutant RAS tumors treated with chemotherapy/antiangiogenic, longitudinal analysis of RAS ctDNA mirrored response to treatment, being an early predictor of response. In patients RAS wt, longitudinal monitoring of RAS ctDNA revealed that OncoBEAM was useful to detect emergence of RAS mutations during anti-EGFR treatment. Conclusion The high overall agreement in RAS mutational assessment between plasma and tissue supports blood-based testing with OncoBEAM™ as a viable alternative for genotyping RAS of mCRC patients in routine clinical practice. Our study describes practical clinico-pathological specifications to optimize RAS ctDNA determination. Moreover, OncoBEAM™ is useful to monitor RAS in patients undergoing systemic therapy to detect resistance and evaluate the efficacy of particular treatments. PMID:28419195

Comprehensive analysis of DNA polymerase III α subunits and their homologs in bacterial genomes

PubMed Central

Timinskas, Kęstutis; Balvočiūtė, Monika; Timinskas, Albertas; Venclovas, Česlovas

2014-01-01

The analysis of ∼2000 bacterial genomes revealed that they all, without a single exception, encode one or more DNA polymerase III α-subunit (PolIIIα) homologs. Classified into C-family of DNA polymerases they come in two major forms, PolC and DnaE, related by ancient duplication. While PolC represents an evolutionary compact group, DnaE can be further subdivided into at least three groups (DnaE1-3). We performed an extensive analysis of various sequence, structure and surface properties of all four polymerase groups. Our analysis suggests a specific evolutionary pathway leading to PolC and DnaE from the last common ancestor and reveals important differences between extant polymerase groups. Among them, DnaE1 and PolC show the highest conservation of the analyzed properties. DnaE3 polymerases apparently represent an ‘impaired’ version of DnaE1. Nonessential DnaE2 polymerases, typical for oxygen-using bacteria with large GC-rich genomes, have a number of features in common with DnaE3 polymerases. The analysis of polymerase distribution in genomes revealed three major combinations: DnaE1 either alone or accompanied by one or more DnaE2s, PolC + DnaE3 and PolC + DnaE1. The first two combinations are present in Escherichia coli and Bacillus subtilis, respectively. The third one (PolC + DnaE1), found in Clostridia, represents a novel, so far experimentally uncharacterized, set. PMID:24106089

Autoclave sterilization of instruments used on women with cervical neoplasia is an effective method of eradicating residual human papillomavirus DNA: a polymerase chain reaction-based evaluation.

PubMed

Estes, Jacob M; Kirby, Tyler O; Huh, Warner K

2007-01-01

To determine whether autoclave sterilization eradicates human papillomavirus (HPV) DNA on specula and instruments used to treat women with cervical neoplasia. Specula and instruments used in two referral colposcopy clinics were evaluated to determine the PGMY9/11 primer system's ability to amplify residual HPV DNA. Each speculum and instrument was sampled with a Dacron swab and stored in PreservCyt solution (Cytyc Corporation, Marlborough, MA) at 4 degrees C. DNA amplification was performed under standard conditions with appropriate controls followed by HPV typing using the reverse line blot test (Roche Molecular Systems, Alameda, CA). Once validated, the same polymerase chain reaction method was used on autoclave-sterilized specula and biopsy instruments and heated glass bead- and Cidex bath (Johnson & Johnson, New Brunswick, NJ)-sterilized instruments. All results, with appropriate positive and negative controls, were confirmed in triplicate. A total of 140 instruments (70 used and 70 autoclaved) were sampled for residual HPV DNA. Five samples in the contaminated specula arm were excluded from analysis secondary to insufficient sampling. Of the remaining samples, 52.3% (34/65) of contaminated instruments-both specula and biopsy instruments-had detectable HPV DNA. Fifty-five percent of contaminated biopsy instruments (11/20) were positive and 51.1% of contaminated specula (23/45) were positive. All 70 autoclaved samples (50 specula and 20 biopsy instruments) were negative for residual HPV DNA or beta-globin. One instrument in the glass bead and Cidex group that was presumed sterile was positive for HPV 16 DNA. The PGMY9/11 primer system is an effective method to detect residual HPV DNA. Autoclave sterilization appears to eradicate HPV DNA to levels undetectable with this sensitive assay, whereas heated glass beads followed by Cidex bath appears to be inadequate methods. These results suggest that autoclave sterilization is effective when using nondisposable instruments and should be the method of choice in studies using polymerase chain reaction-based amplification of HPV DNA.

Nanopore detection of DNA molecules in crowded neutral polymer solutions

NASA Astrophysics Data System (ADS)

Sharma, Rajesh Kumar; Dai, Liang; Doyle, Patrick; Garaj, Slaven

Nanopore sensing is a precise technique for analysis of the structure and dynamics of individual biomolecules in different environments, and has even become a prominent technique for next-gen DNA sequencing. In the nanopore sensor, an individual DNA molecule is electrophoretically translocated through a single, nanometer-scaled pore in a solid-state membrane separating two chambers filled with electrolyte. The conformation of the molecule is deduced from modulations in the ionic current through the pore during the translocation event. Using nanopores, we investigated the dynamics of the DNA molecules in a crowded solution of neutral polymers of different sizes and concentrations. The translocation dynamics depends significantly on the size and concentration of the polymers, as different contributions to the electrophoretic and entropic forces on the DNA molecules come into play. This setup offers an excellent, tuneable model-system for probing biologically relevant questions regarding the behaviour of DNA molecules in highly confined and crowded environments. Singapore-MIT Alliance for Research and Technology.

Identification and DNA annotation of a plasmid isolated from Chromobacterium violaceum.

PubMed

Lima, Daniel C; Nyberg, Lena K; Westerlund, Fredrik; Batistuzzo de Medeiros, Silvia R

2018-03-28

Chromobacterium violaceum is a ß-proteobacterium found widely worldwide with important biotechnological properties and is associated to lethal sepsis in immune-depressed individuals. In this work, we report the discover, complete sequence and annotation of a plasmid detected in C. violaceum that has been unnoticed until now. We used DNA single-molecule analysis to confirm that the episome found was a circular molecule and then proceeded with NGS sequencing. After DNA annotation, we found that this extra-chromosomal DNA is probably a defective bacteriophage of approximately 44 kilobases, with 39 ORFs comprising, mostly hypothetical proteins. We also found DNA sequences that ensure proper plasmid replication and partitioning as well as a toxin addiction system. This report sheds light on the biology of this important species, helping us to understand the mechanisms by which C. violaceum endures to several harsh conditions. This discovery could also be a first step in the development of a DNA manipulation tool in this bacterium.

Analysis of mutational spectra by denaturant capillary electrophoresis

PubMed Central

Ekstrøm, Per O.; Khrapko, Konstantin; Li-Sucholeiki, Xiao-Cheng; Hunter, Ian W.; Thilly, William G.

2009-01-01

Numbers and kinds of point mutant within DNA from cells, tissues and human population may be discovered for nearly any 75–250bp DNA sequence. High fidelity DNA amplification incorporating a thermally stable DNA “clamp” is followed by separation by denaturing capillary electrophoresis (DCE). DCE allows for peak collection and verification sequencing. DCE in a mode of cycling temperature, e.g.+/− 5°C, CyDCE, permits high resolution of mutant sequences using computer defined analytes without preliminary optimization experiments. DNA sequencers have been modified to permit higher throughput CyDCE and a massively parallel,~25,000 capillary system, has been designed for pangenomic scans in large human populations. DCE has been used to define quantitative point mutational spectra for study a wide variety of genetic phenomena: errors of DNA polymerases, mutations induced in human cells by chemicals and irradiation, testing of human gene-common disease associations and the discovery of origins of point mutations in human development and carcinogenesis. PMID:18600220

Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

PubMed

Villalobos, Michael J; Betti, Christopher J; Vaughan, Andrew T M

2006-01-01

Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location.

Analysis of JC virus DNA replication using a quantitative and high-throughput assay

PubMed Central

Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

2015-01-01

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

Genome organization of Tobacco leaf curl Zimbabwe virus, a new, distinct monopartite begomovirus associated with subgenomic defective DNA molecules.

PubMed

Paximadis, M; Rey, M E

2001-12-01

The complete DNA A of the begomovirus Tobacco leaf curl Zimbabwe virus (TbLCZWV) was sequenced: it comprises 2767 nucleotides with six major open reading frames encoding proteins with molecular masses greater than 9 kDa. Full-length TbLCZWV DNA A tandem dimers, cloned in binary vectors (pBin19 and pBI121) and transformed into Agrobacterium tumefaciens, were systemically infectious upon agroinoculation of tobacco and tomato. Efforts to identify a DNA B component were unsuccessful. These findings suggest that TbLCZWV is a new member of the monopartite group of begomoviruses. Phylogenetic analysis identified TbLCZWV as a distinct begomovirus with its closest relative being Chayote mosaic virus. Abutting primer PCR amplified ca. 1300 bp molecules, and cloning and sequencing of two of these molecules revealed them to be subgenomic defective DNA molecules originating from TbLCZWV DNA A. Variable symptom severity associated with tobacco leaf curl disease and TbLCZWV is discussed.

Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

PubMed

Brightwell, Gale; Boerema, Jackie; Mills, John; Mowat, Eilidh; Pulford, David

2006-05-25

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

Comparative reactivity of mismatched and unpaired bases in relation to their type and surroundings. Chemical cleavage of DNA mismatches in mutation detection analysis.

PubMed

Yakubovskaya, Marianna G; Belyakova, Anna A; Gasanova, Viktoria K; Belitsky, Gennady A; Dolinnaya, Nina G

2010-07-01

Systematic study of chemical reactivity of non-Watson-Crick base pairs depending on their type and microenvironment was performed on a model system that represents two sets of synthetic DNA duplexes with all types of mismatched and unmatched bases flanked by T.A or G.C pairs. Using comparative cleavage pattern analysis, we identified the main and additional target bases and performed quantitative study of the time course and efficacy of DNA modification caused by potassium permanganate or hydroxylamine. Potassium permanganate in combination with tetraethylammonium chloride was shown to induce DNA cleavage at all mismatched or bulged T residues, as well as at thymines of neighboring canonical pairs. Other mispaired (bulged) bases and thymine residues located on the second position from the mismatch site were not the targets for KMnO(4) attack. In contrast, hydroxylamine cleaved only heteroduplexes containing mismatched or unmatched C residues, and did not modify adjacent cytosines. However when G.C pairs flank bulged C residue, neighboring cytosines are also attacked by hydroxylamine due to defect migration. Chemical reactivity of target bases was shown to correlate strongly with the local disturbance of DNA double helix at mismatch or bulge site. With our model system, we were able to prove the absence of false-negative and false-positive results. Portion of heteroduplex reliably revealed in a mixture with corresponding homoduplex consists of 5% for bulge bases and "open" non-canonical pairs, and 10% for wobble base pairs giving minimal violations in DNA structure. This study provides a complete understanding of the principles of mutation detection methodology based on chemical cleavage of mismatches and clarifies the advantages and limitations of this approach in various biological and conformational studies of DNA. Copyright 2010 Elsevier Masson SAS. All rights reserved.

In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling*

PubMed Central

Lindsey-Boltz, Laura A.; Reardon, Joyce T.; Wold, Marc S.; Sancar, Aziz

2012-01-01

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair. PMID:22948311

In vitro analysis of the role of replication protein A (RPA) and RPA phosphorylation in ATR-mediated checkpoint signaling.

PubMed

Lindsey-Boltz, Laura A; Reardon, Joyce T; Wold, Marc S; Sancar, Aziz

2012-10-19

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.

A Fluorescent Tile DNA Diagnocode System for In Situ Rapid and Selective Diagnosis of Cytosolic RNA Cancer Markers

PubMed Central

Park, Kyung Soo; Shin, Seung Won; Jang, Min Su; Shin, Woojung; Yang, Kisuk; Min, Junhong; Cho, Seung-Woo; Oh, Byung-Keun; Bae, Jong Wook; Jung, Sunghwan; Choi, Jeong-Woo; Um, Soong Ho

2015-01-01

Accurate cancer diagnosis often requires extraction and purification of genetic materials from cells, and sophisticated instrumentations that follow. Otherwise in order to directly treat the diagnostic materials to cells, multiple steps to optimize dose concentration and treatment time are necessary due to diversity in cellular behaviors. These processes may offer high precision but hinder fast analysis of cancer, especially in clinical situations that need rapid detection and characterization of cancer. Here we present a novel fluorescent tile DNA nanostructure delivered to cancer cytosol by employing nanoparticle technology. Its structural anisotropicity offers easy manipulation for multifunctionalities, enabling the novel DNA nanostructure to detect intracellular cancer RNA markers with high specificity within 30 minutes post treatment, while the nanoparticle property bypasses the requirement of treatment optimization, effectively reducing the complexity of applying the system for cancer diagnosis. Altogether, the system offers a precise and rapid detection of cancer, suggesting the future use in the clinical fields. PMID:26678430

An optimized rapid bisulfite conversion method with high recovery of cell-free DNA.

PubMed

Yi, Shaohua; Long, Fei; Cheng, Juanbo; Huang, Daixin

2017-12-19

Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA. We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods. The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

PubMed Central

Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

2016-01-01

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence. PMID:27031831

Visualization of DNA Replication in the Vertebrate Model System DT40 using the DNA Fiber Technique

PubMed Central

Schwab, Rebekka A.V.; Niedzwiedz, Wojciech

2011-01-01

Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development. Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope. PMID:22064662

Laser-induced fluorescence microscopic system using an optical parametric oscillator for tunable detection in microchip analysis.

PubMed

Kumemura, Momoko; Odake, Tamao; Korenaga, Takashi

2005-06-01

A laser-induced fluorescence microscopic system based on optical parametric oscillation has been constructed as a tunable detector for microchip analysis. The detection limit of sulforhodamine B (Ex. 520 nm, Em. 570 nm) was 0.2 mumol, which was approximately eight orders of magnitude better than with a conventional fluorophotometer. The system was applied to the determination of fluorescence-labeled DNA (Ex. 494 nm, Em. 519 nm) in a microchannel and the detection limit reached a single molecule. These results showed the feasibility of this system as a highly sensitive and tunable fluorescence detector for microchip analysis.

ddPCRclust - An R package and Shiny app for automated analysis of multiplexed ddPCR data.

PubMed

Brink, Benedikt G; Meskas, Justin; Brinkman, Ryan R

2018-03-09

Droplet digital PCR (ddPCR) is an emerging technology for quantifying DNA. By partitioning the target DNA into ∼20000 droplets, each serving as its own PCR reaction compartment, a very high sensitivity of DNA quantification can be achieved. However, manual analysis of the data is time consuming and algorithms for automated analysis of non-orthogonal, multiplexed ddPCR data are unavailable, presenting a major bottleneck for the advancement of ddPCR transitioning from low-throughput to high- throughput. ddPCRclust is an R package for automated analysis of data from Bio-Rad's droplet digital PCR systems (QX100 and QX200). It can automatically analyse and visualise multiplexed ddPCR experiments with up to four targets per reaction. Results are on par with manual analysis, but only take minutes to compute instead of hours. The accompanying Shiny app ddPCRvis provides easy access to the functionalities of ddPCRclust through a web-browser based GUI. R package: https://github.com/bgbrink/ddPCRclust; Interface: https://github.com/bgbrink/ddPCRvis/; Web: https://bibiserv.cebitec.uni-bielefeld.de/ddPCRvis/. bbrink@cebitec.uni-bielefeld.de.

A high-throughput method for GMO multi-detection using a microfluidic dynamic array.

PubMed

Brod, Fábio Cristiano Angonesi; van Dijk, Jeroen P; Voorhuijzen, Marleen M; Dinon, Andréia Zilio; Guimarães, Luis Henrique S; Scholtens, Ingrid M J; Arisi, Ana Carolina Maisonnave; Kok, Esther J

2014-02-01

The ever-increasing production of genetically modified crops generates a demand for high-throughput DNA-based methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis by DNA based methods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.

Partial DNA-guided Cas9 enables genome editing with reduced off-target activity

PubMed Central

Yin, Hao; Song, Chun-Qing; Suresh, Sneha; Kwan, Suet-Yan; Wu, Qiongqiong; Walsh, Stephen; Ding, Junmei; Bogorad, Roman L; Zhu, Lihua Julie; Wolfe, Scot A; Koteliansky, Victor; Xue, Wen; Langer, Robert; Anderson, Daniel G

2018-01-01

CRISPR–Cas9 is a versatile RNA-guided genome editing tool. Here we demonstrate that partial replacement of RNA nucleotides with DNA nucleotides in CRISPR RNA (crRNA) enables efficient gene editing in human cells. This strategy of partial DNA replacement retains on-target activity when used with both crRNA and sgRNA, as well as with multiple guide sequences. Partial DNA replacement also works for crRNA of Cpf1, another CRISPR system. We find that partial DNA replacement in the guide sequence significantly reduces off-target genome editing through focused analysis of off-target cleavage, measurement of mismatch tolerance and genome-wide profiling of off-target sites. Using the structure of the Cas9–sgRNA complex as a guide, the majority of the 3′ end of crRNA can be replaced with DNA nucleotide, and the 5 - and 3′-DNA-replaced crRNA enables efficient genome editing. Cas9 guided by a DNA–RNA chimera may provide a generalized strategy to reduce both the cost and the off-target genome editing in human cells. PMID:29377001

Mitochondrial sequence analysis for forensic identification using pyrosequencing technology.

PubMed

Andréasson, H; Asp, A; Alderborn, A; Gyllensten, U; Allen, M

2002-01-01

Over recent years, requests for mtDNA analysis in the field of forensic medicine have notably increased, and the results of such analyses have proved to be very useful in forensic cases where nuclear DNA analysis cannot be performed. Traditionally, mtDNA has been analyzed by DNA sequencing of the two hypervariable regions, HVI and HVII, in the D-loop. DNA sequence analysis using the conventional Sanger sequencing is very robust but time consuming and labor intensive. By contrast, mtDNA analysis based on the pyrosequencing technology provides fast and accurate results from the human mtDNA present in many types of evidence materials in forensic casework. The assay has been developed to determine polymorphic sites in the mitochondrial D-loop as well as the coding region to further increase the discrimination power of mtDNA analysis. The pyrosequencing technology for analysis of mtDNA polymorphisms has been tested with regard to sensitivity, reproducibility, and success rate when applied to control samples and actual casework materials. The results show that the method is very accurate and sensitive; the results are easily interpreted and provide a high success rate on casework samples. The panel of pyrosequencing reactions for the mtDNA polymorphisms were chosen to result in an optimal discrimination power in relation to the number of bases determined.

Phylogenetic reconstruction in the Order Nymphaeales: ITS2 secondary structure analysis and in silico testing of maturase k (matK) as a potential marker for DNA bar coding

PubMed Central

2012-01-01

Background The Nymphaeales (waterlilly and relatives) lineage has diverged as the second branch of basal angiosperms and comprises of two families: Cabombaceae and Nymphaceae. The classification of Nymphaeales and phylogeny within the flowering plants are quite intriguing as several systems (Thorne system, Dahlgren system, Cronquist system, Takhtajan system and APG III system (Angiosperm Phylogeny Group III system) have attempted to redefine the Nymphaeales taxonomy. There have been also fossil records consisting especially of seeds, pollen, stems, leaves and flowers as early as the lower Cretaceous. Here we present an in silico study of the order Nymphaeales taking maturaseK (matK) and internal transcribed spacer (ITS2) as biomarkers for phylogeny reconstruction (using character-based methods and Bayesian approach) and identification of motifs for DNA barcoding. Results The Maximum Likelihood (ML) and Bayesian approach yielded congruent fully resolved and well-supported trees using a concatenated (ITS2+ matK) supermatrix aligned dataset. The taxon sampling corroborates the monophyly of Cabombaceae. Nuphar emerges as a monophyletic clade in the family Nymphaeaceae while there are slight discrepancies in the monophyletic nature of the genera Nymphaea owing to Victoria-Euryale and Ondinea grouping in the same node of Nymphaeaceae. ITS2 secondary structures alignment corroborate the primary sequence analysis. Hydatellaceae emerged as a sister clade to Nymphaeaceae and had a basal lineage amongst the water lilly clades. Species from Cycas and Ginkgo were taken as outgroups and were rooted in the overall tree topology from various methods. Conclusions MatK genes are fast evolving highly variant regions of plant chloroplast DNA that can serve as potential biomarkers for DNA barcoding and also in generating primers for angiosperms with identification of unique motif regions. We have reported unique genus specific motif regions in the Order Nymphaeles from matK dataset which can be further validated for barcoding and designing of PCR primers. Our analysis using a novel approach of sequence-structure alignment and phylogenetic reconstruction using molecular morphometrics congrue with the current placement of Hydatellaceae within the early-divergent angiosperm order Nymphaeales. The results underscore the fact that more diverse genera, if not fully resolved to be monophyletic, should be represented by all major lineages. PMID:23282079

Determination of mutated genes in the presence of wild-type DNA by using molecular beacons as probe

NASA Astrophysics Data System (ADS)

Zhang, Yonghua; Ai, Junjie; Gu, Qiaorong; Gao, Qiang; Qi, Honglan; Zhang, Chengxiao

2017-03-01

Low-abundance mutations in the presence of wild-type DNA can be determined using molecular beacon (MB) as probe. A MB is generally used as DNA probe because it can distinguish single-base mismatched target DNA from fully matched target DNA. However, the probe can not determine low-abundance mutations in the presence of wild-type DNA. In this study, this limitation is addressed by enhancing the stability of unpaired base-containing dsDNA with a hydrogen-bonding ligand, which was added after hybridization of the MB to the target DNA. The ligand formed hydrogen bonds with unpaired bases and stabilized the unpaired base-containing dsDNA if target DNA is mutated one. As a result, more MBs were opened by the mutant genes in the presence of the ligand and a further increase in the fluorescence intensity was obtained. By contrast, fluorescence intensity did not change if target DNA is wild-type one. Consequent increase in the fluorescence intensity of the MB was regarded as a signal derived from mutant genes. The proposed method was applied in synthetic template systems to determine point mutation in DNA obtained from PCR analysis. The method also allows rapid and simple discrimination of a signal if it is originated in the presence of mutant gene or alternatively by a lower concentration of wild gene.

High-Throughput Analysis of Global DNA Methylation Using Methyl-Sensitive Digestion.

PubMed

Shiratori, Hiromi; Feinweber, Carmen; Knothe, Claudia; Lötsch, Jörn; Thomas, Dominique; Geisslinger, Gerd; Parnham, Michael J; Resch, Eduard

2016-01-01

DNA methylation is a major regulatory process of gene transcription, and aberrant DNA methylation is associated with various diseases including cancer. Many compounds have been reported to modify DNA methylation states. Despite increasing interest in the clinical application of drugs with epigenetic effects, and the use of diagnostic markers for genome-wide hypomethylation in cancer, large-scale screening systems to measure the effects of drugs on DNA methylation are limited. In this study, we improved the previously established fluorescence polarization-based global DNA methylation assay so that it is more suitable for application to human genomic DNA. Our methyl-sensitive fluorescence polarization (MSFP) assay was highly repeatable (inter-assay coefficient of variation = 1.5%) and accurate (r2 = 0.99). According to signal linearity, only 50-80 ng human genomic DNA per reaction was necessary for the 384-well format. MSFP is a simple, rapid approach as all biochemical reactions and final detection can be performed in one well in a 384-well plate without purification steps in less than 3.5 hours. Furthermore, we demonstrated a significant correlation between MSFP and the LINE-1 pyrosequencing assay, a widely used global DNA methylation assay. MSFP can be applied for the pre-screening of compounds that influence global DNA methylation states and also for the diagnosis of certain types of cancer.

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array.

PubMed

Douglas, Erik S; Hsiao, Sonny C; Onoe, Hiroaki; Bertozzi, Carolyn R; Francis, Matthew B; Mathies, Richard A

2009-07-21

A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min(-1), while primary T cells exhibited only 2 milli-pH min(-1). This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties.

DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array

PubMed Central

Douglas, Erik S.; Hsiao, Sonny C.; Onoe, Hiroaki; Bertozzi, Carolyn R.; Francis, Matthew B.; Mathies, Richard A.

2010-01-01

A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min−1, while primary T cells exhibited only 2 milli-pH min−1. This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties. PMID:19568668

Development of a high-speed real-time PCR system for rapid and precise nucleotide recognition

NASA Astrophysics Data System (ADS)

Terazono, Hideyuki; Takei, Hiroyuki; Hattori, Akihiro; Yasuda, Kenji

2010-04-01

Polymerase chain reaction (PCR) is a common method used to create copies of a specific target region of a DNA sequence and to produce large quantities of DNA. A few DNA molecules, which act as templates, are rapidly amplified by PCR into many billions of copies. PCR is a key technology in genome-based biological analysis, revolutionizing many life science fields such as medical diagnostics, food safety monitoring, and countermeasures against bioterrorism. Thus, many applications have been developed with the thermal cycling. For these PCR applications, one of the most important key factors is reduction in the data acquisition time. To reduce the acquisition time, it is necessary to decrease the temperature transition time between the high and low ends as much as possible. We have developed a novel rapid real-time PCR system based on rapid exchange of media maintained at different temperatures. This system consists of two thermal reservoirs and a reaction chamber for PCR observation. The temperature transition was achieved within 0.3 sec, and good thermal stability was achieved during thermal cycling with rapid exchange of circulating media. This system allows rigorous optimization of the temperatures required for each stage of the PCR processes. Resulting amplicons were confirmed by electrophoresis. Using the system, rapid DNA amplification was accomplished within 3.5 min, including initial heating and complete 50 PCR cycles. It clearly shows that the device could allow us faster temperature switching than the conventional conduction-based heating systems based on Peltier heating/cooling.

A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome.

PubMed

Wang, Hui-Yun; Luo, Minjie; Tereshchenko, Irina V; Frikker, Danielle M; Cui, Xiangfeng; Li, James Y; Hu, Guohong; Chu, Yi; Azaro, Marco A; Lin, Yong; Shen, Li; Yang, Qifeng; Kambouris, Manousos E; Gao, Richeng; Shih, Weichung; Li, Honghua

2005-02-01

A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system. The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.

Azospirillum zeae sp. nov., a diazotrophic bacterium isolated from rhizosphere soil of Zea mays.

PubMed

Mehnaz, Samina; Weselowski, Brian; Lazarovits, George

2007-12-01

Two free-living nitrogen-fixing bacterial strains, N6 and N7(T), were isolated from corn rhizosphere. A polyphasic taxonomic approach, including morphological characterization, Biolog analysis, DNA-DNA hybridization, and 16S rRNA, cpn60 and nifH gene sequence analysis, was taken to analyse the two strains. 16S rRNA gene sequence analysis indicated that strains N6 and N7(T) both belonged to the genus Azospirillum and were closely related to Azospirillum oryzae (98.7 and 98.8 % similarity, respectively) and Azospirillum lipoferum (97.5 and 97.6 % similarity, respectively). DNA-DNA hybridization of strains N6 and N7(T) showed reassociation values of 48 and 37 %, respectively, with A. oryzae and 43 % with A. lipoferum. Sequences of the nifH and cpn60 genes of both strains showed 99 and approximately 95 % similarity, respectively, with those of A. oryzae. Chemotaxonomic characteristics (Q-10 as quinone system, 18 : 1omega7c as major fatty acid) and G+C content of the DNA (67.6 mol%) were also similar to those of members of the genus Azospirillum. Gene sequences and Biolog and fatty acid analysis showed that strains N6 and N7(T) differed from the closely related species A. lipoferum and A. oryzae. On the basis of these results, it is proposed that these nitrogen-fixing strains represent a novel species. The name Azospirillum zeae sp. nov. is suggested, with N7(T) (=NCCB 100147(T)=LMG 23989(T)) as the type strain.

Microfluidic magnetic fluidized bed for DNA analysis in continuous flow mode.

PubMed

Hernández-Neuta, Iván; Pereiro, Iago; Ahlford, Annika; Ferraro, Davide; Zhang, Qiongdi; Viovy, Jean-Louis; Descroix, Stéphanie; Nilsson, Mats

2018-04-15

Magnetic solid phase substrates for biomolecule manipulation have become a valuable tool for simplification and automation of molecular biology protocols. However, the handling of magnetic particles inside microfluidic chips for miniaturized assays is often challenging due to inefficient mixing, aggregation, and the advanced instrumentation required for effective actuation. Here, we describe the use of a microfluidic magnetic fluidized bed approach that enables dynamic, highly efficient and simplified magnetic bead actuation for DNA analysis in a continuous flow platform with minimal technical requirements. We evaluate the performance of this approach by testing the efficiency of individual steps of a DNA assay based on padlock probes and rolling circle amplification. This assay comprises common nucleic acid analysis principles, such as hybridization, ligation, amplification and restriction digestion. We obtained efficiencies of up to 90% for these reactions with high throughput processing up to 120μL of DNA dilution at flow rates ranging from 1 to 5μL/min without compromising performance. The fluidized bed was 20-50% more efficient than a commercially available solution for microfluidic manipulation of magnetic beads. Moreover, to demonstrate the potential of this approach for integration into micro-total analysis systems, we optimized the production of a low-cost polymer based microarray and tested its analytical performance for integrated single-molecule digital read-out. Finally, we provide the proof-of-concept for a single-chamber microfluidic chip that combines the fluidized bed with the polymer microarray for a highly simplified and integrated magnetic bead-based DNA analyzer, with potential applications in diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities.

PubMed

Oldham, Athenia L; Drilling, Heather S; Stamps, Blake W; Stevenson, Bradley S; Duncan, Kathleen E

2012-11-20

The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources.

Automated DNA extraction platforms offer solutions to challenges of assessing microbial biofouling in oil production facilities

PubMed Central

2012-01-01

The analysis of microbial assemblages in industrial, marine, and medical systems can inform decisions regarding quality control or mitigation. Modern molecular approaches to detect, characterize, and quantify microorganisms provide rapid and thorough measures unbiased by the need for cultivation. The requirement of timely extraction of high quality nucleic acids for molecular analysis is faced with specific challenges when used to study the influence of microorganisms on oil production. Production facilities are often ill equipped for nucleic acid extraction techniques, making the preservation and transportation of samples off-site a priority. As a potential solution, the possibility of extracting nucleic acids on-site using automated platforms was tested. The performance of two such platforms, the Fujifilm QuickGene-Mini80™ and Promega Maxwell®16 was compared to a widely used manual extraction kit, MOBIO PowerBiofilm™ DNA Isolation Kit, in terms of ease of operation, DNA quality, and microbial community composition. Three pipeline biofilm samples were chosen for these comparisons; two contained crude oil and corrosion products and the third transported seawater. Overall, the two more automated extraction platforms produced higher DNA yields than the manual approach. DNA quality was evaluated for amplification by quantitative PCR (qPCR) and end-point PCR to generate 454 pyrosequencing libraries for 16S rRNA microbial community analysis. Microbial community structure, as assessed by DGGE analysis and pyrosequencing, was comparable among the three extraction methods. Therefore, the use of automated extraction platforms should enhance the feasibility of rapidly evaluating microbial biofouling at remote locations or those with limited resources. PMID:23168231

Rapid discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) analysis.

PubMed

Zianni, Michael R; Nikbakhtzadeh, Mahmood R; Jackson, Bryan T; Panescu, Jenny; Foster, Woodbridge A

2013-04-01

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software.

Rapid Discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) Analysis

PubMed Central

Zianni, Michael R.; Nikbakhtzadeh, Mahmood R.; Jackson, Bryan T.; Panescu, Jenny; Foster, Woodbridge A.

2013-01-01

There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software. PMID:23543777

Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

PubMed

Greenspoon, S A; Sykes, K L V; Ban, J D; Pollard, A; Baisden, M; Farr, M; Graham, N; Collins, B L; Green, M M; Christenson, C C

2006-12-20

Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the software methods.

DNA and RNA profiling of excavated human remains with varying postmortem intervals.

PubMed

van den Berge, M; Wiskerke, D; Gerretsen, R R R; Tabak, J; Sijen, T

2016-11-01

When postmortem intervals (PMIs) increase such as with longer burial times, human remains suffer increasingly from the taphonomic effects of decomposition processes such as autolysis and putrefaction. In this study, various DNA analysis techniques and a messenger RNA (mRNA) profiling method were applied to examine for trends in nucleic acid degradation and the postmortem interval. The DNA analysis techniques include highly sensitive DNA quantitation (with and without degradation index), standard and low template STR profiling, insertion and null alleles (INNUL) of retrotransposable elements typing and mitochondrial DNA profiling. The used mRNA profiling system targets genes with tissue specific expression for seven human organs as reported by Lindenbergh et al. (Int J Legal Med 127:891-900, 27) and has been applied to forensic evidentiary traces but not to excavated tissues. The techniques were applied to a total of 81 brain, lung, liver, skeletal muscle, heart, kidney and skin samples obtained from 19 excavated graves with burial times ranging from 4 to 42 years. Results show that brain and heart are the organs in which both DNA and RNA remain remarkably stable, notwithstanding long PMIs. The other organ tissues either show poor overall profiling results or vary for DNA and RNA profiling success, with sometimes DNA and other times RNA profiling being more successful. No straightforward relations were observed between nucleic acid profiling results and the PMI. This study shows that not only DNA but also RNA molecules can be remarkably stable and used for profiling of long-buried human remains, which corroborate forensic applications. The insight that the brain and heart tissues tend to provide the best profiling results may change sampling policies in identification cases of degrading cadavers.

PISMA: A Visual Representation of Motif Distribution in DNA Sequences.

PubMed

Alcántara-Silva, Rogelio; Alvarado-Hermida, Moisés; Díaz-Contreras, Gibrán; Sánchez-Barrios, Martha; Carrera, Samantha; Galván, Silvia Carolina

2017-01-01

Because the graphical presentation and analysis of motif distribution can provide insights for experimental hypothesis, PISMA aims at identifying motifs on DNA sequences, counting and showing them graphically. The motif length ranges from 2 to 10 bases, and the DNA sequences range up to 10 kb. The motif distribution is shown as a bar-code-like, as a gene-map-like, and as a transcript scheme. We obtained graphical schemes of the CpG site distribution from 91 human papillomavirus genomes. Also, we present 2 analyses: one of DNA motifs associated with either methylation-resistant or methylation-sensitive CpG islands and another analysis of motifs associated with exosome RNA secretion. PISMA is developed in Java; it is executable in any type of hardware and in diverse operating systems. PISMA is freely available to noncommercial users. The English version and the User Manual are provided in Supplementary Files 1 and 2, and a Spanish version is available at www.biomedicas.unam.mx/wp-content/software/pisma.zip and www.biomedicas.unam.mx/wp-content/pdf/manual/pisma.pdf.

Epigenetics of Ancient DNA.

PubMed

Zhenilo, S V; Sokolov, A S; Prokhortchouk, E B

2016-01-01

Initially, the study of DNA isolated from ancient specimens had been based on the analysis of the primary nucleotide sequence. This approach has allowed researchers to study the evolutionary changes that occur in different populations and determine the influence of the environment on genetic selection. However, the improvement of methodological approaches to genome-wide analysis has opened up new possibilities in the search for the epigenetic mechanisms involved in the regulation of gene expression. It was discovered recently that the methylation status of the regulatory elements of the HOXD cluster and MEIS 1 gene changed during human evolution. Epigenetic changes in these genes played a key role in the evolution of the limbs of modern humans. Recent works have demonstrated that it is possible to determine the transcriptional activity of genes in ancient DNA samples by combining information on DNA methylation and the DNAaseI hypersensitive sequences located at the transcription start sites of genes. In the nearest future, if a preserved fossils brain is found, it will be possible to identify the evolutionary changes in the higher nervous system associated with epigenetic differences.

Correlation dynamics and enhanced signals for the identification of serial biomolecules and DNA bases.

PubMed

Ahmed, Towfiq; Haraldsen, Jason T; Rehr, John J; Di Ventra, Massimiliano; Schuller, Ivan; Balatsky, Alexander V

2014-03-28

Nanopore-based sequencing has demonstrated a significant potential for the development of fast, accurate, and cost-efficient fingerprinting techniques for next generation molecular detection and sequencing. We propose a specific multilayered graphene-based nanopore device architecture for the recognition of single biomolecules. Molecular detection and analysis can be accomplished through the detection of transverse currents as the molecule or DNA base translocates through the nanopore. To increase the overall signal-to-noise ratio and the accuracy, we implement a new 'multi-point cross-correlation' technique for identification of DNA bases or other molecules on the single molecular level. We demonstrate that the cross-correlations between each nanopore will greatly enhance the transverse current signal for each molecule. We implement first-principles transport calculations for DNA bases surveyed across a multilayered graphene nanopore system to illustrate the advantages of the proposed geometry. A time-series analysis of the cross-correlation functions illustrates the potential of this method for enhancing the signal-to-noise ratio. This work constitutes a significant step forward in facilitating fingerprinting of single biomolecules using solid state technology.

PISMA: A Visual Representation of Motif Distribution in DNA Sequences

PubMed Central

Alcántara-Silva, Rogelio; Alvarado-Hermida, Moisés; Díaz-Contreras, Gibrán; Sánchez-Barrios, Martha; Carrera, Samantha; Galván, Silvia Carolina

2017-01-01

Background: Because the graphical presentation and analysis of motif distribution can provide insights for experimental hypothesis, PISMA aims at identifying motifs on DNA sequences, counting and showing them graphically. The motif length ranges from 2 to 10 bases, and the DNA sequences range up to 10 kb. The motif distribution is shown as a bar-code–like, as a gene-map–like, and as a transcript scheme. Results: We obtained graphical schemes of the CpG site distribution from 91 human papillomavirus genomes. Also, we present 2 analyses: one of DNA motifs associated with either methylation-resistant or methylation-sensitive CpG islands and another analysis of motifs associated with exosome RNA secretion. Availability and Implementation: PISMA is developed in Java; it is executable in any type of hardware and in diverse operating systems. PISMA is freely available to noncommercial users. The English version and the User Manual are provided in Supplementary Files 1 and 2, and a Spanish version is available at www.biomedicas.unam.mx/wp-content/software/pisma.zip and www.biomedicas.unam.mx/wp-content/pdf/manual/pisma.pdf. PMID:28469418

[DNA extraction from bones and teeth using AutoMate Express forensic DNA extraction system].

PubMed

Gao, Lin-Lin; Xu, Nian-Lai; Xie, Wei; Ding, Shao-Cheng; Wang, Dong-Jing; Ma, Li-Qin; Li, You-Ying

2013-04-01

To explore a new method in order to extract DNA from bones and teeth automatically. Samples of 33 bones and 15 teeth were acquired by freeze-mill method and manual method, respectively. DNA materials were extracted and quantified from the triturated samples by AutoMate Express forensic DNA extraction system. DNA extraction from bones and teeth were completed in 3 hours using the AutoMate Express forensic DNA extraction system. There was no statistical difference between the two methods in the DNA concentration of bones. Both bones and teeth got the good STR typing by freeze-mill method, and the DNA concentration of teeth was higher than those by manual method. AutoMate Express forensic DNA extraction system is a new method to extract DNA from bones and teeth, which can be applied in forensic practice.

Qualitative and quantitative assessment of Illumina's forensic STR and SNP kits on MiSeq FGx™.

PubMed

Sharma, Vishakha; Chow, Hoi Yan; Siegel, Donald; Wurmbach, Elisa

2017-01-01

Massively parallel sequencing (MPS) is a powerful tool transforming DNA analysis in multiple fields ranging from medicine, to environmental science, to evolutionary biology. In forensic applications, MPS offers the ability to significantly increase the discriminatory power of human identification as well as aid in mixture deconvolution. However, before the benefits of any new technology can be employed, a thorough evaluation of its quality, consistency, sensitivity, and specificity must be rigorously evaluated in order to gain a detailed understanding of the technique including sources of error, error rates, and other restrictions/limitations. This extensive study assessed the performance of Illumina's MiSeq FGx MPS system and ForenSeq™ kit in nine experimental runs including 314 reaction samples. In-depth data analysis evaluated the consequences of different assay conditions on test results. Variables included: sample numbers per run, targets per run, DNA input per sample, and replications. Results are presented as heat maps revealing patterns for each locus. Data analysis focused on read numbers (allele coverage), drop-outs, drop-ins, and sequence analysis. The study revealed that loci with high read numbers performed better and resulted in fewer drop-outs and well balanced heterozygous alleles. Several loci were prone to drop-outs which led to falsely typed homozygotes and therefore to genotype errors. Sequence analysis of allele drop-in typically revealed a single nucleotide change (deletion, insertion, or substitution). Analyses of sequences, no template controls, and spurious alleles suggest no contamination during library preparation, pooling, and sequencing, but indicate that sequencing or PCR errors may have occurred due to DNA polymerase infidelities. Finally, we found utilizing Illumina's FGx System at recommended conditions does not guarantee 100% outcomes for all samples tested, including the positive control, and required manual editing due to low read numbers and/or allele drop-in. These findings are important for progressing towards implementation of MPS in forensic DNA testing.

Growing trend of CE at the omics level: the frontier of systems biology--an update.

PubMed

Ban, Eunmi; Park, Soo Hyun; Kang, Min-Jung; Lee, Hyun-Jung; Song, Eun Joo; Yoo, Young Sook

2012-01-01

Omics is the study of proteins, peptides, genes, and metabolites in living organisms. Systems biology aims to understand the system through the study of the relationship between elements such as genes and proteins in biological system. Recently, systems biology emerged as the result of the advanced development of high-throughput analysis technologies such as DNA sequencers, DNA arrays, and mass spectrometry for omics studies. Among a number of analytical tools and technologies, CE and CE coupled to MS are promising and relatively rapidly developing tools with the potential to provide qualitative and quantitative analyses of biological molecules. With an emphasis on CE for systems biology, this review summarizes the method developments and applications of CE for the genomic, transcriptomic, proteomic, and metabolomic studies focusing on the drug discovery and disease diagnosis and therapies since 2009. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Pre-Steady-State Kinetic Analysis of Single-Nucleotide Incorporation by DNA Polymerases

PubMed Central

Su, Yan; Guengerich, F. Peter

2016-01-01

Pre-steady-state kinetic analysis is a powerful and widely used method to obtain multiple kinetic parameters. This protocol provides a step-by-step procedure for pre-steady-state kinetic analysis of single-nucleotide incorporation by a DNA polymerase. It describes the experimental details of DNA substrate annealing, reaction mixture preparation, handling of the RQF-3 rapid quench-flow instrument, denaturing polyacrylamide DNA gel preparation, electrophoresis, quantitation, and data analysis. The core and unique part of this protocol is the rationale for preparation of the reaction mixture (the ratio of the polymerase to the DNA substrate) and methods for conducting pre-steady-state assays on an RQF-3 rapid quench-flow instrument, as well as data interpretation after analysis. In addition, the methods for the DNA substrate annealing and DNA polyacrylamide gel preparation, electrophoresis, quantitation and analysis are suitable for use in other studies. PMID:27248785

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

PubMed

Keyamura, Kenji; Katayama, Tsutomu

2011-08-19

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

PubMed Central

Keyamura, Kenji; Katayama, Tsutomu

2011-01-01

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

Illustrative cases for monitoring by quantitative analysis of BRAF/NRAS ctDNA mutations in liquid biopsies of metastatic melanoma patients who gained clinical benefits from anti-PD1 antibody therapy.

PubMed

Seremet, Teofila; Planken, Simon; Schreuer, Max; Jansen, Yanina; Delaunoy, Mélanie; El Housni, Hakim; Lienard, Danielle; Del Marmol, Véronique; Heimann, Pierre; Neyns, Bart

2018-02-01

Anti-programmed death 1 (PD-1) monoclonal antibodies improve the survival of metastatic melanoma patients. Predictive or monitoring biomarkers for response to this therapy could improve the clinical management of these patients. To date, no established biomarkers are available for monitoring the response to immunotherapy. Tumor- specific mutations in circulating tumor DNA (ctDNA) such as BRAF and NRAS mutations for melanoma patients have been proposed for monitoring of immunotherapy response. We present seven illustrative cases for the use of ctDNA BRAF and NRAS mutations' monitoring in plasma. The cases described exemplify four distinct clinical benefit patterns: rapid and durable complete response (CR), early progression, followed by CR, CR followed by early progression after interrupting treatment and long-term disease stabilization. These representative cases suggest that comprehensive BRAF/NRAS ctDNA monitoring during anti-PD1 therapy is informative and can be of added value for the monitoring of melanoma patients gaining clinical benefit on anti-PD1 treatment. An important advantage of our approach is that using the cartridge system on the Idylla platform for mutation analysis, the results become available the same day 2 h after plasma collection. Therefore, in the future, the ctDNA level can be an element in the clinical management of the patients.

Abstractions for DNA circuit design.

PubMed

Lakin, Matthew R; Youssef, Simon; Cardelli, Luca; Phillips, Andrew

2012-03-07

DNA strand displacement techniques have been used to implement a broad range of information processing devices, from logic gates, to chemical reaction networks, to architectures for universal computation. Strand displacement techniques enable computational devices to be implemented in DNA without the need for additional components, allowing computation to be programmed solely in terms of nucleotide sequences. A major challenge in the design of strand displacement devices has been to enable rapid analysis of high-level designs while also supporting detailed simulations that include known forms of interference. Another challenge has been to design devices capable of sustaining precise reaction kinetics over long periods, without relying on complex experimental equipment to continually replenish depleted species over time. In this paper, we present a programming language for designing DNA strand displacement devices, which supports progressively increasing levels of molecular detail. The language allows device designs to be programmed using a common syntax and then analysed at varying levels of detail, with or without interference, without needing to modify the program. This allows a trade-off to be made between the level of molecular detail and the computational cost of analysis. We use the language to design a buffered architecture for DNA devices, capable of maintaining precise reaction kinetics for a potentially unbounded period. We test the effectiveness of buffered gates to support long-running computation by designing a DNA strand displacement system capable of sustained oscillations.

Testing the interaction between analytical modules: an example with Roundup Ready® soybean line GTS 40-3-2

PubMed Central

2010-01-01

Background The modular approach to analysis of genetically modified organisms (GMOs) relies on the independence of the modules combined (i.e. DNA extraction and GM quantification). The validity of this assumption has to be proved on the basis of specific performance criteria. Results An experiment was conducted using, as a reference, the validated quantitative real-time polymerase chain reaction (PCR) module for detection of glyphosate-tolerant Roundup Ready® GM soybean (RRS). Different DNA extraction modules (CTAB, Wizard and Dellaporta), were used to extract DNA from different food/feed matrices (feed, biscuit and certified reference material [CRM 1%]) containing the target of the real-time PCR module used for validation. Purity and structural integrity (absence of inhibition) were used as basic criteria that a DNA extraction module must satisfy in order to provide suitable template DNA for quantitative real-time (RT) PCR-based GMO analysis. When performance criteria were applied (removal of non-compliant DNA extracts), the independence of GMO quantification from the extraction method and matrix was statistically proved, except in the case of Wizard applied to biscuit. A fuzzy logic-based procedure also confirmed the relatively poor performance of the Wizard/biscuit combination. Conclusions For RRS, this study recognises that modularity can be generally accepted, with the limitation of avoiding combining highly processed material (i.e. biscuit) with a magnetic-beads system (i.e. Wizard). PMID:20687918

Improved Force Fields for Peptide Nucleic Acids with Optimized Backbone Torsion Parameters.

PubMed

Jasiński, Maciej; Feig, Michael; Trylska, Joanna

2018-06-06

Peptide nucleic acids are promising nucleic acid analogs for antisense therapies as they can form stable duplex and triplex structures with DNA and RNA. Computational studies of PNA-containing duplexes and triplexes are an important component for guiding their design, yet existing force fields have not been well validated and parametrized with modern computational capabilities. We present updated CHARMM and Amber force fields for PNA that greatly improve the stability of simulated PNA-containing duplexes and triplexes in comparison with experimental structures and allow such systems to be studied on microsecond time scales. The force field modifications focus on reparametrized PNA backbone torsion angles to match high-level quantum mechanics reference energies for a model compound. The microsecond simulations of PNA-PNA, PNA-DNA, PNA-RNA, and PNA-DNA-PNA complexes also allowed a comprehensive analysis of hydration and ion interactions with such systems.

Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses.

PubMed

Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; Yano, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

2015-01-01

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources.

Identification of Human N-Myristoylated Proteins from Human Complementary DNA Resources by Cell-Free and Cellular Metabolic Labeling Analyses

PubMed Central

Takamitsu, Emi; Otsuka, Motoaki; Haebara, Tatsuki; Yano, Manami; Matsuzaki, Kanako; Kobuchi, Hirotsugu; Moriya, Koko; Utsumi, Toshihiko

2015-01-01

To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude known human N-myristoylated proteins, 37 cDNA clones were selected as potential human N-myristoylated proteins. The susceptibility of these cDNA clones to protein N-myristoylation was first evaluated using fusion proteins in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. Then, protein N-myristoylation of the gene products of full-length cDNAs was evaluated by metabolic labeling experiments both in an insect cell-free protein synthesis system and in transfected human cells. As a result, the products of 13 cDNA clones (FBXL7, PPM1B, SAMM50, PLEKHN, AIFM3, C22orf42, STK32A, FAM131C, DRICH1, MCC1, HID1, P2RX5, STK32B) were found to be human N-myristoylated proteins. Analysis of the role of protein N-myristoylation on the intracellular localization of SAMM50, a mitochondrial outer membrane protein, revealed that protein N-myristoylation was required for proper targeting of SAMM50 to mitochondria. Thus, the strategy used in this study is useful for the identification of physiologically important human N-myristoylated proteins from human cDNA resources. PMID:26308446

In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse

PubMed Central

Morselli, Marco; Pastor, William A; Montanini, Barbara; Nee, Kevin; Ferrari, Roberto; Fu, Kai; Bonora, Giancarlo; Rubbi, Liudmilla; Clark, Amander T; Ottonello, Simone; Jacobsen, Steven E; Pellegrini, Matteo

2015-01-01

Methylation of cytosines (5meC) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5meC levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. DOI: http://dx.doi.org/10.7554/eLife.06205.001 PMID:25848745

[A fluoride-sensor for kink structure in DNA condensation process].

PubMed

Liu, Yan-Hui; Zhang, Jing; Chen, Ying-Bing; Li, Yu-Pu; Hu, Lin

2014-01-01

Bloomfield has pointed out that the kink structure occurs for sharp bending during DNA condensation process, until now, which has not been proved by experiments. Using UV Spectrophotometer, the effects of fluoride and chlorine on the polyamine-DNA condensation system can be detected. Fluoride and chlorine both belong to the halogen family, but their effects on spermine-DNA condensation system are totally different. Fluoride ions make blue-shift and hyperchromicity appear in the spermine-DNA condensation system, but chlorine ions only make insignificant hyperchromicity happen in this system. Both fluoride ions and chlorine ions only make insignificant hyperchromicity happen in spermidine-DNA condensation system. Based on the distinguished character of fluoride, a fluoride-sensor for "kink" structure in DNA condensation was developed and the second kind of "kink" structure only appear in the spermine-DNA condensation system.

Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria

DOE PAGES

Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.; ...

2016-04-29

Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA genemore » sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.« less

Seasonal succession leads to habitat-dependent differentiation in ribosomal RNA:DNA ratios among freshwater lake bacteria

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

Denef, Vincent J.; Fujimoto, Masanori; Berry, Michelle A.

Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA genemore » sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. Furthermore, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors.« less

Dangers resulting from DNA profiling of biological materials derived from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with regard to forensic genetic analysis.

PubMed

Jacewicz, R; Lewandowski, K; Rupa-Matysek, J; Jędrzejczyk, M; Berent, J

The study documents the risk that comes with DNA analysis of materials derived from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in forensic genetics. DNA chimerism was studied in 30 patients after allo-HSCT, based on techniques applied in contemporary forensic genetics, i.e. real-time PCR and multiplex PCR-STR with the use of autosomal DNA as well as Y-DNA markers. The results revealed that the DNA profile of the recipient's blood was identical with the donor's in the majority of cases. Therefore, blood analysis can lead to false conclusions in personal identification as well as kinship analysis. An investigation of buccal swabs revealed a mixture of DNA in the majority of recipients. Consequently, personal identification on the basis of stain analysis of the same origin may be impossible. The safest (but not ideal) material turned out to be the hair root. Its analysis based on autosomal DNA revealed 100% of the recipient's profile. However, an analysis based on Y-chromosome markers performed in female allo-HSCT recipients with male donors demonstrated the presence of donor DNA in hair cells - similarly to the blood and buccal swabs. In the light of potential risks arising from DNA profiling of biological materials derived from persons after allotransplantation in judicial aspects, certain procedures were proposed to eliminate such dangers. The basic procedures include abandoning the approach based exclusively on blood collection, both for kinship analysis and personal identification; asking persons who are to be tested about their history of allo-HSCT before sample collection and profile entry in the DNA database, and verification of DNA profiling based on hair follicles in uncertain cases.

Cluster analysis of autoantibodies in 852 patients with systemic lupus erythematosus from a single center.

PubMed

Artim-Esen, Bahar; Çene, Erhan; Şahinkaya, Yasemin; Ertan, Semra; Pehlivan, Özlem; Kamali, Sevil; Gül, Ahmet; Öcal, Lale; Aral, Orhan; Inanç, Murat

2014-07-01

Associations between autoantibodies and clinical features have been described in systemic lupus erythematosus (SLE). Herein, we aimed to define autoantibody clusters and their clinical correlations in a large cohort of patients with SLE. We analyzed 852 patients with SLE who attended our clinic. Seven autoantibodies were selected for cluster analysis: anti-DNA, anti-Sm, anti-RNP, anticardiolipin (aCL) immunoglobulin (Ig)G or IgM, lupus anticoagulant (LAC), anti-Ro, and anti-La. Two-step clustering and Kaplan-Meier survival analyses were used. Five clusters were identified. A cluster consisted of patients with only anti-dsDNA antibodies, a cluster of anti-Sm and anti-RNP, a cluster of aCL IgG/M and LAC, and a cluster of anti-Ro and anti-La antibodies. Analysis revealed 1 more cluster that consisted of patients who did not belong to any of the clusters formed by antibodies chosen for cluster analysis. Sm/RNP cluster had significantly higher incidence of pulmonary hypertension and Raynaud phenomenon. DsDNA cluster had the highest incidence of renal involvement. In the aCL/LAC cluster, there were significantly more patients with neuropsychiatric involvement, antiphospholipid syndrome, autoimmune hemolytic anemia, and thrombocytopenia. According to the Systemic Lupus International Collaborating Clinics damage index, the highest frequency of damage was in the aCL/LAC cluster. Comparison of 10 and 20 years survival showed reduced survival in the aCL/LAC cluster. This study supports the existence of autoantibody clusters with distinct clinical features in SLE and shows that forming clinical subsets according to autoantibody clusters may be useful in predicting the outcome of the disease. Autoantibody clusters in SLE may exhibit differences according to the clinical setting or population.

78 FR 70075 - Notice of Permit Applications Received Under the Antarctic Conservation Act of 1978 (Public Law...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-22

... establishment of a permit system for various activities in Antarctica and designation of certain animals and certain geographic areas a requiring special protection. The regulations establish such a permit system to..., stable isotope analysis, and DNA extraction. Data would be used to reconstruct seal population dynamics...

Assessing the potential of surface-immobilized molecular logic machines for integration with solid state technology.

PubMed

Dunn, Katherine E; Trefzer, Martin A; Johnson, Steven; Tyrrell, Andy M

2016-08-01

Molecular computation with DNA has great potential for low power, highly parallel information processing in a biological or biochemical context. However, significant challenges remain for the field of DNA computation. New technology is needed to allow multiplexed label-free readout and to enable regulation of molecular state without addition of new DNA strands. These capabilities could be provided by hybrid bioelectronic systems in which biomolecular computing is integrated with conventional electronics through immobilization of DNA machines on the surface of electronic circuitry. Here we present a quantitative experimental analysis of a surface-immobilized OR gate made from DNA and driven by strand displacement. The purpose of our work is to examine the performance of a simple representative surface-immobilized DNA logic machine, to provide valuable information for future work on hybrid bioelectronic systems involving DNA devices. We used a quartz crystal microbalance to examine a DNA monolayer containing approximately 5×10(11)gatescm(-2), with an inter-gate separation of approximately 14nm, and we found that the ensemble of gates took approximately 6min to switch. The gates could be switched repeatedly, but the switching efficiency was significantly degraded on the second and subsequent cycles when the binding site for the input was near to the surface. Otherwise, the switching efficiency could be 80% or better, and the power dissipated by the ensemble of gates during switching was approximately 0.1nWcm(-2), which is orders of magnitude less than the power dissipated during switching of an equivalent array of transistors. We propose an architecture for hybrid DNA-electronic systems in which information can be stored and processed, either in series or in parallel, by a combination of molecular machines and conventional electronics. In this architecture, information can flow freely and in both directions between the solution-phase and the underlying electronics via surface-immobilized DNA machines that provide the interface between the molecular and electronic domains. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Automated Microfluidic Platform for Serial Polymerase Chain Reaction and High-Resolution Melting Analysis.

PubMed

Cao, Weidong; Bean, Brian; Corey, Scott; Coursey, Johnathan S; Hasson, Kenton C; Inoue, Hiroshi; Isano, Taisuke; Kanderian, Sami; Lane, Ben; Liang, Hongye; Murphy, Brian; Owen, Greg; Shinoda, Nobuhiko; Zeng, Shulin; Knight, Ivor T

2016-06-01

We report the development of an automated genetic analyzer for human sample testing based on microfluidic rapid polymerase chain reaction (PCR) with high-resolution melting analysis (HRMA). The integrated DNA microfluidic cartridge was used on a platform designed with a robotic pipettor system that works by sequentially picking up different test solutions from a 384-well plate, mixing them in the tips, and delivering mixed fluids to the DNA cartridge. A novel image feedback flow control system based on a Canon 5D Mark II digital camera was developed for controlling fluid movement through a complex microfluidic branching network without the use of valves. The same camera was used for measuring the high-resolution melt curve of DNA amplicons that were generated in the microfluidic chip. Owing to fast heating and cooling as well as sensitive temperature measurement in the microfluidic channels, the time frame for PCR and HRMA was dramatically reduced from hours to minutes. Preliminary testing results demonstrated that rapid serial PCR and HRMA are possible while still achieving high data quality that is suitable for human sample testing. © 2015 Society for Laboratory Automation and Screening.

Development of micropump-actuated negative pressure pinched injection for parallel electrophoresis on array microfluidic chip.

PubMed

Li, Bowei; Jiang, Lei; Xie, Hua; Gao, Yan; Qin, Jianhua; Lin, Bingcheng

2009-09-01

A micropump-actuated negative pressure pinched injection method is developed for parallel electrophoresis on a multi-channel LIF detection system. The system has a home-made device that could individually control 16-port solenoid valves and a high-voltage power supply. The laser beam is excitated and distributes to the array separation channels for detection. The hybrid Glass-PDMS microfluidic chip comprises two common reservoirs, four separation channels coupled to their respective pneumatic micropumps and two reference channels. Due to use of pressure as a driving force, the proposed method has no sample bias effect for separation. There is only one high-voltage supply needed for separation without relying on the number of channels, which is significant for high-throughput analysis, and the time for sample loading is shortened to 1 s. In addition, the integrated micropumps can provide the versatile interface for coupling with other function units to satisfy the complicated demands. The performance is verified by separation of DNA marker and Hepatitis B virus DNA samples. And this method is also expected to show the potential throughput for the DNA analysis in the field of disease diagnosis.

Phylogeny and classification of bacteria in the genera Clavibacter and Rathayibacter on the basis of 16s rRNA gene sequence analyses.

PubMed

Lee, I M; Bartoszyk, I M; Gundersen-Rindal, D E; Davis, R E

1997-07-01

A phylogenetic analysis by parsimony of 16S rRNA gene sequences (16S rDNA) revealed that species and subspecies of Clavibacter and Rathayibacter form a discrete monophyletic clade, paraphyletic to Corynebacterium species. Within the Clavibacter-Rathayibacter clade, four major phylogenetic groups (subclades) with a total of 10 distinct taxa were recognized: (I) species C. michiganensis; (II) species C. xyli; (III) species R. iranicus and R. tritici; and (IV) species R. rathayi. The first three groups form a monophyletic cluster, paraphyletic to R. rathayi. On the basis of the phylogeny inferred, reclassification of members of Clavibacter-Rathayibacter group is proposed. A system for classification of taxa in Clavibacter and Rathayibacter was developed based on restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S rDNA sequences. The groups delineated on the basis of RFLP patterns of 16S rDNA coincided well with the subclades delineated on the basis of phylogeny. In contrast to previous classification systems, which are based primarily on phenotypic properties and are laborious, the RFLP analyses allow for rapid differentiation among species and subspecies in the two genera.

Targeting human c-Myc promoter duplex DNA with actinomycin D by use of multi-way analysis of quantum-dot-mediated fluorescence resonance energy transfer.

PubMed

Gholami, Somayeh; Kompany-Zareh, Mohsen

2013-07-01

Actinomycin D (Act D), an oncogenic c-Myc promoter binder, interferes with the action of RNA polymerase. There is great demand for high-throughput technology able to monitor the activity of DNA-binding drugs. To this end, binding of 7-aminoactinomycin D (7AAD) to the duplex c-Myc promoter was investigated by use of 2D-photoluminescence emission (2D-PLE), and the resulting data were subjected to analysis by use of convenient and powerful multi-way approaches. Fluorescence measurements were performed by use of the quantum dot (QD)-conjugated c-Myc promoter. Intercalation of 7AAD within duplex base pairs resulted in efficient energy transfer from drug to QD via fluorescence resonance energy transfer (FRET). Multi-way analysis of the three-way data array obtained from titration experiments was performed by use of restricted Tucker3 and hard trilinear decomposition (HTD). These techniques enable analysis of high-dimensional and complex data from nanobiological systems which include several spectrally overlapped structures. It was almost impossible to obtain robust and meaningful information about the FRET process for such high overlap data by use of classical analysis. The soft approach had the important advantage over univariate classical methods of enabling us to investigate the source of variance in the fluorescence signal of the DNA-drug complex. It was established that hard trilinear decomposition analysis of FRET-measured data overcomes the problem of rank deficiency, enabling calculation of concentration profiles and pure spectra for all species, including non-fluorophores. The hard modeling approach was also used for determination of equilibrium constants for the hybridization and intercalation equilibria, using nonlinear fit data analysis. The intercalation constant 3.6 × 10(6) mol(-1) L and hybridization stability 1.0 × 10(8) mol(-1) L obtained were in good agreement with values reported in the literature. The analytical concentration of the QD-labeled DNA was determined by use of nonlinear fitting, without using external standard calibration samples. This study was a successful application of multi-way chemometric methods to investigation of nano-biotechnological systems where several overlapped species coexist in solution.

Traceability of biotech-derived animals: application of DNA technology.

PubMed

Loftus, R

2005-04-01

Traceability is increasingly becoming standard across the agri-food industry, largely driven by recent food crises and the consequent demands for transparency within the food chain. This is leading to the development of a range of traceability concepts and technologies adapted to different industry needs. Experience with genetically modified plants has shown that traceability can play a role in increasing public confidence in biotechnology, and might similarly help allay concerns relating to the development of animal biotechnology. Traceability also forms an essential component of any risk management strategy and is a key requirement for post-marketing surveillance. Given the diversity of traceability concepts and technologies available, consideration needs to be given to the scope and precision of traceability systems for animal biotechnology. Experience to date has shown that conventional tagging and labelling systems can incorporate levels of error and may not have sufficient precision for biotech-derived animals. Deoxyribonucleic acid (DNA) technology can overcome these difficulties by tracing animals and animal by-products through their DNA code rather than an associated label. This offers the possibility of tracing some by-products of animal biotechnology through the supply chain back to source animals, offering unprecedented levels of traceability. Developments in both DNA sampling and analysis technology are making large-scale applications of DNA traceability increasingly cost effective and feasible, and are likely to lead to a broader uptake of DNA traceability concepts.

Multicenter Evaluation of Epidemiological Typing of Methicillin-Resistant Staphylococcus aureus Strains by Repetitive-Element PCR Analysis

PubMed Central

Deplano, Ariane; Schuermans, Annette; Van Eldere, Johan; Witte, Wolfgang; Meugnier, Hèléne; Etienne, Jerome; Grundmann, Hajo; Jonas, Daniel; Noordhoek, Gerda T.; Dijkstra, Jolanda; van Belkum, Alex; van Leeuwen, Willem; Tassios, Panayotis T.; Legakis, Nicholas J.; van der Zee, Anneke; Bergmans, Anneke; Blanc, Dominique S.; Tenover, Fred C.; Cookson, Barry C.; O'Neil, Gael; Struelens, Marc J.

2000-01-01

Rapid and efficient epidemiologic typing systems would be useful to monitor transmission of methicillin-resistant Staphylococcus aureus (MRSA) at both local and interregional levels. To evaluate the intralaboratory performance and interlaboratory reproducibility of three recently developed repeat-element PCR (rep-PCR) methods for the typing of MRSA, 50 MRSA strains characterized by pulsed-field gel electrophoresis (PFGE) (SmaI) analysis and epidemiological data were blindly typed by inter-IS256, 16S-23S ribosomal DNA (rDNA), and MP3 PCR in 12 laboratories in eight countries using standard reagents and protocols. Performance of typing was defined by reproducibility (R), discriminatory power (D), and agreement with PFGE analysis. Interlaboratory reproducibility of pattern and type classification was assessed visually and using gel analysis software. Each typing method showed a different performance level in each center. In the center performing best with each method, inter-IS256 PCR typing achieved R = 100% and D = 100%; 16S-23S rDNA PCR, R = 100% and D = 82%; and MP3 PCR, R = 80% and D = 83%. Concordance between rep-PCR type and PFGE type ranged by center: 70 to 90% for inter-IS256 PCR, 44 to 57% for 16S-23S rDNA PCR, and 53 to 54% for MP3 PCR analysis. In conclusion, the performance of inter-IS256 PCR typing was similar to that of PFGE analysis in some but not all centers, whereas other rep-PCR protocols showed lower discrimination and intralaboratory reproducibility. None of these assays, however, was sufficiently reproducible for interlaboratory exchange of data. PMID:11015358

Aging effects on DNA methylation modules in human brain and blood tissue

PubMed Central

2012-01-01

Background Several recent studies reported aging effects on DNA methylation levels of individual CpG dinucleotides. But it is not yet known whether aging-related consensus modules, in the form of clusters of correlated CpG markers, can be found that are present in multiple human tissues. Such a module could facilitate the understanding of aging effects on multiple tissues. Results We therefore employed weighted correlation network analysis of 2,442 Illumina DNA methylation arrays from brain and blood tissues, which enabled the identification of an age-related co-methylation module. Module preservation analysis confirmed that this module can also be found in diverse independent data sets. Biological evaluation showed that module membership is associated with Polycomb group target occupancy counts, CpG island status and autosomal chromosome location. Functional enrichment analysis revealed that the aging-related consensus module comprises genes that are involved in nervous system development, neuron differentiation and neurogenesis, and that it contains promoter CpGs of genes known to be down-regulated in early Alzheimer's disease. A comparison with a standard, non-module based meta-analysis revealed that selecting CpGs based on module membership leads to significantly increased gene ontology enrichment, thus demonstrating that studying aging effects via consensus network analysis enhances the biological insights gained. Conclusions Overall, our analysis revealed a robustly defined age-related co-methylation module that is present in multiple human tissues, including blood and brain. We conclude that blood is a promising surrogate for brain tissue when studying the effects of age on DNA methylation profiles. PMID:23034122

DNA Damage and Oxidative Stress in Dyskeratosis Congenita: Analysis of Pathways and Therapeutic Stategies Using CPISPR and iPSC Model Systems

DTIC Science & Technology

2017-06-01

Milestone Achieved: HRPO/ACURO Approval 6 Finished Major Task 2 CRISPR knockout/RNAseq Viral infection/prep 3-6 CRISPR KO virus library prep...finished; RNA-Seq: ~75% Cell manipulation 3-6 CRISPR KO virus infection: 50%; Single cDNA infections: finished Bioinformatics 1 CRISPR KO library...characterization 1-3 Finished Update: production of iPSC clones harboring DC mutations generated by CRISPR : Design 1 Finished Update: production of

Terahertz Sensing of Materials

NASA Astrophysics Data System (ADS)

Xuan, G.; Ghosh, S.; Kim, S.; Lv, P.-C.; Buma, T.; Weng, B.; Barner, K.; Kolodzey, J.

2007-06-01

Biomolecules such as DNA and proteins exhibit a wealth of modes in the Terahertz (THz) range from the rotational, vibrational and stretching modes of biomolecules. Many materials such as drywall that are opaque to human eyes are transparent to THz. Therefore, it can be used as a powerful tool for biomolecular sensing, biomedical analysis and through-the-wall imaging. Experiments were carried out to study the absorption of various materials including DNA and see-through imaging of drywall using FTIR spectrometer and Time Domain Spectroscopy (TDS) system.

Generation of DNA profiles from fingerprints developed with columnar thin film technique.

PubMed

Plazibat, Stephanie L; Roy, Reena; Swiontek, Stephen E; Lakhtakia, Akhlesh

2015-12-01

Partial-bloody fingerprints and partial fingerprints with saliva are often encountered at crime scenes, potentially enabling the combination of fingerprint and DNA analyses for absolute identification, provided that the development technique for fingerprint analysis does not inhibit DNA analysis. 36 partial-bloody fingerprints and 30 fingerprints wetted with saliva, all deposited on brass, were first developed using the columnar-thin-film (CTF) technique and then subjected to short tandem repeat (STR) DNA analysis. Equal numbers of samples were subjected to the same DNA analysis without development. Tris (8-hydroxyquinolinato) aluminum, or Alq3, was evaporated to deposit CTFs for development of the prints. DNA was extracted from all 132 samples, quantified, and amplified with AmpFlSTR(®) Identifiler Plus Amplification Kit. Additionally, DNA analyses were conducted on four blood smears on un-fingerprinted brass that had been subjected to CTF deposition and four blood smears on un-fingerprinted brass that had not been subjected to CTF deposition. Complete and concordant autosomal STR profiles of the same quality were obtained from both undeveloped and CTF-developed fingerprints, indicating that CTF development of fingerprints preserves DNA and does not inhibit subsequent DNA analysis. Even when there were no fingerprints, CTF deposition did not lead to inhibition of DNA analysis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Cytology smears as diagnostic material for EGFR gene testing in non-small cell lung cancer.

PubMed

Powrózek, Tomasz; Krawczyk, Paweł; Pankowski, Juliusz; Reszka, Katarzyna; Jakubiak, Magdalena; Obrochta, Anna; Wojas-Krawczyk, Kamila; Buczkowski, Jarosław; Milanowski, Janusz

2015-11-14

Cytology smears can be effectively used for EGFR mutation testing in the qualification of NSCLC patients for EGFR tyrosine kinase inhibitor therapy. However, tissue specimens are preferred for EGFR mutation analysis. The aim of this study was to estimate the effectiveness of the real-time PCR method for EGFR testing in histology and cytology materials obtained simultaneously from NSCLC patients. Fourteen adenocarcinoma patients with EGFR-mutation-positive primary tumor tissues were included in the study. Corresponding cytological smears of metastatic lymph nodes obtained by EBUS-TBNA were examined. EGFR Mutation Analysis Kit (EntroGen, USA) and real-time PCR (m2000rt system, Abbott, USA) were used for EGFR mutation analysis in both types of material. In primary tumor tissues, 12 deletions in exon 19 and 2 substitutions in exon 21 (L858R mutation) of the EGFR gene were found. Except for 1 deletion in exon 19, the same EGFR gene mutations were detected in all corresponding cytology samples. The percentage of tumor cells, DNA concentration, percentage of mutated DNA as well as ΔCt values were similar in cytology slides and histology material. In both types of materials, no significant correlations were found between the percentage of tumor cells and the percentage of mutated DNA nor between the DNA concentration and the percentage of mutated DNA. We demonstrated the high effectiveness of a sensitive real-time PCR method in EGFR gene mutation detection in cytology smears.

DNA-mediated gold nanoparticle signal transducers for combinatorial logic operations and heavy metal ions sensing.

PubMed

Zhang, Yuhuan; Liu, Wei; Zhang, Wentao; Yu, Shaoxuan; Yue, Xiaoyue; Zhu, Wenxin; Zhang, Daohong; Wang, Yanru; Wang, Jianlong

2015-10-15

Herein, the structure of two DNA strands which are complementary except fourteen T-T and C-C mismatches was programmed for the design of the combinatorial logic operation by utilizing the different protective capacities of single chain DNA, part-hybridized DNA and completed-hybridized DNA on unmodified gold nanoparticles. In the presence of either Hg(2+) or Ag(+), the T-Hg(2+)-T or C-Ag(+)-C coordination chemistry could lead to the formation of part-hybridized DNA which keeps gold nanoparticles from clumping after the addition of 40 μL 0.2M NaClO4 solution, but the protection would be screened by 120 μL 0.2M NaClO4 solution. While the coexistence of Hg(2+), Ag(+) caused the formation of completed-hybridized DNA and the protection for gold nanoparticles lost in either 40 μL or 120 μL NaClO4 solutions. Benefiting from sharing of the same inputs of Hg(2+) and Ag(+), OR and AND logic gates were easily integrated into a simple colorimetric combinatorial logic operation in one system, which make it possible to execute logic gates in parallel to mimic arithmetic calculations on a binary digit. Furthermore, two other logic gates including INHIBIT1 and INHIBIT2 were realized to integrated with OR logic gate both for simultaneous qualitative discrimination and quantitative determination of Hg(2+) and Ag(+). Results indicate that the developed logic system based on the different protective capacities of DNA structure on gold nanoparticles provides a new pathway for the design of the combinatorial logic operation in one system and presents a useful strategy for development of advanced sensors, which may have potential applications in multiplex chemical analysis and molecular-scale computer design. Copyright © 2015 Elsevier B.V. All rights reserved.

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

PubMed Central

Leontiou, Chrysanthia A.; Hadjidaniel, Michael D.; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C.

2015-01-01

Introduction Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Methods Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. Results The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Conclusion Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions. PMID:26247357

Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing.

PubMed

Leontiou, Chrysanthia A; Hadjidaniel, Michael D; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C

2015-01-01

Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite amplicon sequencing is a suitable approach for methylation analysis of targeted regions.

Performance comparison of two commercial human whole-exome capture systems on formalin-fixed paraffin-embedded lung adenocarcinoma samples.

PubMed

Bonfiglio, Silvia; Vanni, Irene; Rossella, Valeria; Truini, Anna; Lazarevic, Dejan; Dal Bello, Maria Giovanna; Alama, Angela; Mora, Marco; Rijavec, Erika; Genova, Carlo; Cittaro, Davide; Grossi, Francesco; Coco, Simona

2016-08-30

Next Generation Sequencing (NGS) has become a valuable tool for molecular landscape characterization of cancer genomes, leading to a better understanding of tumor onset and progression, and opening new avenues in translational oncology. Formalin-fixed paraffin-embedded (FFPE) tissue is the method of choice for storage of clinical samples, however low quality of FFPE genomic DNA (gDNA) can limit its use for downstream applications. To investigate the FFPE specimen suitability for NGS analysis and to establish the performance of two solution-based exome capture technologies, we compared the whole-exome sequencing (WES) data of gDNA extracted from 5 fresh frozen (FF) and 5 matched FFPE lung adenocarcinoma tissues using: SeqCap EZ Human Exome v.3.0 (Roche NimbleGen) and SureSelect XT Human All Exon v.5 (Agilent Technologies). Sequencing metrics on Illumina HiSeq were optimal for both exome systems and comparable among FFPE and FF samples, with a slight increase of PCR duplicates in FFPE, mainly in Roche NimbleGen libraries. Comparison of single nucleotide variants (SNVs) between FFPE-FF pairs reached overlapping values >90 % in both systems. Both WES showed high concordance with target re-sequencing data by Ion PGM™ in 22 lung-cancer genes, regardless the source of samples. Exon coverage of 623 cancer-related genes revealed high coverage efficiency of both kits, proposing WES as a valid alternative to target re-sequencing. High-quality and reliable data can be successfully obtained from WES of FFPE samples starting from a relatively low amount of input gDNA, suggesting the inclusion of NGS-based tests into clinical contest. In conclusion, our analysis suggests that the WES approach could be extended to a translational research context as well as to the clinic (e.g. to study rare malignancies), where the simultaneous analysis of the whole coding region of the genome may help in the detection of cancer-linked variants.

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.

Resolving DNA-ligand intercalation in the entropic stretching regime

NASA Astrophysics Data System (ADS)

Almaqwashi, Ali A.

Single molecule studies of DNA intercalation are typically conducted by applying stretching forces to obtain force-dependent DNA elongation measurements. The zero-force properties of DNA intercalation are determined by equilibrium and kinetic force-analysis. However, the applied stretching forces that are above the entropic regime (>5 pN) prevent DNA-DNA contact which may eliminate competitive DNA-ligand interactions. In particular, it is noted that cationic mono-intercalators investigated by single molecule force spectroscopy are mostly found to intercalate DNA with single rate, while bulk studies reported additional slower rates. Here, a proposed framework quantifies DNA intercalation by cationic ligands in competition with relatively rapid kinetic DNA-ligand aggregation. At a constant applied force in the entropic stretching regime, the analysis illustrates that DNA intercalation would be measurably optimized only within a narrow range of low ligand concentrations. As DNA intercalators are considered for potential DNA-targeted therapeutics, this analysis provides insights in tuning ligand concertation to maximize therapeutics efficiency.

New concepts of fluorescent probes for specific detection of DNA sequences: bis-modified oligonucleotides in excimer and exciplex detection.

PubMed

Gbaj, A; Bichenkova, Ev; Walsh, L; Savage, He; Sardarian, Ar; Etchells, Ll; Gulati, A; Hawisa, S; Douglas, Kt

2009-12-01

The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5'-bispyrene and 3'-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5'-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5'-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.

Separation of {sup 32}P-postlabeled DNA adducts of polycyclic aromatic hydrocarbons and nitrated polycyclic aromatic hydrocarbons by HPLC

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

King, L.C.; Gallagher, J.E.; Lewtas, J.

The {sup 32}P-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO{sub 2}-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a]anthracene. The NO{sub 2}-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO{sub 2}-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. Amore » second NO{sub 2}-PAH HPLC gradient system was developed to separate NO{sub 2}-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO{sub 2}-PAH-DNA adducts were compared using both adduct enhancement versions of the {sup 32}P-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO{sub 2}-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the {sup 32}P-postlabeled DNA adduct standards (PAHs and NO{sub 2}-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO{sub 2}-PAH standards used in this study. HPLC analyses of the NO{sub 2}-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts.« less

Genome-wide DNA methylation analysis in lung fibroblasts co-cultured with silica-exposed alveolar macrophages.

PubMed

Li, Juan; Yao, Wu; Zhang, Lin; Bao, Lei; Chen, Huiting; Wang, Di; Yue, Zhongzheng; Li, Yiping; Zhang, Miao; Hao, Changfu

2017-05-12

Exposure to crystalline silica is considered to increase the risk of lung fibrosis. The primary effector cell, the myofibroblast, plays an important role in the deposition of extracellular matrix (ECM). DNA methylation change is considered to have a potential effect on myofibroblast differentiation. Therefore, the present study was designed to investigate the genome-wide DNA methylation profiles of lung fibroblasts co-cultured with alveolar macrophages exposed to crystalline silica in vitro. AM/fibroblast co-culture system was established. CCK8 was used to assess the toxicity of AMs. mRNA and protein expression of collagen I, α-SMA, MAPK9 and TGF-β1 of fibroblasts after AMs exposed to 100 μg /ml SiO 2 for 0-, 24-, or 48 h were determined by means of quantitative real-time PCR, immunoblotting and immunohistochemistry. Genomic DNA of fibroblasts was isolated using MeDIP-Seq to sequence. R software, GO, KEGG and Cytoscape were used to analyze the data. SiO 2 exposure increased the expression of collagen I and α-SMA in fibroblasts in co-culture system. Analysis of fibroblast methylome identified extensive methylation changes involved in several signaling pathways, such as the MAPK signaling pathway and metabolic pathways. Several candidates, including Tgfb1 and Mapk9, are hubs who can connect the gene clusters. MAPK9 mRNA expression was significantly higher in fibroblast exposed to SiO 2 in co-culture system for 48 h. MAPK9 protein expression was increased at both 24-h and 48-h treatment groups. TGF-β1 mRNA expression of fibroblast has a time-dependent manner, but we didn't observe the TGF-β1 protein expression. Tgfb1 and Mapk9 are helpful to explore the mechanism of myofibroblast differentiation. The genome-wide DNA methylation profiles of fibroblasts in this experimental silicosis model will be useful for future studies on epigenetic gene regulation during myofibroblast differentiation.

Beyond the cold hit: measuring the impact of the national DNA data bank on public safety at the city and county level.

PubMed

Gabriel, Matthew; Boland, Cherisse; Holt, Cydne

2010-01-01

Over the past decade, the Combined DNA Index System (CODIS) has increased solvability of violent crimes by linking evidence DNA profiles to known offenders. At present, an in-depth analysis of the United States National DNA Data Bank effort has not assessed the success of this national public safety endeavor. Critics of this effort often focus on laboratory and police investigators unable to provide timely investigative support as a root cause(s) of CODIS' failure to increase public safety. By studying a group of nearly 200 DNA cold hits obtained in SFPD criminal investigations from 2001-2006, three key performance metrics (Significance of Cold Hits, Case Progression & Judicial Resolution, and Potential Reduction of Future Criminal Activity) provide a proper context in which to define the impact of CODIS at the City and County level. Further, the analysis of a recidivist group of cold hit offenders and their past interaction with law enforcement established five noteworthy criminal case resolution trends; these trends signify challenges to CODIS in achieving meaningful case resolutions. CODIS' effectiveness and critical activities to support case resolutions are the responsibility of all criminal justice partners in order to achieve long-lasting public safety within the United States.

Skeletal Remains from World War II Mass Grave: from Discovery to Identification

PubMed Central

Definis Gojanović, Marija; Sutlović, Davorka

2007-01-01

Aim To present the process of identification of skeletal remains from a mass grave found on a Dalmatian mountain-range in 2005, which allegedly contained the remains of civilians from Herzegovina killed in the World War II, including a group of 8 Franciscan monks. Methods Excavation of the site in Dalmatian hinterland, near the village of Zagvozd, was accomplished according to archeological procedures. Anthropological analysis was performed to estimate sex, age at death, and height of the individuals, as well as pathological and traumatic changes of the bones. Due to the lack of ante-mortem data, DNA typing using Y-chromosome was performed. DNA was isolated from bones and teeth samples using standard phenol/chloroform/isoamyl alcohol extraction. Two Y-chromosome short tandem repeats (STR) systems were used for DNA quantification and amplification. Typing of polymerase chain reaction (PCR) products was performed on an ABI Prism 310 Genetic Analyzer. PCR typing results were matched with results from DNA analysis of samples collected from the relatives of supposed victims – blood samples from the living relatives and bone samples collected during further exhumation of died parents or relatives of the supposed victims. Results The remains contained 18 almost complete skeletons, with considerable post-mortal damage. All remains were men, mainly middle-aged, with gunshot wounds to the head. DNA analysis and cross-matching of the results with relatives’ data resulted in three positive identifications using the Y-chromosomal short tandem repeat (Y-STR) systems. All of the positively identified remains belonged to the Franciscan friars allegedly killed in Herzegovina and buried at the analyzed site. Conclusion Our analysis of remains from a mass grave from the World War II confirmed the value of patrilineal lineage based on Y-STRs, even when missing persons had left no offspring, as was the case with Franciscan monks. Although this report is primarily focused on the identification of remains from a mass grave, it also emphasizes the role of forensic approach in documenting human right violations. PMID:17696307

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

21 CFR 864.7280 - Factor V Leiden DNA mutation detection systems.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Factor V Leiden DNA mutation detection systems....7280 Factor V Leiden DNA mutation detection systems. (a) Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and...

Analysis of a DNA simulation model through hairpin melting experiments.

PubMed

Linak, Margaret C; Dorfman, Kevin D

2010-09-28

We compare the predictions of a two-bead Brownian dynamics simulation model to melting experiments of DNA hairpins with complementary AT or GC stems and noninteracting loops in buffer A. This system emphasizes the role of stacking and hydrogen bonding energies, which are characteristics of DNA, rather than backbone bending, stiffness, and excluded volume interactions, which are generic characteristics of semiflexible polymers. By comparing high throughput data on the open-close transition of various DNA hairpins to the corresponding simulation data, we (1) establish a suitable metric to compare the simulations to experiments, (2) find a conversion between the simulation and experimental temperatures, and (3) point out several limitations of the model, including the lack of G-quartets and cross stacking effects. Our approach and experimental data can be used to validate similar coarse-grained simulation models.

Illuminating Cell Biology

NASA Technical Reports Server (NTRS)

2002-01-01

NASA's Ames Research Center awarded Ciencia, Inc., a Small Business Innovation Research contract to develop the Cell Fluorescence Analysis System (CFAS) to address the size, mass, and power constraints of using fluorescence spectroscopy in the International Space Station's Life Science Research Facility. The system will play an important role in studying biological specimen's long-term adaptation to microgravity. Commercial applications for the technology include diverse markets such as food safety, in situ environmental monitoring, online process analysis, genomics and DNA chips, and non-invasive diagnostics. Ciencia has already sold the system to the private sector for biosensor applications.

Improved programs for DNA and protein sequence analysis on the IBM personal computer and other standard computer systems.

PubMed Central

Mount, D W; Conrad, B

1986-01-01

We have previously described programs for a variety of types of sequence analysis (1-4). These programs have now been integrated into a single package. They are written in the standard C programming language and run on virtually any computer system with a C compiler, such as the IBM/PC and other computers running under the MS/DOS and UNIX operating systems. The programs are widely distributed and may be obtained from the authors as described below. PMID:3753780

DNA Fingerprinting in a Forensic Teaching Experiment

ERIC Educational Resources Information Center

Wagoner, Stacy A.; Carlson, Kimberly A.

2008-01-01

This article presents an experiment designed to provide students, in a classroom laboratory setting, a hands-on demonstration of the steps used in DNA forensic analysis by performing DNA extraction, DNA fingerprinting, and statistical analysis of the data. This experiment demonstrates how DNA fingerprinting is performed and how long it takes. It…

Evaluation of the Environmental DNA Method for Estimating Distribution and Biomass of Submerged Aquatic Plants

PubMed Central

Matsuhashi, Saeko; Doi, Hideyuki; Fujiwara, Ayaka; Watanabe, Sonoko; Minamoto, Toshifumi

2016-01-01

The environmental DNA (eDNA) method has increasingly been recognized as a powerful tool for monitoring aquatic animal species; however, its application for monitoring aquatic plants is limited. To evaluate eDNA analysis for estimating the distribution of aquatic plants, we compared its estimated distributions with eDNA analysis, visual observation, and past distribution records for the submerged species Hydrilla verticillata. Moreover, we conducted aquarium experiments using H. verticillata and Egeria densa and analyzed the relationships between eDNA concentrations and plant biomass to investigate the potential for biomass estimation. The occurrences estimated by eDNA analysis closely corresponded to past distribution records, and eDNA detections were more frequent than visual observations, indicating that the method is potentially more sensitive. The results of the aquarium experiments showed a positive relationship between plant biomass and eDNA concentration; however, the relationship was not always significant. The eDNA concentration peaked within three days of the start of the experiment in most cases, suggesting that plants do not release constant amounts of DNA. These results showed that eDNA analysis can be used for distribution surveys, and has the potential to estimate the biomass of aquatic plants. PMID:27304876

Evaluation of the Environmental DNA Method for Estimating Distribution and Biomass of Submerged Aquatic Plants.

PubMed

Matsuhashi, Saeko; Doi, Hideyuki; Fujiwara, Ayaka; Watanabe, Sonoko; Minamoto, Toshifumi

2016-01-01

The environmental DNA (eDNA) method has increasingly been recognized as a powerful tool for monitoring aquatic animal species; however, its application for monitoring aquatic plants is limited. To evaluate eDNA analysis for estimating the distribution of aquatic plants, we compared its estimated distributions with eDNA analysis, visual observation, and past distribution records for the submerged species Hydrilla verticillata. Moreover, we conducted aquarium experiments using H. verticillata and Egeria densa and analyzed the relationships between eDNA concentrations and plant biomass to investigate the potential for biomass estimation. The occurrences estimated by eDNA analysis closely corresponded to past distribution records, and eDNA detections were more frequent than visual observations, indicating that the method is potentially more sensitive. The results of the aquarium experiments showed a positive relationship between plant biomass and eDNA concentration; however, the relationship was not always significant. The eDNA concentration peaked within three days of the start of the experiment in most cases, suggesting that plants do not release constant amounts of DNA. These results showed that eDNA analysis can be used for distribution surveys, and has the potential to estimate the biomass of aquatic plants.

The First Chameleon Transcriptome: Comparative Genomic Analysis of the OXPHOS System Reveals Loss of COX8 in Iguanian Lizards

PubMed Central

Bar-Yaacov, Dan; Bouskila, Amos; Mishmar, Dan

2013-01-01

Recently, we found dramatic mitochondrial DNA divergence of Israeli Chamaeleo chamaeleon populations into two geographically distinct groups. We aimed to examine whether the same pattern of divergence could be found in nuclear genes. However, no genomic resource is available for any chameleon species. Here we present the first chameleon transcriptome, obtained using deep sequencing (SOLiD). Our analysis identified 164,000 sequence contigs of which 19,000 yielded unique BlastX hits. To test the efficacy of our sequencing effort, we examined whether the chameleon and other available reptilian transcriptomes harbored complete sets of genes comprising known biochemical pathways, focusing on the nDNA-encoded oxidative phosphorylation (OXPHOS) genes as a model. As a reference for the screen, we used the human 86 (including isoforms) known structural nDNA-encoded OXPHOS subunits. Analysis of 34 publicly available vertebrate transcriptomes revealed orthologs for most human OXPHOS genes. However, OXPHOS subunit COX8 (Cytochrome C oxidase subunit 8), including all its known isoforms, was consistently absent in transcriptomes of iguanian lizards, implying loss of this subunit during the radiation of this suborder. The lack of COX8 in the suborder Iguania is intriguing, since it is important for cellular respiration and ATP production. Our sequencing effort added a new resource for comparative genomic studies, and shed new light on the evolutionary dynamics of the OXPHOS system. PMID:24009133

The first Chameleon transcriptome: comparative genomic analysis of the OXPHOS system reveals loss of COX8 in Iguanian lizards.

PubMed

Bar-Yaacov, Dan; Bouskila, Amos; Mishmar, Dan

2013-01-01

Recently, we found dramatic mitochondrial DNA divergence of Israeli Chamaeleo chamaeleon populations into two geographically distinct groups. We aimed to examine whether the same pattern of divergence could be found in nuclear genes. However, no genomic resource is available for any chameleon species. Here we present the first chameleon transcriptome, obtained using deep sequencing (SOLiD). Our analysis identified 164,000 sequence contigs of which 19,000 yielded unique BlastX hits. To test the efficacy of our sequencing effort, we examined whether the chameleon and other available reptilian transcriptomes harbored complete sets of genes comprising known biochemical pathways, focusing on the nDNA-encoded oxidative phosphorylation (OXPHOS) genes as a model. As a reference for the screen, we used the human 86 (including isoforms) known structural nDNA-encoded OXPHOS subunits. Analysis of 34 publicly available vertebrate transcriptomes revealed orthologs for most human OXPHOS genes. However, OXPHOS subunit COX8 (Cytochrome C oxidase subunit 8), including all its known isoforms, was consistently absent in transcriptomes of iguanian lizards, implying loss of this subunit during the radiation of this suborder. The lack of COX8 in the suborder Iguania is intriguing, since it is important for cellular respiration and ATP production. Our sequencing effort added a new resource for comparative genomic studies, and shed new light on the evolutionary dynamics of the OXPHOS system.

Lab-on-a-Drone: Toward Pinpoint Deployment of Smartphone-Enabled Nucleic Acid-Based Diagnostics for Mobile Health Care

PubMed Central

2016-01-01

We introduce a portable biochemical analysis platform for rapid field deployment of nucleic acid-based diagnostics using consumer-class quadcopter drones. This approach exploits the ability to isothermally perform the polymerase chain reaction (PCR) with a single heater, enabling the system to be operated using standard 5 V USB sources that power mobile devices (via battery, solar, or hand crank action). Time-resolved fluorescence detection and quantification is achieved using a smartphone camera and integrated image analysis app. Standard sample preparation is enabled by leveraging the drone’s motors as centrifuges via 3D printed snap-on attachments. These advancements make it possible to build a complete DNA/RNA analysis system at a cost of ∼$50 ($US). Our instrument is rugged and versatile, enabling pinpoint deployment of sophisticated diagnostics to distributed field sites. This capability is demonstrated by successful in-flight replication of Staphylococcus aureus and λ-phage DNA targets in under 20 min. The ability to perform rapid in-flight assays with smartphone connectivity eliminates delays between sample collection and analysis so that test results can be delivered in minutes, suggesting new possibilities for drone-based systems to function in broader and more sophisticated roles beyond cargo transport and imaging. PMID:26898247

Lab-on-a-Drone: Toward Pinpoint Deployment of Smartphone-Enabled Nucleic Acid-Based Diagnostics for Mobile Health Care.

PubMed

Priye, Aashish; Wong, Season; Bi, Yuanpeng; Carpio, Miguel; Chang, Jamison; Coen, Mauricio; Cope, Danielle; Harris, Jacob; Johnson, James; Keller, Alexandra; Lim, Richard; Lu, Stanley; Millard, Alex; Pangelinan, Adriano; Patel, Neal; Smith, Luke; Chan, Kamfai; Ugaz, Victor M

2016-05-03

We introduce a portable biochemical analysis platform for rapid field deployment of nucleic acid-based diagnostics using consumer-class quadcopter drones. This approach exploits the ability to isothermally perform the polymerase chain reaction (PCR) with a single heater, enabling the system to be operated using standard 5 V USB sources that power mobile devices (via battery, solar, or hand crank action). Time-resolved fluorescence detection and quantification is achieved using a smartphone camera and integrated image analysis app. Standard sample preparation is enabled by leveraging the drone's motors as centrifuges via 3D printed snap-on attachments. These advancements make it possible to build a complete DNA/RNA analysis system at a cost of ∼$50 ($US). Our instrument is rugged and versatile, enabling pinpoint deployment of sophisticated diagnostics to distributed field sites. This capability is demonstrated by successful in-flight replication of Staphylococcus aureus and λ-phage DNA targets in under 20 min. The ability to perform rapid in-flight assays with smartphone connectivity eliminates delays between sample collection and analysis so that test results can be delivered in minutes, suggesting new possibilities for drone-based systems to function in broader and more sophisticated roles beyond cargo transport and imaging.

Identification of forensic samples by using an infrared-based automatic DNA sequencer.

PubMed

Ricci, Ugo; Sani, Ilaria; Klintschar, Michael; Cerri, Nicoletta; De Ferrari, Francesco; Giovannucci Uzielli, Maria Luisa

2003-06-01

We have recently introduced a new protocol for analyzing all core loci of the Federal Bureau of Investigation's (FBI) Combined DNA Index System (CODIS) with an infrared (IR) automatic DNA sequencer (LI-COR 4200). The amplicons were labeled with forward oligonucleotide primers, covalently linked to a new infrared fluorescent molecule (IRDye 800). The alleles were displayed as familiar autoradiogram-like images with real-time detection. This protocol was employed for paternity testing, population studies, and identification of degraded forensic samples. We extensively analyzed some simulated forensic samples and mixed stains (blood, semen, saliva, bones, and fixed archival embedded tissues), comparing the results with donor samples. Sensitivity studies were also performed for the four multiplex systems. Our results show the efficiency, reliability, and accuracy of the IR system for the analysis of forensic samples. We also compared the efficiency of the multiplex protocol with ultraviolet (UV) technology. Paternity tests, undegraded DNA samples, and real forensic samples were analyzed with this approach based on IR technology and with UV-based automatic sequencers in combination with commercially-available kits. The comparability of the results with the widespread UV methods suggests that it is possible to exchange data between laboratories using the same core group of markers but different primer sets and detection methods.

Self-powered switch-controlled nucleic acid extraction system.

PubMed

Han, Kyungsup; Yoon, Yong-Jin; Shin, Yong; Park, Mi Kyoung

2016-01-07

Over the past few decades, lab-on-a-chip (LOC) technologies have played a great role in revolutionizing the way in vitro medical diagnostics are conducted and transforming bulky and expensive laboratory instruments and labour-intensive tests into easy to use, cost-effective miniaturized systems with faster analysis time, which can be used for near-patient or point-of-care (POC) tests. Fluidic pumps and valves are among the key components for LOC systems; however, they often require on-line electrical power or batteries and make the whole system bulky and complex, therefore limiting its application to POC testing especially in low-resource setting. This is particularly problematic for molecular diagnostics where multi-step sample processing (e.g. lysing, washing, elution) is necessary. In this work, we have developed a self-powered switch-controlled nucleic acid extraction system (SSNES). The main components of SSNES are a powerless vacuum actuator using two disposable syringes and a switchgear made of PMMA blocks and an O-ring. In the vacuum actuator, an opened syringe and a blocked syringe are bound together and act as a working syringe and an actuating syringe, respectively. The negative pressure in the opened syringe is generated by a restoring force of the compressed air inside the blocked syringe and utilized as the vacuum source. The Venus symbol shape of the switchgear provides multiple functions including being a reagent reservoir, a push-button for the vacuum actuator, and an on-off valve. The SSNES consists of three sets of vacuum actuators, switchgears and microfluidic components. The entire system can be easily fabricated and is fully disposable. We have successfully demonstrated DNA extraction from a urine sample using a dimethyl adipimidate (DMA)-based extraction method and the performance of the DNA extraction has been confirmed by genetic (HRAS) analysis of DNA biomarkers from the extracted DNAs using the SSNES. Therefore, the SSNES can be widely used as a powerless and disposable system for DNA extraction and the syringe-based vacuum actuator would be easily utilized for diverse applications with various microchannels as a powerless fluidic pump.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

NASA Astrophysics Data System (ADS)

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-01

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection.

PubMed

Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

2016-10-14

The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

Characterization of the gene encoding component C3 of the complement system from the spider Loxosceles laeta venom glands: Phylogenetic implications.

PubMed

Myamoto, D T; Pidde-Queiroz, G; Pedroso, A; Gonçalves-de-Andrade, R M; van den Berg, C W; Tambourgi, D V

2016-09-01

A transcriptome analysis of the venom glands of the spider Loxosceles laeta, performed by our group, in a previous study (Fernandes-Pedrosa et al., 2008), revealed a transcript with a sequence similar to the human complement component C3. Here we present the analysis of this transcript. cDNA fragments encoding the C3 homologue (Lox-C3) were amplified from total RNA isolated from the venom glands of L. laeta by RACE-PCR. Lox-C3 is a 5178 bps cDNA sequence encoding a 190kDa protein, with a domain configuration similar to human C3. Multiple alignments of C3-like proteins revealed two processing sites, suggesting that Lox-C3 is composed of three chains. Furthermore, the amino acids consensus sequences for the thioester was found, in addition to putative sequences responsible for FB binding. The phylogenetic analysis showed that Lox-C3 belongs to the same group as two C3 isoforms from the spider Hasarius adansoni (Family Salcitidae), showing 53% homology with these. This is the first characterization of a Loxosceles cDNA sequence encoding a human C3 homologue, and this finding, together with our previous finding of the expression of a FB-like molecule, suggests that this spider species also has a complement system. This work will help to improve our understanding of the innate immune system in these spiders and the ancestral structure of C3. Copyright © 2016 Elsevier GmbH. All rights reserved.

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction.

PubMed

Patel, Palak G; Selvarajah, Shamini; Boursalie, Suzanne; How, Nathan E; Ejdelman, Joshua; Guerard, Karl-Philippe; Bartlett, John M; Lapointe, Jacques; Park, Paul C; Okello, John B A; Berman, David M

2016-08-21

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

Quantitative analysis of small molecule-nucleic acid interactions with a biosensor surface and surface plasmon resonance detection.

PubMed

Liu, Yang; Wilson, W David

2010-01-01

Surface plasmon resonance (SPR) technology with biosensor surfaces has become a widely-used tool for the study of nucleic acid interactions without any labeling requirements. The method provides simultaneous kinetic and equilibrium characterization of the interactions of biomolecules as well as small molecule-biopolymer binding. SPR monitors molecular interactions in real time and provides significant advantages over optical or calorimetic methods for systems with strong binding coupled to small spectroscopic signals and/or reaction heats. A detailed and practical guide for nucleic acid interaction analysis using SPR-biosensor methods is presented. Details of the SPR technology and basic fundamentals are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples, as well as extensive information on experimental design, quantitative and qualitative data analysis and presentation. A specific example of the interaction of a minor-groove-binding agent with DNA is evaluated by both kinetic and steady-state SPR methods to illustrate the technique. Since the molecules that bind cooperatively to specific DNA sequences are attractive for many applications, a cooperative small molecule-DNA interaction is also presented.

Probing planetary biodiversity with DNA barcodes: The Noctuoidea of North America

PubMed Central

Lafontaine, J. Donald; Schmidt, B. Christian; deWaard, Jeremy R.; Zakharov, Evgeny V.; Hebert, Paul D. N.

2017-01-01

This study reports the assembly of a DNA barcode reference library for species in the lepidopteran superfamily Noctuoidea from Canada and the USA. Based on the analysis of 69,378 specimens, the library provides coverage for 97.3% of the noctuoid fauna (3565 of 3664 species). In addition to verifying the strong performance of DNA barcodes in the discrimination of these species, the results indicate close congruence between the number of species analyzed (3565) and the number of sequence clusters (3816) recognized by the Barcode Index Number (BIN) system. Distributional patterns across 12 North American ecoregions are examined for the 3251 species that have GPS data while BIN analysis is used to quantify overlap between the noctuoid faunas of North America and other zoogeographic regions. This analysis reveals that 90% of North American noctuoids are endemic and that just 7.5% and 1.8% of BINs are shared with the Neotropics and with the Palearctic, respectively. One third (29) of the latter species are recent introductions and, as expected, they possess low intraspecific divergences. PMID:28570635

A novel image encryption algorithm based on the chaotic system and DNA computing

NASA Astrophysics Data System (ADS)

Chai, Xiuli; Gan, Zhihua; Lu, Yang; Chen, Yiran; Han, Daojun

A novel image encryption algorithm using the chaotic system and deoxyribonucleic acid (DNA) computing is presented. Different from the traditional encryption methods, the permutation and diffusion of our method are manipulated on the 3D DNA matrix. Firstly, a 3D DNA matrix is obtained through bit plane splitting, bit plane recombination, DNA encoding of the plain image. Secondly, 3D DNA level permutation based on position sequence group (3DDNALPBPSG) is introduced, and chaotic sequences generated from the chaotic system are employed to permutate the positions of the elements of the 3D DNA matrix. Thirdly, 3D DNA level diffusion (3DDNALD) is given, the confused 3D DNA matrix is split into sub-blocks, and XOR operation by block is manipulated to the sub-DNA matrix and the key DNA matrix from the chaotic system. At last, by decoding the diffused DNA matrix, we get the cipher image. SHA 256 hash of the plain image is employed to calculate the initial values of the chaotic system to avoid chosen plaintext attack. Experimental results and security analyses show that our scheme is secure against several known attacks, and it can effectively protect the security of the images.

Structural and Functional Analysis of the Signal-Transducing Linker in the pH-Responsive One-Component System CadC of Escherichia coli.

PubMed

Buchner, Sophie; Schlundt, Andreas; Lassak, Jürgen; Sattler, Michael; Jung, Kirsten

2015-07-31

The pH-responsive one-component signaling system CadC in Escherichia coli belongs to the family of ToxR-like proteins, whose members share a conserved modular structure, with an N-terminal cytoplasmic winged helix-turn-helix DNA-binding domain being followed by a single transmembrane helix and a C-terminal periplasmic pH-sensing domain. In E. coli CadC, a cytoplasmic linker comprising approximately 50 amino acids is essential for transmission of the signal from the sensor to the DNA-binding domain. However, the mechanism of transduction is poorly understood. Using NMR spectroscopy, we demonstrate here that the linker region is intrinsically disordered in solution. Furthermore, mutational analyses showed that it tolerates a range of amino acid substitutions (altering polarity, rigidity and α-helix-forming propensity), is robust to extension but is sensitive to truncation. Indeed, truncations either reversed the expression profile of the target operon cadBA or decoupled expression from external pH altogether. CadC dimerizes via its periplasmic domain, but light-scattering analysis provided no evidence for dimerization of the isolated DNA-binding domain, with or without the linker region. However, bacterial two-hybrid analysis revealed that CadC forms stable dimers in a stimulus- and linker-dependent manner, interacting only at pH<6.8. Strikingly, a variant with inversed cadBA expression profile, which lacks most of the linker, dimerizes preferentially at higher pH. Thus, we propose that the disordered CadC linker is required for transducing the pH-dependent response of the periplasmic sensor into a structural rearrangement that facilitates dimerization of the cytoplasmic CadC DNA-binding domain. Copyright © 2015 Elsevier Ltd. All rights reserved.

Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale.

PubMed

Gutzweiler, Ludwig; Gleichmann, Tobias; Tanguy, Laurent; Koltay, Peter; Zengerle, Roland; Riegger, Lutz

2017-07-01

Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on-demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary- and chip-based methods offering low consumable costs (<0.1$) circumventing cost-intensive microfluidic chip fabrication, short process times (5 min per analysis) and high sensitivity (4 ng/μL dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2-4 μL in volume, a semi-contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non-contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR-grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi-circular cross section of adaptable width (∼200-600 μm) and height (∼30-80 μm) as well as a typical length of 15-55 mm. Layout of such liquid structures is adaptable on-demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label-free DNA ladder (100-1000 bp) at 100 V/cm via in-line staining and laser induced fluorescent end-point detection using an automated prototype. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computer-based image analysis of one-dimensional electrophoretic gels used for the separation of DNA restriction fragments.

PubMed Central

Gray, A J; Beecher, D E; Olson, M V

1984-01-01

A stand-alone, interactive computer system has been developed that automates the analysis of ethidium bromide-stained agarose and acrylamide gels on which DNA restriction fragments have been separated by size. High-resolution digital images of the gels are obtained using a camera that contains a one-dimensional, 2048-pixel photodiode array that is mechanically translated through 2048 discrete steps in a direction perpendicular to the gel lanes. An automatic band-detection algorithm is used to establish the positions of the gel bands. A color-video graphics system, on which both the gel image and a variety of operator-controlled overlays are displayed, allows the operator to visualize and interact with critical stages of the analysis. The principal interactive steps involve defining the regions of the image that are to be analyzed and editing the results of the band-detection process. The system produces a machine-readable output file that contains the positions, intensities, and descriptive classifications of all the bands, as well as documentary information about the experiment. This file is normally further processed on a larger computer to obtain fragment-size assignments. Images PMID:6320097

The xeroderma pigmentosum group B protein ERCC3 produced in the baculovirus system exhibits DNA helicase activity.

PubMed Central

Ma, L; Siemssen, E D; Noteborn, H M; van der Eb, A J

1994-01-01

The XPB/ERCC3 gene corrects the nucleotide excision-repair defect in the human hereditary disease xeroderma pigmentosum group B and encodes the largest subunit of the basal transcription factor BTF2/TFIIH. The primary sequence of the XPB/ERCC3 protein features the hallmarks of seven helicase motifs found in many known and putative helicases or helicase-related proteins. Recently, the multiprotein BTF2/TFIIH complex has been found to be associated with DNA helicase activity. To explore the properties and functions of XPB/ERCC3, we have used the baculovirus/insect-cell expression system to produce recombinant protein. We report here the construction and analysis of recombinant baculovirus expressing XPB/ERCC3. The XPB/ERCC3 protein is synthesized at a relatively high level in baculovirus-infected insect cells. While the majority of XPB/ERCC3 end up in the insoluble fraction of insect cell lysates, a minor fraction of recombinant protein is present in soluble form which can be purified under native conditions. We have found that a DNA helicase activity is associated with the purified XPB/ERCC3 protein, suggesting that XPB/ERCC3 may function as a DNA helicase in local unwinding of DNA template both in the context of transcription and nucleotide excision repair. Images PMID:7937133

Application of nanostructured biochips for efficient cell transfection microarrays

NASA Astrophysics Data System (ADS)

Akkamsetty, Yamini; Hook, Andrew L.; Thissen, Helmut; Hayes, Jason P.; Voelcker, Nicolas H.

2007-01-01

Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart materials possessing both switchable and patterned surface properties. A system had been developed to spatially manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced. However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by contact printing has also been investigated.

Is the kinetoplast DNA a percolating network of linked rings at its critical point?

NASA Astrophysics Data System (ADS)

Michieletto, Davide; Marenduzzo, Davide; Orlandini, Enzo

2015-05-01

In this work we present a computational study of the kinetoplast genome, modelled as a large number of semiflexible unknotted loops, which are allowed to link with each other. As the DNA density increases, the systems shows a percolation transition between a gas of unlinked rings and a network of linked loops which spans the whole system. Close to the percolation transition, we find that the mean valency of the network, i.e. the average number of loops which are linked to any one loop, is around three, as found experimentally for the kinetoplast DNA (kDNA). Even more importantly, by simulating the digestion of the network by a restriction enzyme, we show that the distribution of oligomers, i.e. structures formed by a few loops which remain linked after digestion, quantitatively matches experimental data obtained from gel electrophoresis, provided that the density is, once again, close to the percolation transition. With respect to previous work, our analysis builds on a reduced number of assumptions, yet can still fully explain the experimental data. Our findings suggest that the kDNA can be viewed as a network of linked loops positioned very close to the percolation transition, and we discuss the possible biological implications of this remarkable fact.

In situ Localization of the Human Multidrug‐resistance Gene mRNA Using Thymine‐Thymine Dimerized Single‐stranded cDNA

PubMed Central

Koji, Takehiko; Ueda, Kazumitsu; Pastan, Ira; Gottesman, Michael M.; Nakane, Paul K.; Mori, Shigeo

1990-01-01

In order to detect the mRNA transcribed from the multidrug‐resistance gene (MDR1), thymine‐thymine (T‐T) dimerized single‐stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T‐T dimerized single‐stranded DNA (300–400 bases) so that they could penetrate well into the cytoplasm. The hybridized T‐T DNA was detected immunohisto‐chemically using rabbit anti‐T‐T DNA antibody (Ab) and peroxidase‐labeled goat anti‐rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug‐resistant cell lines K562/ADM, CEM/VLB, 2780ad, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene. PMID:1977730