[Efficiency of 27-plex single nucleotide polymorphism multiplex system for ancestry inference in different populations].

PubMed

Feng, Xing-Ling; Sun, Qi-Fan; Liu, Hong; Wei, Yi-Liang; DU, Wei-An; Li, Cai-Xia; Chen, Ling; Liu, Chao

2016-04-20

To validate the efficiency of 27-plex single nucleotide polymorphism (SNP) multiplex system for ancestry inference. The 27-plex SNP system was validated for its sensitivity and species specificity. A total of 533 samples were collected from African, Southern Chinese Han, China's ethic minorities (Yi, Hui, Miao, Tibet, and Uygur), European, Central Asian, Western Asian, Southern Asian, Southeast Asian and South American populations for clustering analysis of the genotypes by citing 3 representative continental ancestral groups [East Asia (CHB), Europe (CEU), and Africa (YRI)] from HapMap database. The system sensitivity is 0.125 ng. Twenty and six genotypes were detected in chimpanzee and monkeys, respectively. Except in rs10496971, no more products were found in other animals. The system was capable of differentiating intercontinental populations but not of distinguishing between East Asian and Southeast Asian population or between Southern Chinese Han population and Chinese Ethnic populations (Hui, Miao, Yi and Tibet). This system achieved a 100% accuracy for intercontinental population source inference for 46 blind test samples. 27-plex SNPs multiplex system has a high sensitivity and species specificity and can correctly differentiate the ancestry origins of individuals from African, European and East Asian for criminal case investigation. But this system is not capable of distinguishing subpopulation groups and more specific ancestry-informative markers are needed to improve its recognition of Southeast Asian and Chinese ethnic populations.

Multiplexed resequencing analysis to identify rare variants in pooled DNA with barcode indexing using next-generation sequencer.

PubMed

Mitsui, Jun; Fukuda, Yoko; Azuma, Kyo; Tozaki, Hirokazu; Ishiura, Hiroyuki; Takahashi, Yuji; Goto, Jun; Tsuji, Shoji

2010-07-01

We have recently found that multiple rare variants of the glucocerebrosidase gene (GBA) confer a robust risk for Parkinson disease, supporting the 'common disease-multiple rare variants' hypothesis. To develop an efficient method of identifying rare variants in a large number of samples, we applied multiplexed resequencing using a next-generation sequencer to identification of rare variants of GBA. Sixteen sets of pooled DNAs from six pooled DNA samples were prepared. Each set of pooled DNAs was subjected to polymerase chain reaction to amplify the target gene (GBA) covering 6.5 kb, pooled into one tube with barcode indexing, and then subjected to extensive sequence analysis using the SOLiD System. Individual samples were also subjected to direct nucleotide sequence analysis. With the optimization of data processing, we were able to extract all the variants from 96 samples with acceptable rates of false-positive single-nucleotide variants.

Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

PubMed

Gopaul, Krishna K; Sells, Jessica; Lee, Robin; Beckstrom-Sternberg, Stephen M; Foster, Jeffrey T; Whatmore, Adrian M

2014-12-11

The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

A high density integrated genetic linkage map of soybean and the development of a 1,536 Universal Soy Linkage Panel for QTL mapping

USDA-ARS?s Scientific Manuscript database

Single nucleotide polymorphisms (SNPs) are the marker of choice for many researchers due to their abundance and the high-throughput methods available for their multiplex analysis. Only recently have SNP markers been available to researchers in soybean [Glycine max (L.) Merr.] with the release of th...

Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection

PubMed Central

KC, R.; Srivastava, A.; Wilkowski, J. M.; Richter, C. E.; Shavit, J. A.; Burke, D. T.; Bielas, S. L.

2016-01-01

CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. PMID:27557703

Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

PubMed Central

Fuller, Carl W.; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P. Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T.; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J.; Kasianowicz, John J.; Davis, Randy; Roever, Stefan; Church, George M.; Ju, Jingyue

2016-01-01

DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. PMID:27091962

Multiplex Droplet Digital PCR Quantification of Recurrent Somatic Mutations in Diffuse Large B-Cell and Follicular Lymphoma.

PubMed

Alcaide, Miguel; Yu, Stephen; Bushell, Kevin; Fornika, Daniel; Nielsen, Julie S; Nelson, Brad H; Mann, Koren K; Assouline, Sarit; Johnson, Nathalie A; Morin, Ryan D

2016-09-01

A plethora of options to detect mutations in tumor-derived DNA currently exist but each suffers limitations in analytical sensitivity, cost, or scalability. Droplet digital PCR (ddPCR) is an appealing technology for detecting the presence of specific mutations based on a priori knowledge and can be applied to tumor biopsies, including formalin-fixed paraffin embedded (FFPE) tissues. More recently, ddPCR has gained popularity in its utility in quantifying circulating tumor DNA. We have developed a suite of novel ddPCR assays for detecting recurrent mutations that are prevalent in common B-cell non-Hodgkin lymphomas (NHLs), including diffuse large B-cell lymphoma, follicular lymphoma, and lymphoplasmacytic lymphoma. These assays allowed the differentiation and counting of mutant and wild-type molecules using one single hydrolysis probe. We also implemented multiplexing that allowed the simultaneous detection of distinct mutations and an "inverted" ddPCR assay design, based on employing probes matching wild-type alleles, capable of detecting the presence of multiple single nucleotide polymorphisms. The assays successfully detected and quantified somatic mutations commonly affecting enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) (Y641) and signal transducer and activator of transcription 6 (STAT6) (D419) hotspots in fresh tumor, FFPE, and liquid biopsies. The "inverted" ddPCR approach effectively reported any single nucleotide variant affecting either of these 2 hotspots as well. Finally, we could effectively multiplex hydrolysis probes targeting 2 additional lymphoma-related hotspots: myeloid differentiation primary response 88 (MYD88; L265P) and cyclin D3 (CCND3; I290R). Our suite of ddPCR assays provides sufficient analytical sensitivity and specificity for either the invasive or noninvasive detection of multiple recurrent somatic mutations in B-cell NHLs. © 2016 American Association for Clinical Chemistry.

Continuously tunable nucleic acid hybridization probes.

PubMed

Wu, Lucia R; Wang, Juexiao Sherry; Fang, John Z; Evans, Emily R; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J; Beechem, Joseph; Zhang, David Yu

2015-12-01

In silico-designed nucleic acid probes and primers often do not achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. We present a novel, on-the-fly method of tuning probe affinity and selectivity by adjusting the stoichiometry of auxiliary species, which allows for independent and decoupled adjustment of the hybridization yield for different probes in multiplexed assays. Using this method, we achieved near-continuous tuning of probe effective free energy. To demonstrate our approach, we enforced uniform capture efficiency of 31 DNA molecules (GC content, 0-100%), maximized the signal difference for 11 pairs of single-nucleotide variants and performed tunable hybrid capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples.

Interferometric Reflectance Imaging Sensor (IRIS)—A Platform Technology for Multiplexed Diagnostics and Digital Detection

PubMed Central

Avci, Oguzhan; Lortlar Ünlü, Nese; Yalçın Özkumur, Ayça; Ünlü, M. Selim

2015-01-01

Over the last decade, the growing need in disease diagnostics has stimulated rapid development of new technologies with unprecedented capabilities. Recent emerging infectious diseases and epidemics have revealed the shortcomings of existing diagnostics tools, and the necessity for further improvements. Optical biosensors can lay the foundations for future generation diagnostics by providing means to detect biomarkers in a highly sensitive, specific, quantitative and multiplexed fashion. Here, we review an optical sensing technology, Interferometric Reflectance Imaging Sensor (IRIS), and the relevant features of this multifunctional platform for quantitative, label-free and dynamic detection. We discuss two distinct modalities for IRIS: (i) low-magnification (ensemble biomolecular mass measurements) and (ii) high-magnification (digital detection of individual nanoparticles) along with their applications, including label-free detection of multiplexed protein chips, measurement of single nucleotide polymorphism, quantification of transcription factor DNA binding, and high sensitivity digital sensing and characterization of nanoparticles and viruses. PMID:26205273

Pigment phenotype and biogeographical ancestry from ancient skeletal remains: inferences from multiplexed autosomal SNP analysis.

PubMed

Bouakaze, Caroline; Keyser, Christine; Crubézy, Eric; Montagnon, Daniel; Ludes, Bertrand

2009-07-01

In the present study, a multiplexed genotyping assay for ten single nucleotide polymorphisms (SNPs) located within six pigmentation candidate genes was developed on modern biological samples and applied to DNA retrieved from 25 archeological human remains from southern central Siberia dating from the Bronze and Iron Ages. SNP genotyping was successful for the majority of ancient samples and revealed that most probably had typical European pigment features, i.e., blue or green eye color, light hair color and skin type, and were likely of European individual ancestry. To our knowledge, this study reports for the first time the multiplexed typing of autosomal SNPs on aged and degraded DNA. By providing valuable information on pigment traits of an individual and allowing individual biogeographical ancestry estimation, autosomal SNP typing can improve ancient DNA studies and aid human identification in some forensic casework situations when used to complement conventional molecular markers.

Genotyping of Single Nucleotide Polymorphisms in DNA Isolated from Serum Using Sequenom MassARRAY Technology.

PubMed

Clendenen, Tess V; Rendleman, Justin; Ge, Wenzhen; Koenig, Karen L; Wirgin, Isaac; Currie, Diane; Shore, Roy E; Kirchhoff, Tomas; Zeleniuch-Jacquotte, Anne

2015-01-01

Large epidemiologic studies have the potential to make valuable contributions to the assessment of gene-environment interactions because they prospectively collected detailed exposure data. Some of these studies, however, have only serum or plasma samples as a low quantity source of DNA. We examined whether DNA isolated from serum can be used to reliably and accurately genotype single nucleotide polymorphisms (SNPs) using Sequenom multiplex SNP genotyping technology. We genotyped 81 SNPs using samples from 158 participants in the NYU Women's Health Study. Each participant had DNA from serum and at least one paired DNA sample isolated from a high quality source of DNA, i.e. clots and/or cell precipitates, for comparison. We observed that 60 of the 81 SNPs (74%) had high call frequencies (≥95%) using DNA from serum, only slightly lower than the 85% of SNPs with high call frequencies in DNA from clots or cell precipitates. Of the 57 SNPs with high call frequencies for serum, clot, and cell precipitate DNA, 54 (95%) had highly concordant (>98%) genotype calls across all three sample types. High purity was not a critical factor to successful genotyping. Our results suggest that this multiplex SNP genotyping method can be used reliably on DNA from serum in large-scale epidemiologic studies.

Development of a multiplex polymerase chain reaction-sequence-specific primer method for NKG2D and NKG2F single-nucleotide polymorphism typing using isothermal multiple displacement amplification products.

PubMed

Kaewmanee, M; Phoksawat, W; Romphruk, A; Romphruk, A V; Jumnainsong, A; Leelayuwat, C

2013-06-01

Natural killer group 2 member D (NKG2D) on immune effector cells recognizes multiple stress-inducible ligands. NKG2D single-nucleotide polymorphism (SNP) haplotypes were related to the levels of cytotoxic activity of peripheral blood mononuclear cells. Indeed, these polymorphisms were also located in NKG2F. Isothermal multiple displacement amplification (IMDA) is used for whole genome amplification (WGA) that can amplify very small genomic DNA templates into microgram with whole genome coverage. This is particularly useful in the cases of limited amount of valuable DNA samples requiring multi-locus genotyping. In this study, we evaluated the quality and applicability of IMDA to genetic studies in terms of sensitivity, efficiency of IMDA re-amplification and stability of IMDA products. The smallest amount of DNA to be effectively amplified by IMDA was 200 pg yielding final DNA of approximately 16 µg within 1.5 h. IMDA could be re-amplified only once (second round of amplification), and could be kept for 5 months at 4°C and more than a year at -20°C without loosing genome coverage. The amplified products were used successfully to setup a multiplex polymerase chain reaction-sequence-specific primer for SNP typing of the NKG2D/F genes. The NKG2D/F multiplex polymerase chain reaction (PCR) contained six PCR mixtures for detecting 10 selected SNPs, including 8 NKG2D/F SNP haplotypes and 2 additional NKG2D coding SNPs. This typing procedure will be applicable in both clinical and research laboratories. Thus, our data provide useful information and limitations for utilization of genome-wide amplification using IMDA and its application for multiplex NKG2D/F typing. © 2013 John Wiley & Sons Ltd.

Multiplex nucleic acid sequence-based amplification for simultaneous detection of several enteric viruses in model ready-to-eat foods.

PubMed

Jean, Julie; D'Souza, Doris H; Jaykus, Lee-Ann

2004-11-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10(-1) reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 10(0) to 10(2) reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods.

Multiplexed droplet single-cell RNA-sequencing using natural genetic variation.

PubMed

Kang, Hyun Min; Subramaniam, Meena; Targ, Sasha; Nguyen, Michelle; Maliskova, Lenka; McCarthy, Elizabeth; Wan, Eunice; Wong, Simon; Byrnes, Lauren; Lanata, Cristina M; Gate, Rachel E; Mostafavi, Sara; Marson, Alexander; Zaitlen, Noah; Criswell, Lindsey A; Ye, Chun Jimmie

2018-01-01

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples.

Multiplexed direct genomic selection (MDiGS): a pooled BAC capture approach for highly accurate CNV and SNP/INDEL detection.

PubMed

Alvarado, David M; Yang, Ping; Druley, Todd E; Lovett, Michael; Gurnett, Christina A

2014-06-01

Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of ∼ 550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5' cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A valveless rotary microfluidic device for multiplex point mutation identification based on ligation-rolling circle amplification.

PubMed

Heo, Hyun Young; Chung, Soyi; Kim, Yong Tae; Kim, Do Hyun; Seo, Tae Seok

2016-04-15

Genetic variations such as single nucleotide polymorphism (SNP) and point mutations are important biomarkers to monitor disease prognosis and diagnosis. In this study, we developed a novel rotary microfluidic device which can perform multiplex SNP typing on the mutation sites of TP53 genes. The microdevice consists of three glass layers: a channel wafer, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a rotary plate in which twelve reaction chambers were fabricated. A series of sample injection, ligation-rolling circle amplification (L-RCA) reaction, and fluorescence detection of the resultant amplicons could be executed by rotating the top rotary plate, identifying five mutation points related with cancer prognosis. The use of the rotary plate eliminates the necessity of microvalves and micropumps to control the microfluidic flow in the channel, simplifying the chip design and chip operation for multiplex SNP detection. The proposed microdevice provides an advanced genetic analysis platform in terms of multiplexity, simplicity, and portability in the fields of biomedical diagnostics. Copyright © 2015 Elsevier B.V. All rights reserved.

The SNPforID Assay as a Supplementary Method in Kinship and Trace Analysis

PubMed Central

Schwark, Thorsten; Meyer, Patrick; Harder, Melanie; Modrow, Jan-Hendrick; von Wurmb-Schwark, Nicole

2012-01-01

Objective Short tandem repeat (STR) analysis using commercial multiplex PCR kits is the method of choice for kinship testing and trace analysis. However, under certain circumstances (deficiency testing, mutations, minute DNA amounts), STRs alone may not suffice. Methods We present a 50-plex single nucleotide polymorphism (SNP) assay based on the SNPs chosen by the SNPforID consortium as an additional method for paternity and for trace analysis. The new assay was applied to selected routine paternity and trace cases from our laboratory. Results and Conclusions Our investigation shows that the new SNP multiplex assay is a valuable method to supplement STR analysis, and is a powerful means to solve complicated genetic analyses. PMID:22851934

Screening of a Brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assays

PubMed Central

2013-01-01

Background Efficient screening of bacterial artificial chromosome (BAC) libraries with polymerase chain reaction (PCR)-based markers is feasible provided that a multidimensional pooling strategy is implemented. Single nucleotide polymorphisms (SNPs) can be screened in multiplexed format, therefore this marker type lends itself particularly well for medium- to high-throughput applications. Combining the power of multiplex-PCR assays with a multidimensional pooling system may prove to be especially challenging in a polyploid genome. In polyploid genomes two classes of SNPs need to be distinguished, polymorphisms between accessions (intragenomic SNPs) and those differentiating between homoeologous genomes (intergenomic SNPs). We have assessed whether the highly parallel Illumina GoldenGate® Genotyping Assay is suitable for the screening of a BAC library of the polyploid Brassica napus genome. Results A multidimensional screening platform was developed for a Brassica napus BAC library which is composed of almost 83,000 clones. Intragenomic and intergenomic SNPs were included in Illumina’s GoldenGate® Genotyping Assay and both SNP classes were used successfully for screening of the multidimensional BAC pools of the Brassica napus library. An optimized scoring method is proposed which is especially valuable for SNP calling of intergenomic SNPs. Validation of the genotyping results by independent methods revealed a success of approximately 80% for the multiplex PCR-based screening regardless of whether intra- or intergenomic SNPs were evaluated. Conclusions Illumina’s GoldenGate® Genotyping Assay can be efficiently used for screening of multidimensional Brassica napus BAC pools. SNP calling was specifically tailored for the evaluation of BAC pool screening data. The developed scoring method can be implemented independently of plant reference samples. It is demonstrated that intergenomic SNPs represent a powerful tool for BAC library screening of a polyploid genome. PMID:24010766

L-RCA (ligation-rolling circle amplification): a general method for genotyping of single nucleotide polymorphisms (SNPs)

PubMed Central

Qi, Xiaoquan; Bakht, Saleha; Devos, Katrien M.; Gale, Mike D.; Osbourn, Anne

2001-01-01

A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays. PMID:11713336

Warfarin genotyping in a single PCR reaction for microchip electrophoresis.

PubMed

Poe, Brian L; Haverstick, Doris M; Landers, James P

2012-04-01

Warfarin is the most commonly prescribed oral anticoagulant medication but also is the second leading cause of emergency room visits for adverse drug reactions. Genetic testing for warfarin sensitivity may reduce hospitalization rates, but prospective genotyping is impeded in part by the turnaround time and costs of genotyping. Microfluidics-based assays can reduce reagent consumption and analysis time; however, no current assay has integrated multiplexed allele-specific PCR for warfarin genotyping with electrophoretic microfluidics hardware. Ideally, such an assay would use a single PCR reaction and, without further processing, a single microchip electrophoresis (ME) run to determine the 3 single-nucleotide polymorphisms (SNPs) affecting warfarin sensitivity [i.e., CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) *2, CYP2C9 *3, and the VKORC1 (vitamin K epoxide reductase complex 1) A/B haplotype]. We designed and optimized primers for a fully multiplexed assay to examine 3 biallelic SNPs with the tetraprimer amplification refractory mutation system (T-ARMS). The assay was developed with conventional PCR equipment and demonstrated for microfluidic infrared-mediated PCR. Genotypes were determined by ME on the basis of the pattern of PCR products. Thirty-five samples of human genomic DNA were analyzed with this multiplex T-ARMS assay, and 100% of the genotype determinations agreed with the results obtained by other validated methods. The sample population included several genotypes conferring warfarin sensitivity, with both homozygous and heterozygous genotypes for each SNP. Total analysis times for the PCR and ME were approximately 75 min (1-sample run) and 90 min (12-sample run). This multiplexed T-ARMS assay coupled with microfluidics hardware constitutes a promising avenue for an inexpensive and rapid platform for warfarin genotyping.

Multiplex pyrosequencing assay using AdvISER-MH-PYRO algorithm: a case for rapid and cost-effective genotyping analysis of prostate cancer risk-associated SNPs.

PubMed

Ambroise, Jérôme; Butoescu, Valentina; Robert, Annie; Tombal, Bertrand; Gala, Jean-Luc

2015-06-25

Single Nucleotide Polymorphisms (SNPs) identified in Genome Wide Association Studies (GWAS) have generally moderate association with related complex diseases. Accordingly, Multilocus Genetic Risk Scores (MGRSs) have been computed in previous studies in order to assess the cumulative association of multiple SNPs. When several SNPs have to be genotyped for each patient, using successive uniplex pyrosequencing reactions increases analytical reagent expenses and Turnaround Time (TAT). While a set of several pyrosequencing primers could theoretically be used to analyze multiplex amplicons, this would generate overlapping primer-specific pyro-signals that are visually uninterpretable. In the current study, two multiplex assays were developed consisting of a quadruplex (n=4) and a quintuplex (n=5) polymerase chain reaction (PCR) each followed by multiplex pyrosequencing analysis. The aim was to reliably but rapidly genotype a set of prostate cancer-related SNPs (n=9). The nucleotide dispensation order was selected using SENATOR software. Multiplex pyro-signals were analyzed using the new AdvISER-MH-PYRO software based on a sparse representation of the signal. Using uniplex assays as gold standard, the concordance between multiplex and uniplex assays was assessed on DNA extracted from patient blood samples (n = 10). All genotypes (n=90) generated with the quadruplex and the quintuplex pyroquencing assays were perfectly (100 %) concordant with uniplex pyrosequencing. Using multiplex genotyping approach for analyzing a set of 90 patients allowed reducing TAT by approximately 75 % (i.e., from 2025 to 470 min) while reducing reagent consumption and cost by approximately 70 % (i.e., from ~229 US$ /patient to ~64 US$ /patient). This combination of quadruplex and quintuplex pyrosequencing and PCR assays enabled to reduce the amount of DNA required for multi-SNP analysis, and to lower the global TAT and costs of SNP genotyping while providing results as reliable as uniplex analysis. Using this combined multiplex approach also substantially reduced the production of waste material. These genotyping assays appear therefore to be biologically, economically and ecologically highly relevant, being worth to be integrated in genetic-based predictive strategies for better selecting patients at risk for prostate cancer. In addition, the same approach could now equally be transposed to other clinical/research applications relying on the computation of MGRS based on multi-SNP genotyping.

Continuously Tunable Nucleic Acid Hybridization Probes

PubMed Central

Wu, Lucia R.; Wang, J. Sherry; Fang, John Z.; Reiser, Emily; Pinto, Alessandro; Pekker, Irena; Boykin, Richard; Ngouenet, Celine; Webster, Philippa J.; Beechem, Joseph; Zhang, David Yu

2015-01-01

In silico designed nucleic acid probes and primers often fail to achieve favorable specificity and sensitivity tradeoffs on the first try, and iterative empirical sequence-based optimization is needed, particularly in multiplexed assays. Here, we present a novel, on-the-fly method of tuning probe affinity and selectivity via the stoichiometry of auxiliary species, allowing independent and decoupled adjustment of hybridization yield for different probes in multiplexed assays. Using this method, we achieve near-continuous tuning of probe effective free energy (0.03 kcal·mol−1 granularity). As applications, we enforced uniform capture efficiency of 31 DNA molecules (GC content 0% – 100%), maximized signal difference for 11 pairs of single nucleotide variants, and performed tunable hybrid-capture of mRNA from total RNA. Using the Nanostring nCounter platform, we applied stoichiometric tuning to simultaneously adjust yields for a 24-plex assay, and we show multiplexed quantitation of RNA sequences and variants from formalin-fixed, paraffin-embedded samples (FFPE). PMID:26480474

Multiplex Nucleic Acid Sequence-Based Amplification for Simultaneous Detection of Several Enteric Viruses in Model Ready-To-Eat Foods†

PubMed Central

Jean, Julie; D'Souza, Doris H.; Jaykus, Lee-Ann

2004-01-01

Human enteric viruses are currently recognized as one of the most important causes of food-borne disease. Implication of enteric viruses in food-borne outbreaks can be difficult to confirm due to the inadequacy of the detection methods available. In this study, a nucleic acid sequence-based amplification (NASBA) method was developed in a multiplex format for the specific, simultaneous, and rapid detection of epidemiologically relevant human enteric viruses. Three previously reported primer sets were used in a single reaction for the amplification of RNA target fragments of 474, 371, and 165 nucleotides for the detection of hepatitis A virus and genogroup I and genogroup II noroviruses, respectively. Amplicons were detected by agarose gel electrophoresis and confirmed by electrochemiluminescence and Northern hybridization. Endpoint detection sensitivity for the multiplex NASBA assay was approximately 10−1 reverse transcription-PCR-detectable units (or PFU, as appropriate) per reaction. When representative ready-to-eat foods (deli sliced turkey and lettuce) were inoculated with various concentrations of each virus and processed for virus detection with the multiplex NASBA method, all three human enteric viruses were simultaneously detected at initial inoculum levels of 100 to 102 reverse transcription-PCR-detectable units (or PFU)/9 cm2 in both food commodities. The multiplex NASBA system provides rapid and simultaneous detection of clinically relevant food-borne viruses in a single reaction tube and may be a promising alternative to reverse transcription-PCR for the detection of viral contamination of foods. PMID:15528524

[Application of multiplex PCR for the screening of genotyping system for the rare blood groups Fy(a-), s-,k-,Di(b-) and Js(b-)].

PubMed

Jiao, Wei; Xie, Li; Li, Hailan; Lan, Jiao; Mo, Zhuning; Yang, Ziji; Liu, Fei; Xiao, Ruiping; He, Yunlei; Ye, Luyi; Zhu, Ziyan

2014-04-01

To screen rare blood groups Fy(a-), s-, k-, Di(b-) and Js(b-) in an ethnic Zhuang population. Sequence-specific primers were designed based on single nucleotide polymorphism (SNP) sites of blood group antigens Fy(b) and s. A specific multiplex PCR system I was established. Multiplex PCR system II was applied to detect alleles antigens Di(b), k, Js(b)1910 and Js(b) 2019 at the same time. The two systems was were used to screen for rare blood group antigens in 4490 randomly selected healthy donors of Guangxi Zhuang ethnic origin. We successfully made the multiplex PCR system I. We detected the rare blood group antigens using the two PCR system. There are five Fy(a-), three s(-), two Di(b-) in 4490 Guangxi zhuang random samples. The multiplex PCR system I has achieved good accuracy and stability. With multiplex PCR systems I and II, 4490 samples were screened. Five Fy(a-), three s(-) and two Di(b-) samples were discovered. Multiplex PCR is an effective methods, which can be used for high throughput screening of rare blood groups. The rare blood types of Guangxi Zhuang ethnic origin obtained through the screening can provide valuable information for compatible blood transfusion. Through screening we obtained precious rare blood type materials which can be used to improve the capability of compatible infusion and reduce the transfusion reactions.

Multiplexed SNP genotyping using the Qbead™ system: a quantum dot-encoded microsphere-based assay

PubMed Central

Xu, Hongxia; Sha, Michael Y.; Wong, Edith Y.; Uphoff, Janet; Xu, Yanzhang; Treadway, Joseph A.; Truong, Anh; O’Brien, Eamonn; Asquith, Steven; Stubbins, Michael; Spurr, Nigel K.; Lai, Eric H.; Mahoney, Walt

2003-01-01

We have developed a new method using the Qbead™ system for high-throughput genotyping of single nucleotide polymorphisms (SNPs). The Qbead system employs fluorescent Qdot™ semiconductor nanocrystals, also known as quantum dots, to encode microspheres that subsequently can be used as a platform for multiplexed assays. By combining mixtures of quantum dots with distinct emission wavelengths and intensities, unique spectral ‘barcodes’ are created that enable the high levels of multiplexing required for complex genetic analyses. Here, we applied the Qbead system to SNP genotyping by encoding microspheres conjugated to allele-specific oligonucleotides. After hybridization of oligonucleotides to amplicons produced by multiplexed PCR of genomic DNA, individual microspheres are analyzed by flow cytometry and each SNP is distinguished by its unique spectral barcode. Using 10 model SNPs, we validated the Qbead system as an accurate and reliable technique for multiplexed SNP genotyping. By modifying the types of probes conjugated to microspheres, the Qbead system can easily be adapted to other assay chemistries for SNP genotyping as well as to other applications such as analysis of gene expression and protein–protein interactions. With its capability for high-throughput automation, the Qbead system has the potential to be a robust and cost-effective platform for a number of applications. PMID:12682378

Optimized MOL-PCR for Characterization of Microbial Pathogens.

PubMed

Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

2016-01-06

Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium. Copyright © 2016 John Wiley & Sons, Inc.

A LDR-PCR approach for multiplex polymorphisms genotyping of severely degraded DNA with fragment sizes <100 bp.

PubMed

Zhang, Zhen; Wang, Bao-Jie; Guan, Hong-Yu; Pang, Hao; Xuan, Jin-Feng

2009-11-01

Reducing amplicon sizes has become a major strategy for analyzing degraded DNA typical of forensic samples. However, amplicon sizes in current mini-short tandem repeat-polymerase chain reaction (PCR) and mini-sequencing assays are still not suitable for analysis of severely degraded DNA. In this study, we present a multiplex typing method that couples ligase detection reaction with PCR that can be used to identify single nucleotide polymorphisms and small-scale insertion/deletions in a sample of severely fragmented DNA. This method adopts thermostable ligation for allele discrimination and subsequent PCR for signal enhancement. In this study, four polymorphic loci were used to assess the ability of this technique to discriminate alleles in an artificially degraded sample of DNA with fragment sizes <100 bp. Our results showed clear allelic discrimination of single or multiple loci, suggesting that this method might aid in the analysis of extremely degraded samples in which allelic drop out of larger fragments is observed.

Rapid Genotyping of Single Nucleotide Polymorphisms Influencing Warfarin Drug Response by Surface-Enhanced Laser Desorption and Ionization Time-of-Flight (SELDI-TOF) Mass Spectrometry

PubMed Central

Yang, Shangbin; Xu, LiHui; Wu, Haifeng M.

2010-01-01

Warfarin exhibits significant interindividual variability in dosing requirements. Different drug responses are partly attributed to the single nucleotide polymorphisms (SNPs) that influence either drug action or drug metabolism. Rapid genotyping of these SNPs helps clinicians to choose appropriate initial doses to quickly achieve anticoagulation effects and to prevent complications. We report a novel application of surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the rapid genotyping of SNPs that impact warfarin efficacy. The SNPs were first amplified by PCR and then underwent single base extension to generate the specific SNP product. Next, genetic variants displaying different masses were bound to Q10 anionic proteinChips and then genotyped by using SELDI-TOF MS in a multiplex fashion. SELDI-TOF MS offered unique properties of on-chip sample enrichment and clean-ups, which streamlined the testing procedures and eliminated many tedious experimental steps required by the conventional MS-based method. The turn-around time for genotyping three known warfarin-related SNPs, CYP2C9*2, CYP2C9*3, and VKORC1 3673G>A by SELDI-TOF MS was less than 5 hours. The analytical accuracy of this method was confirmed both by bidirectional DNA sequencing and by comparing the genotype results (n = 189) obtained by SELDI-TOF MS to reports from a clinical reference laboratory. This new multiplex genotyping method provides an excellent clinical laboratory platform to promote personalized medicine in warfarin therapy. PMID:20075209

Rapid genotyping of single nucleotide polymorphisms influencing warfarin drug response by surface-enhanced laser desorption and ionization time-of-flight (SELDI-TOF) mass spectrometry.

PubMed

Yang, Shangbin; Xu, LiHui; Wu, Haifeng M

2010-03-01

Warfarin exhibits significant interindividual variability in dosing requirements. Different drug responses are partly attributed to the single nucleotide polymorphisms (SNPs) that influence either drug action or drug metabolism. Rapid genotyping of these SNPs helps clinicians to choose appropriate initial doses to quickly achieve anticoagulation effects and to prevent complications. We report a novel application of surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the rapid genotyping of SNPs that impact warfarin efficacy. The SNPs were first amplified by PCR and then underwent single base extension to generate the specific SNP product. Next, genetic variants displaying different masses were bound to Q10 anionic proteinChips and then genotyped by using SELDI-TOF MS in a multiplex fashion. SELDI-TOF MS offered unique properties of on-chip sample enrichment and clean-ups, which streamlined the testing procedures and eliminated many tedious experimental steps required by the conventional MS-based method. The turn-around time for genotyping three known warfarin-related SNPs, CYP2C9*2, CYP2C9*3, and VKORC1 3673G>A by SELDI-TOF MS was less than 5 hours. The analytical accuracy of this method was confirmed both by bidirectional DNA sequencing and by comparing the genotype results (n = 189) obtained by SELDI-TOF MS to reports from a clinical reference laboratory. This new multiplex genotyping method provides an excellent clinical laboratory platform to promote personalized medicine in warfarin therapy.

High-Throughput Block Optical DNA Sequence Identification.

PubMed

Sagar, Dodderi Manjunatha; Korshoj, Lee Erik; Hanson, Katrina Bethany; Chowdhury, Partha Pratim; Otoupal, Peter Britton; Chatterjee, Anushree; Nagpal, Prashant

2018-01-01

Optical techniques for molecular diagnostics or DNA sequencing generally rely on small molecule fluorescent labels, which utilize light with a wavelength of several hundred nanometers for detection. Developing a label-free optical DNA sequencing technique will require nanoscale focusing of light, a high-throughput and multiplexed identification method, and a data compression technique to rapidly identify sequences and analyze genomic heterogeneity for big datasets. Such a method should identify characteristic molecular vibrations using optical spectroscopy, especially in the "fingerprinting region" from ≈400-1400 cm -1 . Here, surface-enhanced Raman spectroscopy is used to demonstrate label-free identification of DNA nucleobases with multiplexed 3D plasmonic nanofocusing. While nanometer-scale mode volumes prevent identification of single nucleobases within a DNA sequence, the block optical technique can identify A, T, G, and C content in DNA k-mers. The content of each nucleotide in a DNA block can be a unique and high-throughput method for identifying sequences, genes, and other biomarkers as an alternative to single-letter sequencing. Additionally, coupling two complementary vibrational spectroscopy techniques (infrared and Raman) can improve block characterization. These results pave the way for developing a novel, high-throughput block optical sequencing method with lossy genomic data compression using k-mer identification from multiplexed optical data acquisition. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR.

PubMed

Zhong, Qun; Bhattacharya, Smiti; Kotsopoulos, Steven; Olson, Jeff; Taly, Valérie; Griffiths, Andrew D; Link, Darren R; Larson, Jonathan W

2011-07-07

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented. This journal is © The Royal Society of Chemistry 2011

High-throughput multiplex HLA-typing by ligase detection reaction (LDR) and universal array (UA) approach.

PubMed

Consolandi, Clarissa

2009-01-01

One major goal of genetic research is to understand the role of genetic variation in living systems. In humans, by far the most common type of such variation involves differences in single DNA nucleotides, and is thus termed single nucleotide polymorphism (SNP). The need for improvement in throughput and reliability of traditional techniques makes it necessary to develop new technologies. Thus the past few years have witnessed an extraordinary surge of interest in DNA microarray technology. This new technology offers the first great hope for providing a systematic way to explore the genome. It permits a very rapid analysis of thousands genes for the purpose of gene discovery, sequencing, mapping, expression, and polymorphism detection. We generated a series of analytical tools to address the manufacturing, detection and data analysis components of a microarray experiment. In particular, we set up a universal array approach in combination with a PCR-LDR (polymerase chain reaction-ligation detection reaction) strategy for allele identification in the HLA gene.

Typing and comparative genome analysis of Brucella melitensis isolated from Lebanon.

PubMed

Abou Zaki, Natalia; Salloum, Tamara; Osman, Marwan; Rafei, Rayane; Hamze, Monzer; Tokajian, Sima

2017-10-16

Brucella melitensis is the main causative agent of the zoonotic disease brucellosis. This study aimed at typing and characterizing genetic variation in 33 Brucella isolates recovered from patients in Lebanon. Bruce-ladder multiplex PCR and PCR-RFLP of omp31, omp2a and omp2b were performed. Sixteen representative isolates were chosen for draft-genome sequencing and analyzed to determine variations in virulence, resistance, genomic islands, prophages and insertion sequences. Comparative whole-genome single nucleotide polymorphism analysis was also performed. The isolates were confirmed to be B. melitensis. Genome analysis revealed multiple virulence determinants and efflux pumps. Genome comparisons and single nucleotide polymorphisms divided the isolates based on geographical distribution but revealed high levels of similarity between the strains. Sequence divergence in B. melitensis was mainly due to lateral gene transfer of mobile elements. This is the first report of an in-depth genomic characterization of B. melitensis in Lebanon. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A multiplex single nucleotide polymorphism typing assay for detecting mutations that result in decreased fluoroquinolone susceptibility in Salmonella enterica serovars Typhi and Paratyphi A

PubMed Central

Song, Yajun; Roumagnac, Philippe; Weill, François-Xavier; Wain, John; Dolecek, Christiane; Mazzoni, Camila J.; Holt, Kathryn E.; Achtman, Mark

2010-01-01

Objectives Decreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers. Methods By using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (NalR) and/or decreased susceptibility to fluoroquinolones. Results This assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (NalR = 223 and NalS = 69) and 106 isolates of Salmonella Paratyphi A (NalR = 24 and NalS = 82). All of the 247 NalR Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143/223 for Salmonella Typhi and 18/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight NalS Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies. Conclusions The assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes. PMID:20511368

Kneallhazia (=Thelohania) Solenopsae infection rate of Pseudacteon Curvatus flies determined by multiplex PCR

USDA-ARS?s Scientific Manuscript database

A multiplex PCR method was developed and utilized to determine the Kneallhazia solenopsae infection rate of individual Pseudacteon curvatus flies in north-central Florida. Among P. curvatus flies infected with K. solenopsae, two amplicons were produced, one of 800 nucleotides from the P. curvatus 1...

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

PubMed Central

Girard, Laurie D.; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G.

2014-01-01

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets. PMID:25489607

Detection of clinically relevant copy number alterations in oral cancer progression using multiplexed droplet digital PCR.

PubMed

Hughesman, Curtis B; Lu, X J David; Liu, Kelly Y P; Zhu, Yuqi; Towle, Rebecca M; Haynes, Charles; Poh, Catherine F

2017-09-19

Copy number alterations (CNAs), a common genomic event during carcinogenesis, are known to affect a large fraction of the genome. Common recurrent gains or losses of specific chromosomal regions occur at frequencies that they may be considered distinctive features of tumoral cells. Here we introduce a novel multiplexed droplet digital PCR (ddPCR) assay capable of detecting recurrent CNAs that drive tumorigenesis of oral squamous cell carcinoma. Applied to DNA extracted from oral cell lines and clinical samples of various disease stages, we found good agreement between CNAs detected by our ddPCR assay with those previously reported using comparative genomic hybridization or single nucleotide polymorphism arrays. Furthermore, we demonstrate that the ability to target specific locations of the genome permits detection of clinically relevant oncogenic events such as small, submicroscopic homozygous deletions. Additional capabilities of the multiplexed ddPCR assay include the ability to infer ploidy level, quantify the change in copy number of target loci with high-level gains, and simultaneously assess the status and viral load for high-risk human papillomavirus types 16 and 18. This novel multiplexed ddPCR assay therefore may have clinical value in differentiating between benign oral lesions from those that are at risk of progressing to oral cancer.

Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

PubMed Central

Bernardo, Amy; Wang, Shan; St. Amand, Paul; Bai, Guihua

2015-01-01

With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat ( Triticum aestivum L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat. PMID:26625271

Analysis of LDLR mutations in familial hypercholesterolemia patients in Greece by use of the NanoChip microelectronic array technology.

PubMed

Laios, Eleftheria; Drogari, Euridiki

2006-12-01

Three mutations in the low density lipoprotein receptor (LDLR) gene account for 49% of familial hypercholesterolemia (FH) cases in Greece. We used the microelectronic array technology of the NanoChip Molecular Biology Workstation to develop a multiplex method to analyze these single-nucleotide polymorphisms (SNPs). Primer pairs amplified the region encompassing each SNP. The biotinylated PCR amplicon was electronically addressed to streptavidin-coated microarray sites. Allele-specific fluorescently labeled oligonucleotide reporters were designed and used for detection of wild-type and SNP sequences. Genotypes were compared to PCR-restriction fragment length polymorphism (PCR-RFLP). We developed three monoplex assays (1 SNP/site) and an optimized multiplex assay (3SNPs/site). We performed 92 Greece II, 100 Genoa, and 98 Afrikaner-2 NanoChip monoplex assays (addressed to duplicate sites and analyzed separately). Of the 580 monoplex genotypings (290 samples), 579 agreed with RFLP. Duplicate sites of one sample were not in agreement with each other. Of the 580 multiplex genotypings, 576 agreed with the monoplex results. Duplicate sites of three samples were not in agreement with each other, indicating requirement for repetition upon which discrepancies were resolved. The multiplex assay detects common LDLR mutations in Greek FH patients and can be extended to accommodate additional mutations.

High-Performance Multiplex SNP Analysis of Three Hemochromatosis-Related Mutations With Capillary Array Electrophoresis Microplates

PubMed Central

Medintz, Igor; Wong, Wendy W.; Berti, Lorenzo; Shiow, Lawrence; Tom, Jennifer; Scherer, James; Sensabaugh, George; Mathies, Richard A.

2001-01-01

An assay is described for high-throughput single nucleotide polymorphism (SNP) genotyping on a microfabricated capillary array electrophoresis (CAE) microchip. The assay targets the three common variants at the HFE locus associated with the genetic disease hereditary hemochromatosis (HHC). The assay employs allele-specific PCR (ASPCR) for the C282Y (845g->a), H63D (187c->g), and S65C (193a->t) variants using fluorescently-labeled energy-transfer (ET) allele-specific primers. Using a 96-channel radial CAE microplate, the labeled ASPCR products generated from 96 samples in a reference Caucasian population are simultaneously separated with single-base-pair resolution and genotyped in under 10 min. Detection is accomplished with a laser-excited rotary four-color fluorescence scanner. The allele-specific amplicons are differentiated on the basis of both their size and the color of the label emission. This study is the first demonstration of the combined use of ASPCR with ET primers and microfabricated radial CAE microplates to perform multiplex SNP analyses in a clinically relevant population. PMID:11230165

Single-tube, non-isotopic, multiplex PCR/OLA assay and sequence-coded separation for simultaneous screening of 31 cystic fibrosis mutations

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

Brinson, E.C.; Adriano, T.; Bloch, W.

1994-09-01

We have developed a rapid, single-tube, non-isotopic assay that screens a patient sample for the presence of 31 cystic fibrosis (CF) mutations. This assay can identify these mutations in a single reaction tube and a single electrophoresis run. Sample preparation is a simple, boil-and-go procedure, completed in less than an hour. The assay is composed of a 15-plex PCR, followed by a 61-plex oligonucleotide ligation assay (OLA), and incorporates a novel detection scheme, Sequence Coded Separation. Initially, the multiplex PCR amplifies 15 relevant segments of the CFTR gene, simultaneously. These PCR amplicons serve as templates for the multiplex OLA, whichmore » detects the normal or mutant allele at all loci, simultaneously. Each polymorphic site is interrogated by three oligonucleotide probes, a common probe and two allele-specific probes. Each common probe is tagged with a fluorescent dye, and the competing normal and mutant allelic probes incorporate different, non-nucleotide, mobility modifiers. These modifiers are composed of hexaethylene oxide (HEO) units, incorporated as HEO phosphoramidite monomers during automated DNA synthesis. The OLA is based on both probe hybridization and the ability of DNA ligase to discriminate single base mismatches at the junction between paired probes. Each single tube assay is electrophoresed in a single gel lane of a 4-color fluorescent DNA sequencer (Applied Biosystems, Model 373A). Each of the ligation products is identified by its unique combination of electrophoretic mobility and one of three colors. The fourth color is reserved for the in-lane size standard, used by GENESCAN{sup TM} software (Applied Biosystems) to size the OLA electrophoresis products. The Genotyper{sub TM} software (Applied Biosystems) decodes these Sequence-Coded-Separation data to create a patient summary report for all loci tested.« less

Clinical Relevance of Multiple Single-Nucleotide Polymorphisms in Pneumocystis jirovecii Pneumonia: Development of a Multiplex PCR-Single-Base-Extension Methodology▿

PubMed Central

Esteves, F.; Gaspar, J.; De Sousa, B.; Antunes, F.; Mansinho, K.; Matos, O.

2011-01-01

Pneumocystis jirovecii pneumonia (PcP) is a major cause of respiratory illness in patients with AIDS. The identification of multiple single-nucleotide polymorphisms (SNPs) at three distinct P. jirovecii loci encoding dihydrofolate reductase (DHFR), mitochondrial large-subunit rRNA (mtLSU rRNA), and superoxide dismutase (SOD) was achieved using multiplex-PCR (MPCR) followed by direct sequencing and two single-base extension (SBE) techniques. Four SNPs (DHFR312, mt85, SOD215, and SOD110), correlated previously with parameters of disease, were amplified and genotyped simultaneously. The concordance of results between the standard sequencing technique (direct sequencing) and SBE analysis was 96.9% for the acrylamide gel electrophoresis and 98.4% for the capillary electrophoresis. The cross-genetic analysis established several statistical associations among the SNPs studied: mt85C-SOD110T, SOD110T-SOD215C, and SOD110C-SOD215T. These results were confirmed by cluster analysis. Data showed that among the isolates with low to moderate parasite burden, the highest percentages of DHFR312C, mt85C, SOD110T, and SOD215C were detected, whereas for high parasite burden cases the highest frequencies were observed among isolates with DHFR312T, mt85T, SOD110C, and SOD215T. The polymorphisms studied were shown to be suitable genetic targets potentially correlated with PcP clinical data that can be used as predictors of outcome in further studies to help clinical decision-making in the management of PcP. The MPCR/SBE protocol described for the first time in the present study was shown to be a rapid, highly accurate method for genotyping P. jirovecii SNPs encoded by different loci that could be used for epidemiological studies and as an additional procedure for the prognostic classification and diagnosis of PcP. PMID:21389160

Clinical relevance of multiple single-nucleotide polymorphisms in Pneumocystis jirovecii Pneumonia: development of a multiplex PCR-single-base-extension methodology.

PubMed

Esteves, F; Gaspar, J; De Sousa, B; Antunes, F; Mansinho, K; Matos, O

2011-05-01

Pneumocystis jirovecii pneumonia (PcP) is a major cause of respiratory illness in patients with AIDS. The identification of multiple single-nucleotide polymorphisms (SNPs) at three distinct P. jirovecii loci encoding dihydrofolate reductase (DHFR), mitochondrial large-subunit rRNA (mtLSU rRNA), and superoxide dismutase (SOD) was achieved using multiplex-PCR (MPCR) followed by direct sequencing and two single-base extension (SBE) techniques. Four SNPs (DHFR312, mt85, SOD215, and SOD110), correlated previously with parameters of disease, were amplified and genotyped simultaneously. The concordance of results between the standard sequencing technique (direct sequencing) and SBE analysis was 96.9% for the acrylamide gel electrophoresis and 98.4% for the capillary electrophoresis. The cross-genetic analysis established several statistical associations among the SNPs studied: mt85C-SOD110T, SOD110T-SOD215C, and SOD110C-SOD215T. These results were confirmed by cluster analysis. Data showed that among the isolates with low to moderate parasite burden, the highest percentages of DHFR312C, mt85C, SOD110T, and SOD215C were detected, whereas for high parasite burden cases the highest frequencies were observed among isolates with DHFR312T, mt85T, SOD110C, and SOD215T. The polymorphisms studied were shown to be suitable genetic targets potentially correlated with PcP clinical data that can be used as predictors of outcome in further studies to help clinical decision-making in the management of PcP. The MPCR/SBE protocol described for the first time in the present study was shown to be a rapid, highly accurate method for genotyping P. jirovecii SNPs encoded by different loci that could be used for epidemiological studies and as an additional procedure for the prognostic classification and diagnosis of PcP.

Highly multiplexed subcellular RNA sequencing in situ

PubMed Central

Lee, Je Hyuk; Daugharthy, Evan R.; Scheiman, Jonathan; Kalhor, Reza; Ferrante, Thomas C.; Yang, Joyce L.; Terry, Richard; Jeanty, Sauveur S. F.; Li, Chao; Amamoto, Ryoji; Peters, Derek T.; Turczyk, Brian M.; Marblestone, Adam H.; Inverso, Samuel A.; Bernard, Amy; Mali, Prashant; Rios, Xavier; Aach, John; Church, George M.

2014-01-01

Understanding the spatial organization of gene expression with single nucleotide resolution requires localizing the sequences of expressed RNA transcripts within a cell in situ. Here we describe fluorescent in situ RNA sequencing (FISSEQ), in which stably cross-linked cDNA amplicons are sequenced within a biological sample. Using 30-base reads from 8,742 genes in situ, we examined RNA expression and localization in human primary fibroblasts using a simulated wound healing assay. FISSEQ is compatible with tissue sections and whole mount embryos, and reduces the limitations of optical resolution and noisy signals on single molecule detection. Our platform enables massively parallel detection of genetic elements, including gene transcripts and molecular barcodes, and can be used to investigate cellular phenotype, gene regulation, and environment in situ. PMID:24578530

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.

Direct Multiplex PCR (dmPCR) for the Identification of Six Phlebotomine Sand Fly Species (Diptera: Psychodidae), Including Major Leishmania Vectors of the Mediterranean.

PubMed

Giantsis, Ioannis A; Chaskopoulou, Alexandra; Claude Bon, Marie

2017-02-01

Sand flies (Diptera: Psychodidae, subfamily Phlebotominae) are hematophagous insects that are known to transmit several anthroponotic and zoonotic diseases. Reliable identification of sand flies at species level is crucial for their surveillance, the detection and spread of their pathogens, and the implementation of targeted pest control strategies. Here, we designed a novel, time-saving, cost-effective and easy-to-apply molecular methodology, which avoids sequencing, for the identification of the following six Eastern Mediterranean sand fly species: Phebotomus perfiliewi Parrot, Phebotomus simici Theodor, Phebotomus tobbi Adler and Theodor, Phebotomus papatasi Scopoli, Sergentomyia dentata Sinton, and Sergentomyia minuta Theodor. This methodology, which is a multiplex PCR assay using one common and six diagnostic primers, is based on species-specific single-nucleotide polymorphisms of the nuclear 18S rRNA gene. Amplification products were easily and reliably separated in agarose gel yielding one single clear band of diagnostic size for each species. Further, we verified its successful application on tissue samples that were immersed directly to the PCR mix, skipping DNA extraction. The direct multiplex PCR can be completed in < 3 h, including all operating procedures, and costing no more than a simple PCR. The applicability of this methodology in the detection of hybrids is an additional considerable benefit. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Thirtyfold multiplex genotyping of the p53 gene using solid phase capturable dideoxynucleotides and mass spectrometry.

PubMed

Kim, Sobin; Ulz, Michael E; Nguyen, Tuan; Li, Chi-Ming; Sato, Takaaki; Tycko, Benjamin; Ju, Jingyue

2004-05-01

A mass spectrometry (MS) based multiplex genotyping method using solid phase capturable (SPC) dideoxynucleotides and single base extension (SBE), named the SPC-SBE, has been developed for mutation detection. We report here the simultaneous genotyping of 30 potential point mutation sites in exons 5, 7, and 8 of the human p53 gene in one tube using the SPC-SBE method. The 30 mutation sites, including the most frequently mutated p53 codons, were chosen to explore the high multiplexing scope of the SPC-SBE method. Thirty primers specific to each potential mutation site were designed to yield SBE products with sufficient mass differences. This was achieved by tuning the mass of some primers using modified nucleotides. Genomic DNA was amplified by multiplex PCR to produce amplicons of the three p53 exons. The 30 primers were combined with the PCR products and biotinylated dideoxynucleotides for SBE to generate 3'-biotinylated extension DNA products. These products were then captured by streptavidin-coated magnetic beads, while the unextended primers and other components in the reaction were washed away. The pure extension DNA products were subsequently released from the solid phase and analyzed with MS. We simultaneously genotyped 30 potential mutation sites in the p53 gene from Wilms' tumor, head and neck tumor, and colorectal tumor. Both homozygous and heterozygous genotypes were accurately determined with digital resolution. This is the highest level of multiplex genotyping reported thus far using MS, indicating that the approach might be applicable to screening a repertoire of genotypes in candidate genes as potential disease markers.

Identification of Forensically Important Calliphoridae and Sarcophagidae Species Collected in Korea Using SNaPshot Multiplex System Targeting the Cytochrome c Oxidase Subunit I Gene

PubMed Central

Park, Ji Hye

2018-01-01

Estimation of postmortem interval (PMI) is paramount in modern forensic investigation. After the disappearance of the early postmortem phenomena conventionally used to estimate PMI, entomologic evidence provides important indicators for PMI estimation. The age of the oldest fly larvae or pupae can be estimated to pinpoint the time of oviposition, which is considered the minimum PMI (PMImin). The development rate of insects is usually temperature dependent and species specific. Therefore, species identification is mandatory for PMImin estimation using entomological evidence. The classical morphological identification method cannot be applied when specimens are damaged or have not yet matured. To overcome this limitation, some investigators employ molecular identification using mitochondrial cytochrome c oxidase subunit I (COI) nucleotide sequences. The molecular identification method commonly uses Sanger's nucleotide sequencing and molecular phylogeny, which are complex and time consuming and constitute another obstacle for forensic investigators. In this study, instead of using conventional Sanger's nucleotide sequencing, single-nucleotide polymorphisms (SNPs) in the COI gene region, which are unique between fly species, were selected and targeted for single-base extension (SBE) technology. These SNPs were genotyped using a SNaPshot® kit. Eleven Calliphoridae and seven Sarcophagidae species were covered. To validate this genotyping, fly DNA samples (103 adults, 84 larvae, and 4 pupae) previously confirmed by DNA barcoding were used. This method worked quickly with minimal DNA, providing a potential alternative to conventional DNA barcoding. Consisting of only a few simple electropherogram peaks, the results were more straightforward compared with those of the conventional DNA barcoding produced by Sanger's nucleotide sequencing. PMID:29682531

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

Technologies in the Whole-Genome Age: MALDI-TOF-Based Genotyping.

PubMed

Vogel, Nicolas; Schiebel, Katrin; Humeny, Andreas

2009-01-01

With the decipherment of the human genome, new questions have moved into the focus of today's research. One key aspect represents the discovery of DNA variations capable to influence gene transcription, RNA splicing, or regulating processes, and their link to pathology. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) is a powerful tool for the qualitative investigation and relative quantification of variations like single nucleotide polymorphisms, DNA methylation, microsatellite instability, or loss of heterozygosity. After its introduction into proteomics, efforts were made to adopt this technique to DNA analysis. Initially intended for peptide/protein analysis, it held several difficulties for application to nucleic acids. Today, MALDI-TOF-MS has reached worldwide acceptance and application in nucleic acid research, with a wide spectrum of methods being available. One of the most versatile approaches relies on primer extension to genotype single alleles, microsatellite repeat lengths or the methylation status of a given cytosine. Optimized methods comprising intelligent primer design and proper nucleotide selection for primer extension enabled multiplexing of reactions, rendering the analysis more economic due to parallel genotyping of several alleles in a single experiment. Laboratories equipped with MALDI-TOF-MS possess a universal technical platform for the analysis of a large variety of different molecules.

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

NASA Astrophysics Data System (ADS)

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Qiang, Yu; Liu, Ke; Cui, Chengjun; Chen, Jianhua; Huang, Qing; Hu, Jun; Wang, Lianhui; Huang, Wei; Shi, Yongyong; Fan, Chunhai

2017-04-01

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ~10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level.

DNA origami-based shape IDs for single-molecule nanomechanical genotyping

PubMed Central

Zhang, Honglu; Chao, Jie; Pan, Dun; Liu, Huajie; Qiang, Yu; Liu, Ke; Cui, Chengjun; Chen, Jianhua; Huang, Qing; Hu, Jun; Wang, Lianhui; Huang, Wei; Shi, Yongyong; Fan, Chunhai

2017-01-01

Variations on DNA sequences profoundly affect how we develop diseases and respond to pathogens and drugs. Atomic force microscopy (AFM) provides a nanomechanical imaging approach for genetic analysis with nanometre resolution. However, unlike fluorescence imaging that has wavelength-specific fluorophores, the lack of shape-specific labels largely hampers widespread applications of AFM imaging. Here we report the development of a set of differentially shaped, highly hybridizable self-assembled DNA origami nanostructures serving as shape IDs for magnified nanomechanical imaging of single-nucleotide polymorphisms. Using these origami shape IDs, we directly genotype single molecules of human genomic DNA with an ultrahigh resolution of ∼10 nm and the multiplexing ability. Further, we determine three types of disease-associated, long-range haplotypes in samples from the Han Chinese population. Single-molecule analysis allows robust haplotyping even for samples with low labelling efficiency. We expect this generic shape ID-based nanomechanical approach to hold great potential in genetic analysis at the single-molecule level. PMID:28382928

Protocadherin α (PCDHA) as a novel susceptibility gene for autism

PubMed Central

Anitha, Ayyappan; Thanseem, Ismail; Nakamura, Kazuhiko; Yamada, Kazuo; Iwayama, Yoshimi; Toyota, Tomoko; Iwata, Yasuhide; Suzuki, Katsuaki; Sugiyama, Toshiro; Tsujii, Masatsugu; Yoshikawa, Takeo; Mori, Norio

2013-01-01

Background Synaptic dysfunction has been shown to be involved in the pathogenesis of autism. We hypothesized that the protocadherin α gene cluster (PCDHA), which is involved in synaptic specificity and in serotonergic innervation of the brain, could be a suitable candidate gene for autism. Methods We examined 14 PCDHA single nucleotide polymorphisms (SNPs) for genetic association with autism in DNA samples of 3211 individuals (841 families, including 574 multiplex families) obtained from the Autism Genetic Resource Exchange. Results Five SNPs (rs251379, rs1119032, rs17119271, rs155806 and rs17119346) showed significant associations with autism. The strongest association (p < 0.001) was observed for rs1119032 (z score of risk allele G = 3.415) in multiplex families; SNP associations withstand multiple testing correction in multiplex families (p = 0.041). Haplotypes involving rs1119032 showed very strong associations with autism, withstanding multiple testing corrections. In quantitative transmission disequilibrium testing of multiplex families, the G allele of rs1119032 showed a significant association (p = 0.033) with scores on the Autism Diagnostic Interview–Revised (ADI-R)_D (early developmental abnormalities). We also found a significant difference in the distribution of ADI-R_A (social interaction) scores between the A/A, A/G and G/G genotypes of rs17119346 (p = 0.002). Limitations Our results should be replicated in an independent population and/or in samples of different racial backgrounds. Conclusion Our study provides strong genetic evidence of PCDHA as a potential candidate gene for autism. PMID:23031252

Developing a new nonbinary SNP fluorescent multiplex detection system for forensic application in China.

PubMed

Liu, Yanfang; Liao, Huidan; Liu, Ying; Guo, Juanjuan; Sun, Yi; Fu, Xiaoliang; Xiao, Ding; Cai, Jifeng; Lan, Lingmei; Xie, Pingli; Zha, Lagabaiyila

2017-04-01

Nonbinary single-nucleotide polymorphisms (SNPs) are potential forensic genetic markers because their discrimination power is greater than that of normal binary SNPs, and that they can detect highly degraded samples. We previously developed a nonbinary SNP multiplex typing assay. In this study, we selected additional 20 nonbinary SNPs from the NCBI SNP database and verified them through pyrosequencing. These 20 nonbinary SNPs were analyzed using the fluorescent-labeled SNaPshot multiplex SNP typing method. The allele frequencies and genetic parameters of these 20 nonbinary SNPs were determined among 314 unrelated individuals from Han populations from China. The total power of discrimination was 0.9999999999994, and the cumulative probability of exclusion was 0.9986. Moreover, the result of the combination of this 20 nonbinary SNP assay with the 20 nonbinary SNP assay we previously developed demonstrated that the cumulative probability of exclusion of the 40 nonbinary SNPs was 0.999991 and that no significant linkage disequilibrium was observed in all 40 nonbinary SNPs. Thus, we concluded that this new system consisting of new 20 nonbinary SNPs could provide highly informative polymorphic data which would be further used in forensic application and would serve as a potentially valuable supplement to forensic DNA analysis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ

PubMed Central

Mignardi, Marco; Mezger, Anja; Qian, Xiaoyan; La Fleur, Linnea; Botling, Johan; Larsson, Chatarina; Nilsson, Mats

2015-01-01

In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ. PMID:26240388

Is α‐T catenin (VR22) an Alzheimer's disease risk gene?

PubMed Central

Bertram, Lars; Mullin, Kristina; Parkinson, Michele; Hsiao, Monica; Moscarillo, Thomas J; Wagner, Steven L; Becker, K David; Velicelebi, Gonul; Blacker, Deborah; Tanzi, Rudolph E

2007-01-01

Background Recently, conflicting reports have been published on the potential role of genetic variants in the α‐T catenin gene (VR22; CTNNA3) on the risk for Alzheimer's disease. In these papers, evidence for association is mostly observed in multiplex families with Alzheimer's disease, whereas case–control samples of sporadic Alzheimer's disease are predominantly negative. Methods After sequencing VR22 in multiplex families with Alzheimer's disease linked to chromosome 10q21, we identified a novel non‐synonymous (Ser596Asn; rs4548513) single nucleotide polymorphism (SNP). This and four non‐coding SNPs were assessed in two independent samples of families with Alzheimer's disease, one with 1439 subjects from 437 multiplex families with Alzheimer's disease and the other with 489 subjects from 217 discordant sibships. Results A weak association with the Ser596Asn SNP in the multiplex sample, predominantly in families with late‐onset Alzheimer's disease (p = 0.02), was observed. However, this association does not seem to contribute substantially to the chromosome 10 Alzheimer's disease linkage signal that we and others have reported previously. No evidence was found of association with any of the four additional SNPs tested in the multiplex families with Alzheimer's disease. Finally, the Ser596Asn change was not associated with the risk for Alzheimer's disease in the independent discordant sibship sample. Conclusions This is the first study to report evidence of an association between a potentially functional, non‐synonymous SNP in VR22 and the risk for Alzheimer's disease. As the underlying effects are probably small, and are only seen in families with multiple affected members, the population‐wide significance of this finding remains to be determined. PMID:17209133

Reinvestigations of six unusual paternity cases by typing of autosomal single-nucleotide polymorphisms.

PubMed

Børsting, Claus; Morling, Niels

2012-02-01

In some relationship cases, the initial investigations of autosomal short tandem repeats (STRs) lead to an ambiguous conclusion and supplementary investigations become necessary. Six unusual paternity cases were previously investigated by other researchers and published as case work examples in forensic journals. Here, the cases were reinvestigated by typing the samples for 49 autosomal single-nucleotide polymorphisms (SNPs) using the SNPforID multiplex assay. Three cases were solved by the SNP investigation without the need for any additional testing. In two cases, the SNP results supported the conclusions based on STRs. In the last case, the SNP results spoke in favor of paternity, and the combined paternity index based on autosomal STRs and SNPs was 12.3 billion. Nevertheless, the alleged father was excluded by X-chromosome typing. The case work examples underline the importance of performing supplementary investigations, and they advocate for the implementation of several panels that may be used in the highly unusual cases. Panels with SNPs or other markers with low mutation probabilities are preferable as supplementary markers, because the risk of detecting (additional) mutations is very low. © 2012 American Association of Blood Banks.

Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis.

PubMed

Smola, Matthew J; Rice, Greggory M; Busan, Steven; Siegfried, Nathan A; Weeks, Kevin M

2015-11-01

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPE probing and to construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles and entire transcriptomes.

A Single Molecular Beacon Probe Is Sufficient for the Analysis of Multiple Nucleic Acid Sequences

PubMed Central

Gerasimova, Yulia V.; Hayson, Aaron; Ballantyne, Jack; Kolpashchikov, Dmitry M.

2010-01-01

Molecular beacon (MB) probes are dual-labeled hairpin-shaped oligodeoxyribonucleotides that are extensively used for real-time detection of specific RNA/DNA analytes. In the MB probe, the loop fragment is complementary to the analyte: therefore, a unique probe is required for the analysis of each new analyte sequence. The conjugation of an oligonucleotide with two dyes and subsequent purification procedures add to the cost of MB probes, thus reducing their application in multiplex formats. Here we demonstrate how one MB probe can be used for the analysis of an arbitrary nucleic acid. The approach takes advantage of two oligonucleotide adaptor strands, each of which contains a fragment complementary to the analyte and a fragment complementary to an MB probe. The presence of the analyte leads to association of MB probe and the two DNA strands in quadripartite complex. The MB probe fluorescently reports the formation of this complex. In this design, the MB does not bind the analyte directly; therefore, the MB sequence is independent of the analyte. In this study one universal MB probe was used to genotype three human polymorphic sites. This approach promises to reduce the cost of multiplex real-time assays and improve the accuracy of single-nucleotide polymorphism genotyping. PMID:20665615

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Li, Qingbo; Lu, Xiandan

1998-04-21

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.; Li, Qingbo; Lu, Xiandan

1996-12-10

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, E.S.; Li, Q.; Lu, X.

1998-04-21

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

Multiplexed capillary electrophoresis system

DOEpatents

Yeung, E.S.; Chang, H.T.; Fung, E.N.; Li, Q.; Lu, X.

1996-12-10

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

OzPythonPlex: An optimised forensic STR multiplex assay set for the Australasian carpet python (Morelia spilota).

PubMed

Ciavaglia, Sherryn; Linacre, Adrian

2018-05-01

Reptile species, and in particular snakes, are protected by national and international agreements yet are commonly handled illegally. To aid in the enforcement of such legislation, we report on the development of three 11-plex assays from the genome of the carpet python to type 24 loci of tetra-nucleotide and penta-nucleotide repeat motifs (pure, compound and complex included). The loci range in size between 70 and 550 bp. Seventeen of the loci are newly characterised with the inclusion of seven previously developed loci to facilitate cross-comparison with previous carpet python genotyping studies. Assays were optimised in accordance with human forensic profiling kits using one nanogram template DNA. Three loci are included in all three of the multiplex reactions as quality assurance markers, to ensure sample identity and genotyping accuracy is maintained across the three profiling assays. Allelic ladders have been developed for the three assays to ensure consistent and precise allele designation. A DNA reference database of allele frequencies is presented based on 249 samples collected from throughout the species native range. A small number of validation tests are conducted to demonstrate the utility of these multiplex assays. We suggest further appropriate validation tests that should be conducted prior to the application of the multiplex assays in criminal investigations involving carpet pythons. Copyright © 2018 Elsevier B.V. All rights reserved.

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, R.B.; Kimball, A.W.; Gesteland, R.F.; Ferguson, F.M.; Dunn, D.M.; Di Sera, L.J.; Cherry, J.L.

1995-11-28

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, the enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots. 9 figs.

Automated hybridization/imaging device for fluorescent multiplex DNA sequencing

DOEpatents

Weiss, Robert B.; Kimball, Alvin W.; Gesteland, Raymond F.; Ferguson, F. Mark; Dunn, Diane M.; Di Sera, Leonard J.; Cherry, Joshua L.

1995-01-01

A method is disclosed for automated multiplex sequencing of DNA with an integrated automated imaging hybridization chamber system. This system comprises an hybridization chamber device for mounting a membrane containing size-fractionated multiplex sequencing reaction products, apparatus for fluid delivery to the chamber device, imaging apparatus for light delivery to the membrane and image recording of fluorescence emanating from the membrane while in the chamber device, and programmable controller apparatus for controlling operation of the system. The multiplex reaction products are hybridized with a probe, then an enzyme (such as alkaline phosphatase) is bound to a binding moiety on the probe, and a fluorogenic substrate (such as a benzothiazole derivative) is introduced into the chamber device by the fluid delivery apparatus. The enzyme converts the fluorogenic substrate into a fluorescent product which, when illuminated in the chamber device with a beam of light from the imaging apparatus, excites fluorescence of the fluorescent product to produce a pattern of hybridization. The pattern of hybridization is imaged by a CCD camera component of the imaging apparatus to obtain a series of digital signals. These signals are converted by the controller apparatus into a string of nucleotides corresponding to the nucleotide sequence an automated sequence reader. The method and apparatus are also applicable to other membrane-based applications such as colony and plaque hybridization and Southern, Northern, and Western blots.

Parallel gene analysis with allele-specific padlock probes and tag microarrays

PubMed Central

Banér, Johan; Isaksson, Anders; Waldenström, Erik; Jarvius, Jonas; Landegren, Ulf; Nilsson, Mats

2003-01-01

Parallel, highly specific analysis methods are required to take advantage of the extensive information about DNA sequence variation and of expressed sequences. We present a scalable laboratory technique suitable to analyze numerous target sequences in multiplexed assays. Sets of padlock probes were applied to analyze single nucleotide variation directly in total genomic DNA or cDNA for parallel genotyping or gene expression analysis. All reacted probes were then co-amplified and identified by hybridization to a standard tag oligonucleotide array. The technique was illustrated by analyzing normal and pathogenic variation within the Wilson disease-related ATP7B gene, both at the level of DNA and RNA, using allele-specific padlock probes. PMID:12930977

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.

Multiplex Allele-Specific Amplification from Whole Blood for Detecting Multiple Polymorphisms Simultaneously

PubMed Central

Zhu, Jianjie; Chen, Lanxin; Mao, Yong; Zhou, Huan

2013-01-01

Allele-specific amplification on the basis of polymerase chain reaction (PCR) has been widely used for single-nucleotide polymorphism (SNP) genotyping. However, the extraction of PCR-compatible genomic DNA from whole blood is usually required. This process is complicated and tedious, and is prone to cause cross-contamination between samples. To facilitate direct PCR amplification from whole blood without the extraction of genomic DNA, we optimized the pH value of PCR solution and the concentrations of magnesium ions and facilitator glycerol. Then, we developed multiplex allele-specific amplifications from whole blood and applied them to a case–control study. In this study, we successfully established triplex, five-plex, and eight-plex allele-specific amplifications from whole blood for determining the distribution of genotypes and alleles of 14 polymorphisms in 97 gastric cancer patients and 141 healthy controls. Statistical analysis results showed significant association of SNPs rs9344, rs1799931, and rs1800629 with the risk of gastric cancer. This method is accurate, time-saving, cost-effective, and easy-to-do, especially suitable for clinical prediction of disease susceptibility. PMID:23072573

Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

PubMed Central

Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

2017-01-01

Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

Single-cell high resolution melting analysis: A novel, generic, pre-implantation genetic diagnosis (PGD) method applied to cystic fibrosis (HRMA CF-PGD).

PubMed

Destouni, A; Poulou, M; Kakourou, G; Vrettou, C; Tzetis, M; Traeger-Synodinos, J; Kitsiou-Tzeli, S

2016-03-01

Institutions offering CF-PGD face the challenge of developing and optimizing single cell genotyping protocols that should cover for the extremely heterogeneous CF mutation spectrum. Here we report the development and successful clinical application of a generic CF-PGD protocol to facilitate direct detection of any CFTR nucleotide variation(s) by HRMA and simultaneous confirmation of diagnosis through haplotype analysis. A multiplex PCR was optimized supporting co-amplification of any CFTR exon-region, along with 6 closely linked STRs. Single cell genotypes were established through HRM analysis following melting of the 2nd round PCR products and were confirmed by STR haplotype analysis of the 1st PCR products. The protocol was validated pre-clinically, by testing 208 single lymphocytes, isolated from whole blood samples from 4 validation family trios. Fifteen PGD cycles were performed and 103 embryos were biopsied. In 15 clinical PGD cycles, genotypes were achieved in 88/93 (94.6%) embryo biopsy samples, of which 57/88 (64.8%) were deemed genetically suitable for embryo transfer. Amplification failed at all loci for 10/103 blastomeres biopsied from poor quality embryos. Six clinical pregnancies were achieved (2 twin, 4 singletons). PGD genotypes were confirmed following conventional amniocentesis or chorionic villus sampling in all achieved pregnancies. The single cell HRMA CF-PGD protocol described herein is a flexible, generic, low cost and robust genotyping method, which facilitates the analysis of any CFTR genotype combination. Single-cell HRMA can be beneficial to other clinical settings, for example the detection of single nucleotide variants in single cells derived from clinical tumor samples. Copyright © 2015 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Melting analysis on microbeads in rapid temperature-gradient inside microchannels for single nucleotide polymorphisms detectiona)

PubMed Central

Li, Kan-Chien; Ding, Shih-Torng; Lin, En-Chung; Wang, Lon (Alex); Lu, Yen-Wen

2014-01-01

A continuous-flow microchip with a temperature gradient in microchannels was utilized to demonstrate spatial melting analysis on microbeads for clinical Single Nucleotide Polymorphisms (SNPs) genotyping on animal genomic DNA. The chip had embedded heaters and thermometers, which created a rapid and yet stable temperature gradient between 60 °C and 85 °C in a short distance as the detection region. The microbeads, which served as mobile supports carrying the target DNA and fluorescent dye, were transported across the temperature gradient. As the surrounding temperature increased, the fluorescence signals of the microbeads decayed with this relationship being acquired as the melting curve. Fast DNA denaturation, as a result of the improved heat transfer and thermal stability due to scaling, was also confirmed. Further, each individual microbead could potentially bear different sequences and pass through the detection region, one by one, for a series of melting analysis, with multiplex, high-throughput capability being possible. A prototype was tested with target DNA samples in different genotypes (i.e., wild and mutant types) with a SNP location from Landrace sows. The melting temperatures were obtained and compared to the ones using a traditional tube-based approach. The results showed similar levels of SNP discrimination, validating our proposed technique for scanning homozygotes and heterozygotes to distinguish single base changes for disease research, drug development, medical diagnostics, agriculture, and animal production. PMID:25553186

Forensic SNP genotyping with SNaPshot: Technical considerations for the development and optimization of multiplexed SNP assays.

PubMed

Fondevila, M; Børsting, C; Phillips, C; de la Puente, M; Consortium, Euroforen-NoE; Carracedo, A; Morling, N; Lareu, M V

2017-01-01

This review explores the key factors that influence the optimization, routine use, and profile interpretation of the SNaPshot single-base extension (SBE) system applied to forensic single-nucleotide polymorphism (SNP) genotyping. Despite being a mainly complimentary DNA genotyping technique to routine STR profiling, use of SNaPshot is an important part of the development of SNP sets for a wide range of forensic applications with these markers, from genotyping highly degraded DNA with very short amplicons to the introduction of SNPs to ascertain the ancestry and physical characteristics of an unidentified contact trace donor. However, this technology, as resourceful as it is, displays several features that depart from the usual STR genotyping far enough to demand a certain degree of expertise from the forensic analyst before tackling the complex casework on which SNaPshot application provides an advantage. In order to provide the basis for developing such expertise, we cover in this paper the most challenging aspects of the SNaPshot technology, focusing on the steps taken to design primer sets, optimize the PCR and single-base extension chemistries, and the important features of the peak patterns observed in typical forensic SNP profiles using SNaPshot. With that purpose in mind, we provide guidelines and troubleshooting for multiplex-SNaPshot-oriented primer design and the resulting capillary electrophoresis (CE) profile interpretation (covering the most commonly observed artifacts and expected departures from the ideal conditions). Copyright © 2017 Central Police University.

Next-Generation Molecular Testing of Newborn Dried Blood Spots for Cystic Fibrosis.

PubMed

Lefterova, Martina I; Shen, Peidong; Odegaard, Justin I; Fung, Eula; Chiang, Tsoyu; Peng, Gang; Davis, Ronald W; Wang, Wenyi; Kharrazi, Martin; Schrijver, Iris; Scharfe, Curt

2016-03-01

Newborn screening for cystic fibrosis enables early detection and management of this debilitating genetic disease. Implementing comprehensive CFTR analysis using Sanger sequencing as a component of confirmatory testing of all screen-positive newborns has remained impractical due to relatively lengthy turnaround times and high cost. Here, we describe CFseq, a highly sensitive, specific, rapid (<3 days), and cost-effective assay for comprehensive CFTR gene analysis from dried blood spots, the common newborn screening specimen. The unique design of CFseq integrates optimized dried blood spot sample processing, a novel multiplex amplification method from as little as 1 ng of genomic DNA, and multiplex next-generation sequencing of 96 samples in a single run to detect all relevant CFTR mutation types. Sequence data analysis utilizes publicly available software supplemented by an expert-curated compendium of >2000 CFTR variants. Validation studies across 190 dried blood spots demonstrated 100% sensitivity and a positive predictive value of 100% for single-nucleotide variants and insertions and deletions and complete concordance across the polymorphic poly-TG and consecutive poly-T tracts. Additionally, we accurately detected both a known exon 2,3 deletion and a previously undetected exon 22,23 deletion. CFseq is thus able to replace all existing CFTR molecular assays with a single robust, definitive assay at significant cost and time savings and could be adapted to high-throughput screening of other inherited conditions. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer

PubMed Central

Vargas, Diana Y.; Kramer, Fred Russell; Tyagi, Sanjay; Marras, Salvatore A. E.

2016-01-01

We describe the use of “SuperSelective” primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies). The design of these deoxyribonucleotide primers incorporates both a relatively long “5' anchor sequence” that hybridizes strongly to target DNA fragments, and a very short, physically and functionally separate, “3' foot sequence” that is perfectly complementary to the mutant target sequence, but mismatches the wild-type sequence. As few as ten mutant fragments can reliably be detected in the presence of 1,000,000 wild-type fragments, even when the difference between the mutant and the wild type is only a single nucleotide polymorphism. Multiplex PCR assays employing a set of SuperSelective primers, and a corresponding set of differently colored molecular beacon probes, can be used in situations where the different mutations, though occurring in different cells, are located in the same codon. These non-symmetric real-time multiplex PCR assays contain limited concentrations of each SuperSelective primer, thereby enabling the simultaneous determination of each mutation’s abundance by comparing its threshold value to the threshold value of a reference gene present in the sample. PMID:27244445

Capillaries for use in a multiplexed capillary electrophoresis system

DOEpatents

Yeung, Edward S.; Chang, Huan-Tsang; Fung, Eliza N.

1997-12-09

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification ("base calling") is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations.

Capillaries for use in a multiplexed capillary electrophoresis system

DOEpatents

Yeung, E.S.; Chang, H.T.; Fung, E.N.

1997-12-09

The invention provides a side-entry optical excitation geometry for use in a multiplexed capillary electrophoresis system. A charge-injection device is optically coupled to capillaries in the array such that the interior of a capillary is imaged onto only one pixel. In Sanger-type 4-label DNA sequencing reactions, nucleotide identification (``base calling``) is improved by using two long-pass filters to split fluorescence emission into two emission channels. A binary poly(ethyleneoxide) matrix is used in the electrophoretic separations. 19 figs.

Multiplexing 200 spatial modes with a single hologram

NASA Astrophysics Data System (ADS)

Rosales-Guzmán, Carmelo; Bhebhe, Nkosiphile; Mahonisi, Nyiku; Forbes, Andrew

2017-11-01

The on-demand tailoring of light's spatial shape is of great relevance in a wide variety of research areas. Computer-controlled devices, such as spatial light modulators (SLMs) or digital micromirror devices, offer a very accurate, flexible and fast holographic means to this end. Remarkably, digital holography affords the simultaneous generation of multiple beams (multiplexing), a tool with numerous applications in many fields. Here, we provide a self-contained tutorial on light beam multiplexing. Through the use of several examples, the readers will be guided step by step in the process of light beam shaping and multiplexing. Additionally, we provide a quantitative analysis on the multiplexing capabilities of SLMs to assess the maximum number of beams that can be multiplexed on a single SLM, showing approximately 200 modes on a single hologram.

Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F-108 tri-block copolymer and capillary electrophoresis.

PubMed

Martin, Lauren; Damaso, Natalie; Mills, DeEtta

2016-10-01

Molecular methods for the detection of mammalian coat color phenotypes have expanded greatly within the past decade. Many phenotypes are associated with a single nucleotide polymorphism mutation in the genetic sequence. Traditionally, these mutations are detected through sequencing, hybridization assays or mini-sequencing. However, these techniques can be expensive and tedious. Previously, CE-SSCP using the F-108 polymer was able to distinguish SNPs for the melanocortin-1 receptor (mc1r) coat color gene in horses (Equus caballus) that differed by one nucleotide substitution. The objective of this study was to expand the detection of coat color SNPs in horses. The genes for the solute carrier family member 2 (slc45a2/matp), type III receptor protein-tyrosine kinase (kit) and mc1r genes using CE-SSCP and F-108 polymer were compared to mini-sequencing with the SNaPshot TM kit. The F-108 polymer reproducibly resolved homozygous and heterozygous individuals for the mc1r and kit markers, but was unable to resolve heterozygous individuals for slc45a2 at 38ºC. The need for temperatures <15ºC, the SNP position being close to the 5'-end, and conformational structures/free energy with similar values resulted in the inability to resolve the secondary structures. Despite this limitation, the CE-SSCP method could be used to provide a rapid phenotypic description for equine forensic investigations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automation of complex assays: pharmacogenetics of warfarin dosing.

PubMed

Wu, Whei-Kuo; Hujsak, Paul G; Kureshy, Fareed

2007-10-01

AutoGenomics, Inc. (Carlsbad, CA, USA) have developed a multiplex microarray assay for genotyping both VKORC1 and CYP2C9 using the INFINITI(™) Analyzer. Multiple alleles in each DNA sample are analyzed by polymerase chain reaction amplification, followed by detection primer extension using the INFINITI Analyzer. The INFINITI Analyzer performs single-nucleotide polymorphism (SNP) analysis using universal oligonucleotides immobilized on the biochip. To genotype broader ethnic groups, genomic DNA from whole blood was tested for nine SNPs for VKORC1 and six for CYP2C9 genotypes. Information related to all 15 SNPs is needed to determine dosing of population of diverse ethnic origin. The INFINITI system provides genotyping information for same day dosing of warfarin.

Universal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophy.

PubMed

Wang, Chun-Chi; Chang, Jan-Gowth; Jong, Yuh-Jyh; Wu, Shou-Mei

2009-04-01

We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease.

A novel multiplex pyrosequencing assay for genotyping functionally relevant CTLA-4 polymorphisms: potential applications in autoimmunity and cancer.

PubMed

Banelli, Barbara; Morabito, Anna; Laurent, Stefania; Piccioli, Patrizia; Dozin, Beatrice; Ghio, Massimo; Ascierto, Paolo Antonio; Monteghirfo, Stefano; Marasco, Antonella; Ottaviano, Vincenzo; Queirolo, Paola; Romani, Massimo; Pistillo, Maria Pia

2014-08-01

CTLA-4 expression/function can be affected by single nucleotide polymorphisms (SNPs) of CTLA-4 gene, which have been widely associated with susceptibility or progression to autoimmune diseases and cancer development. In this study, we analyzed six CTLA-4 SNPs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, CT60G>A) in 197 DNA samples from 43 B-lymphoblastoid cell lines (B-LCLs), 40 systemic sclerosis (SSc) patients, 14 pre-analyzed melanoma patients and 100 Italian healthy subjects. Genotyping of -1661A>G, -1577G>A, -658C>T and CT60G>A was performed by newly developed multiplex pyrosequencing (PSQ) assays, whereas -319C>T and +49A>G by T-ARMS PCR and direct sequencing. Genotype/allele frequency were analyzed using χ(2) or Fisher exact test. Our study provides the first multiplex PSQ method that allows simultaneous genotyping of two CTLA-4 SNP pairs (i.e. -1661A>G/-658C>T and -1577G>A/CT60G>A) by two multiplex PSQ reactions. Herein, we show the CTLA-4 genotype distribution in the B-LCLs providing the first and best characterized cell line panel typed for functionally relevant CTLA-4 SNPs. We also report the significant association of the -1661A/G genotype, -1661 & -319 AC-GT diplotype and -319 & CT60 TG haplotype with susceptibility to SSc without Hashimoto's thyroiditis occurrence. Furthermore, we confirmed previous genotyping data referred to melanoma patients and provided new genotyping data for Italian healthy subjects. Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

A single-electrode electrochemical system for multiplex electrochemiluminescence analysis based on a resistance induced potential difference.

PubMed

Gao, Wenyue; Muzyka, Kateryna; Ma, Xiangui; Lou, Baohua; Xu, Guobao

2018-04-28

Developing low-cost and simple electrochemical systems is becoming increasingly important but still challenged for multiplex experiments. Here we report a single-electrode electrochemical system (SEES) using only one electrode not only for a single experiment but also for multiplex experiments based on a resistance induced potential difference. SEESs for a single experiment and multiplex experiments are fabricated by attaching a self-adhesive label with a hole and multiple holes onto an ITO electrode, respectively. This enables multiplex electrochemiluminescence analysis with high sensitivity at a very low safe voltage using a smartphone as a detector. For the multiplex analysis, the SEES using a single electrode is much simpler, cheaper and more user-friendly than conventional electrochemical systems and bipolar electrochemical systems using electrode arrays. Moreover, SEESs are free from the electrochemiluminescent background problem from driving electrodes in bipolar electrochemical systems. Since numerous electrodes and cover materials can be used to fabricate SEESs readily and electrochemistry is being extensively used, SEESs are very promising for broad applications, such as drug screening and high throughput analysis.

Detection of virulent Escherichia coli O157 strains using multiplex PCR and single base sequencing for SNP characterization.

PubMed

Haugum, K; Brandal, L T; Løbersli, I; Kapperud, G; Lindstedt, B-A

2011-06-01

To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1-3 and 8). We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over-representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic-uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2(EDL933) or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993-2008. We observed that the tir-255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. The detection of virulence clade-specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Evaluation of single-nucleotide polymorphisms as internal controls in prenatal diagnosis of fetal blood groups.

PubMed

Doescher, Andrea; Petershofen, Eduard K; Wagner, Franz F; Schunter, Markus; Müller, Thomas H

2013-02-01

Determination of fetal blood groups in maternal plasma samples critically depends on adequate amplification of fetal DNA. We evaluated the routine inclusion of 52 single-nucleotide polymorphisms (SNPs) as internal reference in our polymerase chain reaction (PCR) settings to obtain a positive internal control for fetal DNA. DNA from 223 plasma samples of pregnant women was screened for RHD Exons 3, 4, 5, and 7 in a multiplex PCR including 52 SNPs divided into four primer pools. Amplicons were analyzed by single-base extension and the GeneScan method in a genetic analyzer. Results of D screening were compared to standard RHD genotyping of amniotic fluid or real-time PCR of fetal DNA from maternal plasma. The vast majority of all samples (97.8%) demonstrated differences in maternal and fetal SNP patterns when tested with four primer pools. These differences were not observed in less than 2.2% of the samples most probably due to an extraction failure for adequate amounts of fetal DNA. Comparison of the fetal genotypes with independent results did not reveal a single false-negative case among samples (n = 42) with positive internal control and negative fetal RHD typing. Coamplification of 52 SNPs with RHD-specific sequences for fetal blood group determination introduces a valid positive control for the amplification of fetal DNA to avoid false-negative results. This new approach does not require a paternal blood sample. It may also be applicable to other assays for fetal genotyping in maternal blood samples. © 2012 American Association of Blood Banks.

Improving mapping and SNP-calling performance in multiplexed targeted next-generation sequencing

PubMed Central

2012-01-01

Background Compared to classical genotyping, targeted next-generation sequencing (tNGS) can be custom-designed to interrogate entire genomic regions of interest, in order to detect novel as well as known variants. To bring down the per-sample cost, one approach is to pool barcoded NGS libraries before sample enrichment. Still, we lack a complete understanding of how this multiplexed tNGS approach and the varying performance of the ever-evolving analytical tools can affect the quality of variant discovery. Therefore, we evaluated the impact of different software tools and analytical approaches on the discovery of single nucleotide polymorphisms (SNPs) in multiplexed tNGS data. To generate our own test model, we combined a sequence capture method with NGS in three experimental stages of increasing complexity (E. coli genes, multiplexed E. coli, and multiplexed HapMap BRCA1/2 regions). Results We successfully enriched barcoded NGS libraries instead of genomic DNA, achieving reproducible coverage profiles (Pearson correlation coefficients of up to 0.99) across multiplexed samples, with <10% strand bias. However, the SNP calling quality was substantially affected by the choice of tools and mapping strategy. With the aim of reducing computational requirements, we compared conventional whole-genome mapping and SNP-calling with a new faster approach: target-region mapping with subsequent ‘read-backmapping’ to the whole genome to reduce the false detection rate. Consequently, we developed a combined mapping pipeline, which includes standard tools (BWA, SAMtools, etc.), and tested it on public HiSeq2000 exome data from the 1000 Genomes Project. Our pipeline saved 12 hours of run time per Hiseq2000 exome sample and detected ~5% more SNPs than the conventional whole genome approach. This suggests that more potential novel SNPs may be discovered using both approaches than with just the conventional approach. Conclusions We recommend applying our general ‘two-step’ mapping approach for more efficient SNP discovery in tNGS. Our study has also shown the benefit of computing inter-sample SNP-concordances and inspecting read alignments in order to attain more confident results. PMID:22913592

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

PubMed

Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O'Brien, Mary; Turner, Nicholas C; Popat, Sanjay; Downward, Julian

2015-01-01

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach

PubMed Central

Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J.; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O’Brien, Mary; Turner, Nicholas C.; Popat, Sanjay; Downward, Julian

2015-01-01

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma. PMID:26413866

Multiplex PCR method to discriminate Artemisia iwayomogi from other Artemisia plants.

PubMed

Doh, Eui Jeong; Oh, Seung-Eun

2012-01-01

Some plants in the genus Artemisia have been used for medicinal purposes. Among them, Artemisia iwayomogi, commonly referred to as "Haninjin," is one of the major medicinal materials used in traditional Korean medicine. By contrast, Artemisia capillaris and both Artemisia argyi and Artemisia princeps, referred to as "Injinho" and "Aeyup," respectively, are used to treat diseases different from those for which "Haninjin" is prescribed. Therefore, the development of a reliable method to differentiate each Artemisia herb is necessary. We found that a random amplified polymorphic DNA (RAPD) method can be used to efficiently discriminate a few Artemisia plants from one another. To improve the reliability of RAPD amplification, we designed primer sets based on the nucleotide sequences of RAPD products to amplify a sequence-characterized amplified region (SCAR) marker of A. iwayomogi. In addition, we designed two other primer sets to amplify SCAR markers of "Aeyup" (A. argyi and A. princeps) along with "Injinho" (A. capillaris) and Artemisia japonica, which are also traded in Korean herbal markets. Using these three primer sets, we developed a multiplex PCR method concurrently not only to discriminate A. iwayomogi from other Artemisia plants, but also to identify Artemisia plants using a single PCR process.

Integrated spatial multiplexing of heralded single-photon sources

PubMed Central

Collins, M.J.; Xiong, C.; Rey, I.H.; Vo, T.D.; He, J.; Shahnia, S.; Reardon, C.; Krauss, T.F.; Steel, M.J.; Clark, A.S.; Eggleton, B.J.

2013-01-01

The non-deterministic nature of photon sources is a key limitation for single-photon quantum processors. Spatial multiplexing overcomes this by enhancing the heralded single-photon yield without enhancing the output noise. Here the intrinsic statistical limit of an individual source is surpassed by spatially multiplexing two monolithic silicon-based correlated photon pair sources in the telecommunications band, demonstrating a 62.4% increase in the heralded single-photon output without an increase in unwanted multipair generation. We further demonstrate the scalability of this scheme by multiplexing photons generated in two waveguides pumped via an integrated coupler with a 63.1% increase in the heralded photon rate. This demonstration paves the way for a scalable architecture for multiplexing many photon sources in a compact integrated platform and achieving efficient two-photon interference, required at the core of optical quantum computing and quantum communication protocols. PMID:24107840

Optofluidic wavelength division multiplexing for single-virus detection

PubMed Central

Ozcelik, Damla; Parks, Joshua W.; Wall, Thomas A.; Stott, Matthew A.; Cai, Hong; Parks, Joseph W.; Hawkins, Aaron R.; Schmidt, Holger

2015-01-01

Optical waveguides simultaneously transport light at different colors, forming the basis of fiber-optic telecommunication networks that shuttle data in dozens of spectrally separated channels. Here, we reimagine this wavelength division multiplexing (WDM) paradigm in a novel context––the differentiated detection and identification of single influenza viruses on a chip. We use a single multimode interference (MMI) waveguide to create wavelength-dependent spot patterns across the entire visible spectrum and enable multiplexed single biomolecule detection on an optofluidic chip. Each target is identified by its time-dependent fluorescence signal without the need for spectral demultiplexing upon detection. We demonstrate detection of individual fluorescently labeled virus particles of three influenza A subtypes in two implementations: labeling of each virus using three different colors and two-color combinatorial labeling. By extending combinatorial multiplexing to three or more colors, MMI-based WDM provides the multiplexing power required for differentiated clinical tests and the growing field of personalized medicine. PMID:26438840

A multiplexed microsatellite fingerprinting set for hazelnut cultivar identification

USDA-ARS?s Scientific Manuscript database

The objective of this study was to develop a robust and cost-effective fingerprinting set for hazelnuts using microsatellite (SSR) markers. Twenty SSRs containing repeat motifs of = three nucleotides distributed throughout the hazelnut genome were screened on eight genetically diverse cultivars to a...

Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus.

PubMed

Rogers, Stephanie M; Payton, Mark; Allen, Robert W; Melcher, Ulrich; Carver, Jesse; Fletcher, Jacqueline

2012-05-17

The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories. single nucleotide polymorphisms, genotyping, plant pathology, viruses, microbial forensics, Single base primer extension, SNaPshot Multiplex Kit.

Method: a single nucleotide polymorphism genotyping method for Wheat streak mosaic virus

PubMed Central

2012-01-01

Background The September 11, 2001 attacks on the World Trade Center and the Pentagon increased the concern about the potential for terrorist attacks on many vulnerable sectors of the US, including agriculture. The concentrated nature of crops, easily obtainable biological agents, and highly detrimental impacts make agroterrorism a potential threat. Although procedures for an effective criminal investigation and attribution following such an attack are available, important enhancements are still needed, one of which is the capability for fine discrimination among pathogen strains. The purpose of this study was to develop a molecular typing assay for use in a forensic investigation, using Wheat streak mosaic virus (WSMV) as a model plant virus. Method This genotyping technique utilizes single base primer extension to generate a genetic fingerprint. Fifteen single nucleotide polymorphisms (SNPs) within the coat protein and helper component-protease genes were selected as the genetic markers for this assay. Assay optimization and sensitivity testing was conducted using synthetic targets. WSMV strains and field isolates were collected from regions around the world and used to evaluate the assay for discrimination. The assay specificity was tested against a panel of near-neighbors consisting of genetic and environmental near-neighbors. Result Each WSMV strain or field isolate tested produced a unique SNP fingerprint, with the exception of three isolates collected within the same geographic location that produced indistinguishable fingerprints. The results were consistent among replicates, demonstrating the reproducibility of the assay. No SNP fingerprints were generated from organisms included in the near-neighbor panel, suggesting the assay is specific for WSMV. Using synthetic targets, a complete profile could be generated from as low as 7.15 fmoles of cDNA. Conclusion The molecular typing method presented is one tool that could be incorporated into the forensic science tool box after a thorough validation study. This method incorporates molecular biology techniques that are already well established in research and diagnostic laboratories, allowing for an easy introduction of this method into existing laboratories. Keywords: single nucleotide polymorphisms, genotyping, plant pathology, viruses, microbial forensics, Single base primer extension, SNaPshot Multiplex Kit PMID:22594601

Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood.

PubMed

Nicolaides, Kypros H; Syngelaki, Argyro; del Mar Gil, Maria; Quezada, Maria Soledad; Zinevich, Yana

2014-01-01

To investigate potential performance of cell-free DNA (cfDNA) testing in maternal blood in detecting fetal triploidy. Plasma and buffy coat samples obtained at 11-13 weeks' gestation from singleton pregnancies with diandric triploidy (n=4), digynic triploidy (n=4), euploid fetuses (n=48) were sent to Natera, Inc. (San Carlos, Calif., USA) for cfDNA testing. Multiplex polymerase chain reaction amplification of cfDNA followed by sequencing of single nucleotide polymorphic loci covering chromosomes 13, 18, 21, X, and Y was performed. Sequencing data were analyzed using the NATUS algorithm which identifies copy number for each of the five chromosomes. cfDNA testing provided a result in 44 (91.7%) of the 48 euploid cases and correctly predicted the fetal sex and the presence of two copies each of chromosome 21, 18 and 13. In diandric triploidy, cfDNA testing identified multiple paternal haplotypes (indicating fetal trisomy 21, trisomy 18 and trisomy 13) suggesting the presence of either triploidy or dizygotic twins. In digynic triploidy the fetal fraction corrected for maternal weight and gestational age was below the 0.5th percentile. cfDNA testing by targeted sequencing and allelic ratio analysis of single nucleotide polymorphisms covering chromosomes 21, 18, 13, X, and Y can detect diandric triploidy and raise the suspicion of digynic triploidy. © 2013 S. Karger AG, Basel.

Microfluidic platform for multiplexed detection in single cells and methods thereof

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

Wu, Meiye; Singh, Anup K.

The present invention relates to a microfluidic device and platform configured to conduct multiplexed analysis within the device. In particular, the device allows multiple targets to be detected on a single-cell level. Also provided are methods of performing multiplexed analyses to detect one or more target nucleic acids, proteins, and post-translational modifications.

Construction and forensic genetic characterization of 11 autosomal haplotypes consisting of 22 tri-allelic indels.

PubMed

Zhao, Xiaohong; Chen, Xiaogang; Zhao, Yuancun; Zhang, Shu; Gao, Zehua; Yang, Yiwen; Wang, Yufang; Zhang, Ji

2018-05-01

Insertion/deletion polymorphisms (indels), which combine the advantages of both short tandem repeats and single-nucleotide polymorphisms, are suitable for parentage testing. To overcome the limitations of the low polymorphism of di-allelic indels, we constructed a set of haplotypes with physically linked, multi-allelic indels. Candidate haplotypes were selected from the 1000 Genomes Project database, and were subject to the following criteria for inclusion: (i) each marker must have a minimum allele frequency (MAF) of ≥0.1 in the Han population of China; (ii) markers must exist in a non-coding region; (iii) the physical distance between a pair of candidate indels must be <500 bp; (iv) the allele length variation of each indel from 1 to 20 bp; (v) different haplotypes must be located on different chromosomes or chromosomal arms, or be more than 10 Mb apart if on the same chromosomal arm; and (vi) they must not be located across a recombination hotspot. A multiplex system with 11 haplotype markers, comprising 22 tri-allelic indel loci distributed over 10 chromosomes was developed. To validate the multiplex panel, we investigated the haplotype distribution in sets of two and three-generation pedigrees. The results demonstrated that the haplotypes consisting of multi-allelic indel markers exhibited higher polymorphism than a single indel locus, and thus provide Supplementary information for forensic kinship identification. Copyright © 2018 Elsevier B.V. All rights reserved.

Simultaneous determination of seven informative Y chromosome SNPs to differentiate East Asian, European, and African populations.

PubMed

Muro, Tomonori; Iida, Reiko; Fujihara, Junko; Yasuda, Toshihiro; Watanabe, Yukina; Imamura, Shinji; Nakamura, Hiroaki; Kimura-Kataoka, Kaori; Yuasa, Isao; Toga, Tomoko; Takeshita, Haruo

2011-05-01

Identification of the population origin of an individual is very useful for crime investigators who need to narrow down a suspect based on specimens left at a crime scene. Single nucleotide polymorphisms of the Y chromosome (Y-SNPs) are a class of markers of interest to forensic investigators because many of the markers indicate regional specificity, thus providing useful information about the geographic origin of a subject. We selected seven informative Y-SNPs (M168, M130, JST021355, M96, P126, P196, and P234) to differentiate the three major population groups (East Asian, European, and African) and used them to develop forensic application. SNP genotyping was carried out by multiplex PCR reaction and multiplex single base extension (MSBE) reaction followed by capillary electrophoresis of extension products. This method can be used to assign a haplogroup from both degraded male DNA samples and DNA samples containing a mixture of female and male DNA through PCR primers that generate small amplicons (less than about 150 bp) and are highly specific for targets on the Y chromosome. The allelic state of each marker was definitively determined from a total of 791 males from the three major population groups. As expected, samples from the three major population groups showed Y-haplogroups common in the region of provenance: Y haplogroups C, D, and O for East Asians; IJ and R1 for Europeans; and AB and E for Africans. Published by Elsevier Ireland Ltd.

Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children.

PubMed

Giri, Sidhartha; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Iturriza-Gomara, Miren; Taniuchi, Mami; John, Jacob; Abraham, Asha Mary; Kang, Gagandeep

2017-08-01

Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant. © 2017 Wiley Periodicals, Inc.

The development and validation of a single SNaPshot multiplex for tiger species and subspecies identification--implications for forensic purposes.

PubMed

Kitpipit, Thitika; Tobe, Shanan S; Kitchener, Andrew C; Gill, Peter; Linacre, Adrian

2012-03-01

The tiger (Panthera tigris) is currently listed on Appendix I of the Convention on the International Trade in Endangered Species of Wild Fauna and Flora; this affords it the highest level of international protection. To aid in the investigation of alleged illegal trade in tiger body parts and derivatives, molecular approaches have been developed to identify biological material as being of tiger in origin. Some countries also require knowledge of the exact tiger subspecies present in order to prosecute anyone alleged to be trading in tiger products. In this study we aimed to develop and validate a reliable single assay to identify tiger species and subspecies simultaneously; this test is based on identification of single nucleotide polymorphisms (SNPs) within the tiger mitochondrial genome. The mitochondrial DNA sequence from four of the five extant putative tiger subspecies that currently exist in the wild were obtained and combined with DNA sequence data from 492 tiger and 349 other mammalian species available on GenBank. From the sequence data a total of 11 SNP loci were identified as suitable for further analyses. Five SNPs were species-specific for tiger and six amplify one of the tiger subspecies-specific SNPs, three of which were specific to P. t. sumatrae and the other three were specific to P. t. tigris. The multiplex assay was able to reliably identify 15 voucher tiger samples. The sensitivity of the test was 15,000 mitochondrial DNA copies (approximately 0.26 pg), indicating that it will work on trace amounts of tissue, bone or hair samples. This simple test will add to the DNA-based methods currently being used to identify the presence of tiger within mixed samples. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality.

PubMed

Dictor, Michael; Skogvall, Ingela; Warenholt, Janina; Rambech, Eva

2007-01-01

Two-colour flow cytometry was compared with multiplex PCR with capillary electrophoresis for clonality determination in specific categories of B-cell lymphoma. FTA cards were evaluated for preserving DNA from node imprints and expediting molecular analysis. A single-tube multiplex PCR targeted IGH and lymphoma-specific translocations in DNA extracted from 180 frozen lymphoid tissues and DNA bound to FTA cards from 192 fresh tissues and 137 aspirates. PCR results were compared with flow cytometry in the extracted and aspirated samples. Overall, single-tube multiplex PCR sensitivity was equivalent in the sample groups (intergroup range 79%-91%). False negatives were associated with tumour origin in the follicle centre. Multiplex PCR and flow cytometry were equally sensitive and together detected 98% of B-cell lymphomas. Additional two-tube targeting of IGK suggested an overall molecular sensitivity >90%. False positive (pseudoclonal) single-tube multiplex PCR was associated with necrosis and sparse lymphocytes. Multiplex PCR using template DNA bound to an FTA card effectively detects B-lymphocyte clonality, obviates DNA extraction and refrigeration, and can be used without diminished sensitivity in fine needle aspirates or node imprints as a replacement for or complement to flow cytometry at any point in the diagnostic work-up.

Multiplexed Microsphere Suspension-Array Assay for Urine Mitochondrial DNA Typing by C-Stretch Length in Hypervariable Regions.

PubMed

Aoki, Kimiko; Tanaka, Hiroyuki; Kawahara, Takashi

2018-07-01

The standard method for personal identification and verification of urine samples in doping control is short tandem repeat (STR) analysis using nuclear DNA (nDNA). The DNA concentration of urine is very low and decreases under most conditions used for sample storage; therefore, the amount of DNA from cryopreserved urine samples may be insufficient for STR analysis. We aimed to establish a multiplexed assay for urine mitochondrial DNA typing containing only trace amounts of DNA, particularly for Japanese populations. A multiplexed suspension-array assay using oligo-tagged microspheres (Luminex MagPlex-TAG) was developed to measure C-stretch length in hypervariable region 1 (HV1) and 2 (HV2), five single nucleotide polymorphisms (SNPs), and one polymorphic indel. Based on these SNPs and the indel, the Japanese population can be classified into five major haplogroups (D4, B, M7a, A, D5). The assay was applied to DNA samples from urine cryopreserved for 1 - 1.5 years (n = 63) and fresh blood (n = 150). The assay with blood DNA enabled Japanese subjects to be categorized into 62 types, exhibiting a discriminatory power of 0.960. The detection limit for cryopreserved urine was 0.005 ng of nDNA. Profiling of blood and urine pairs revealed that 5 of 63 pairs showed different C-stretch patterns in HV1 or HV2. The assay described here yields valuable information in terms of the verification of urine sample sources employing only trace amounts of recovered DNA. However, blood cannot be used as a reference sample.

Optimal percolation on multiplex networks.

PubMed

Osat, Saeed; Faqeeh, Ali; Radicchi, Filippo

2017-11-16

Optimal percolation is the problem of finding the minimal set of nodes whose removal from a network fragments the system into non-extensive disconnected clusters. The solution to this problem is important for strategies of immunization in disease spreading, and influence maximization in opinion dynamics. Optimal percolation has received considerable attention in the context of isolated networks. However, its generalization to multiplex networks has not yet been considered. Here we show that approximating the solution of the optimal percolation problem on a multiplex network with solutions valid for single-layer networks extracted from the multiplex may have serious consequences in the characterization of the true robustness of the system. We reach this conclusion by extending many of the methods for finding approximate solutions of the optimal percolation problem from single-layer to multiplex networks, and performing a systematic analysis on synthetic and real-world multiplex networks.

Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction

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

Day, D.J.; Barany, F.; Speiser, P.W.

Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and themore » remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems. 30 refs., 5 figs.« less

PrimerStation: a highly specific multiplex genomic PCR primer design server for the human genome

PubMed Central

Yamada, Tomoyuki; Soma, Haruhiko; Morishita, Shinichi

2006-01-01

PrimerStation () is a web service that calculates primer sets guaranteeing high specificity against the entire human genome. To achieve high accuracy, we used the hybridization ratio of primers in liquid solution. Calculating the status of sequence hybridization in terms of the stringent hybridization ratio is computationally costly, and no web service checks the entire human genome and returns a highly specific primer set calculated using a precise physicochemical model. To shorten the response time, we precomputed candidates for specific primers using a massively parallel computer with 100 CPUs (SunFire 15 K) about 3 months in advance. This enables PrimerStation to search and output qualified primers interactively. PrimerStation can select highly specific primers suitable for multiplex PCR by seeking a wider temperature range that minimizes the possibility of cross-reaction. It also allows users to add heuristic rules to the primer design, e.g. the exclusion of single nucleotide polymorphisms (SNPs) in primers, the avoidance of poly(A) and CA-repeats in the PCR products, and the elimination of defective primers using the secondary structure prediction. We performed several tests to verify the PCR amplification of randomly selected primers for ChrX, and we confirmed that the primers amplify specific PCR products perfectly. PMID:16845094

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification

PubMed Central

Schouten, Jan P.; McElgunn, Cathal J.; Waaijer, Raymond; Zwijnenburg, Danny; Diepvens, Filip; Pals, Gerard

2002-01-01

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down’s syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50–70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences. PMID:12060695

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.

PubMed

Schouten, Jan P; McElgunn, Cathal J; Waaijer, Raymond; Zwijnenburg, Danny; Diepvens, Filip; Pals, Gerard

2002-06-15

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down's syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50-70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.

Improved Multiplex Ligation-dependent Probe Amplification (i-MLPA) for rapid copy number variant (CNV) detection.

PubMed

Saxena, Sonal; Gowdhaman, Kavitha; Kkani, Poornima; Vennapusa, Bhavyasri; Rama Subramanian, Chellamuthu; Ganesh Kumar, S; Mohan, Kommu Naga

2015-10-23

In Multiplex Ligation-dependent Probe Amplification (MLPA), copy number variants (CNVs) for specific genes are identified after normalization of the amounts of PCR products from ligated reference probes hybridized to genomic regions that are ideally free from normal variation. However, we observed ambiguous calls for two reference probes in an investigation of the human 15q11.2 region by MLPA among 20 controls, due to the presence of single nucleotide polymorphisms (SNPs) in the probe-binding regions. Further in silico analysis revealed that 18 out of 19 reference probes hybridize to regions subject to variation, underlining the requirement for designing new reference probes against variation-free regions. An improved MLPA (i-MLPA) method was developed by generating a new set of reference probes to reduce the chances of ambiguous calls and new reagents that reduce hybridization times to 30 min from 16h to obtain MLPA ratio data within 6h. Using i-MLPA, we screened 240 schizophrenia patients for CNVs in 15q11.2 region. Three deletions and two duplications were identified among the 240 schizophrenia patients. No variation was observed for the new reference probes. Taken together, i-MLPA procedure helps obtaining non-ambiguous CNV calls within 6h without compromising accuracy. Copyright © 2015 Elsevier B.V. All rights reserved.

Array of Synthetic Oligonucleotides to Generate Unique Multi-Target Artificial Positive Controls and Molecular Probe-Based Discrimination of Liposcelis Species

PubMed Central

Arif, Mohammad; Opit, George; Mendoza-Yerbafría, Abigail; Dobhal, Shefali; Li, Zhihong; Kučerová, Zuzana; Ochoa-Corona, Francisco M.

2015-01-01

Several species of the genus Liposcelis are common insect pests that cause serious qualitative and quantitative losses to various stored grains and processed grain products. They also can contaminate foods, transmit pathogenic microorganisms and cause allergies in humans. The common occurrence of multi-species infestations and the fact that it is difficult to identify and discriminate Liposcelis spp. make accurate, rapid detection and discriminatory tools absolutely necessary for confirmation of their identity. In this study, PCR primers and probes specific to different Liposcelis spp. were designed based on nucleotide sequences of the cytochrome oxidase 1 (CO1) gene. Primer sets ObsCo13F/13R, PeaCo15F/14R, BosCO7F/7R, BruCo5F/5R, and DecCo11F/11R were used to specifically detect Liposcelis obscura Broadhead, Liposcelis pearmani Lienhard, Liposcelis bostrychophila Badonnel, Liposcelis brunnea Motschulsky and Liposcelis decolor (Pearman) in multiplex endpoint PCRs, which amplified products of 438-, 351-, 191-, 140-, and 87-bp, respectively. In multiplex TaqMan qPCR assays, orange, yellow, red, crimson and green channels corresponding to reporter dyes 6-ROXN, HEX, Cy5, Quasar705 and 6-FAM specifically detected L. obscura, L. brunnea, L. bostrychophila, L. pearmani and L. decolor, respectively. All developed primer and probe sets allowed specific amplification of corresponding targeted Liposcelis species. The development of multiplex endpoint PCR and multiplex TaqMan qPCR will greatly facilitate psocid identification and their management. The use of APCs will streamline and standardize PCR assays. APC will also provide the opportunity to have all positive controls in a single tube, which reduces maintenance cost and labor, but increases the accuracy and reliability of the assays. These novel methods from our study will have applications in pest management, biosecurity, quarantine, food safety, and routine diagnostics. PMID:26086728

Haplotype Analysis Discriminates Genetic Risk for DR3-Associated Endocrine Autoimmunity and Helps Define Extreme Risk for Addison’s Disease

PubMed Central

Baker, Peter R.; Baschal, Erin E.; Fain, Pam R.; Triolo, Taylor M.; Nanduri, Priyaanka; Siebert, Janet C.; Armstrong, Taylor K.; Babu, Sunanda R.; Rewers, Marian J.; Gottlieb, Peter A.; Barker, Jennifer M.; Eisenbarth, George S.

2010-01-01

Context: Multiple autoimmune disorders (e.g. Addison’s disease, type 1 diabetes, celiac disease) are associated with HLA-DR3, but it is likely that alleles of additional genes in linkage disequilibrium with HLA-DRB1 contribute to disease. Objective: The objective of the study was to characterize major histocompatability complex (MHC) haplotypes conferring extreme risk for autoimmune Addison’s disease (AD). Design, Setting, and Participants: Eighty-six 21-hydroxylase autoantibody-positive, nonautoimmune polyendocrine syndrome type 1, Caucasian individuals collected from 1992 to 2009 with clinical AD from 68 families (12 multiplex and 56 simplex) were genotyped for HLA-DRB1, HLA-DQB1, MICA, HLA-B, and HLA-A as well as high density MHC single-nucleotide polymorphism (SNP) analysis for 34. Main Outcome Measures: AD and genotype were measured. Result: Ninety-seven percent of the multiplex individuals had both HLA-DR3 and HLA-B8 vs. 60% of simplex AD patients (P = 9.72 × 10−4) and 13% of general population controls (P = 3.00 × 10−19). The genotype DR3/DR4 with B8 was present in 85% of AD multiplex patients, 24% of simplex patients, and 1.5% of control individuals (P = 4.92 × 10−191). The DR3-B8 haplotype of AD patients had HLA-A1 less often (47%) than controls (81%, P = 7.00 × 10−5) and type 1 diabetes patients (73%, P = 1.93 × 10−3). Analysis of 1228 SNPs across the MHC for individuals with AD revealed a shorter conserved haplotype (3.8) with the loss of the extended conserved 3.8.1 haplotype approximately halfway between HLA-B and HLA-A. Conclusion: Extreme risk for AD, especially in multiplex families, is associated with haplotypic DR3 variants, in particular a portion (3.8) but not all of the conserved 3.8.1 haplotype. PMID:20631027

Polarization-multiplexing ghost imaging

NASA Astrophysics Data System (ADS)

Dongfeng, Shi; Jiamin, Zhang; Jian, Huang; Yingjian, Wang; Kee, Yuan; Kaifa, Cao; Chenbo, Xie; Dong, Liu; Wenyue, Zhu

2018-03-01

A novel technique for polarization-multiplexing ghost imaging is proposed to simultaneously obtain multiple polarimetric information by a single detector. Here, polarization-division multiplexing speckles are employed for object illumination. The light reflected from the objects is detected by a single-pixel detector. An iterative reconstruction method is used to restore the fused image containing the different polarimetric information by using the weighted sum of the multiplexed speckles based on the correlation coefficients obtained from the detected intensities. Next, clear images of the different polarimetric information are recovered by demultiplexing the fused image. The results clearly demonstrate that the proposed method is effective.

Active temporal multiplexing of indistinguishable heralded single photons

PubMed Central

Xiong, C.; Zhang, X.; Liu, Z.; Collins, M. J.; Mahendra, A.; Helt, L. G.; Steel, M. J.; Choi, D. -Y.; Chae, C. J.; Leong, P. H. W.; Eggleton, B. J.

2016-01-01

It is a fundamental challenge in quantum optics to deterministically generate indistinguishable single photons through non-deterministic nonlinear optical processes, due to the intrinsic coupling of single- and multi-photon-generation probabilities in these processes. Actively multiplexing photons generated in many temporal modes can decouple these probabilities, but key issues are to minimize resource requirements to allow scalability, and to ensure indistinguishability of the generated photons. Here we demonstrate the multiplexing of photons from four temporal modes solely using fibre-integrated optics and off-the-shelf electronic components. We show a 100% enhancement to the single-photon output probability without introducing additional multi-photon noise. Photon indistinguishability is confirmed by a fourfold Hong–Ou–Mandel quantum interference with a 91±16% visibility after subtracting multi-photon noise due to high pump power. Our demonstration paves the way for scalable multiplexing of many non-deterministic photon sources to a single near-deterministic source, which will be of benefit to future quantum photonic technologies. PMID:26996317

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA.

PubMed

Song, Y; Kato, N; Liu, C; Matsumiya, Y; Kato, H; Watanabe, K

2000-06-15

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.

The -(α)(5.2) Deletion Detected in a Uruguayan Family: First Case Report in the Americas.

PubMed

Soler, Ana María; Schelotto, Magdalena; de Oliveira Mota, Natalia; Dorta Ferreira, Roberta; Sonati, Maria de Fatima; da Luz, Julio Abayubá

2016-08-01

In Uruguay, α-thalassemia (α-thal) mutations were introduced predominantly by Mediterranean European immigrant populations and by slave trade of African populations. A patient with anemia with hypochromia and microcytosis, refractory to iron treatment and with normal hemoglobin (Hb) electrophoresis was analyzed for α-thal mutations by multiplex gap-polymerase chain reaction (gap-PCR), automated sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses. Agarose gel electrophoresis of the multiplex gap-PCR showed a band of unexpected size (approximately 700 bp) in the samples from the proband and mother. Automated sequencing of the amplified fragment showed the presence of the -(α)(5.2) deletion (NG_000006.1: g.32867_38062del5196) [an α-thal-1 deletion of 5196 nucleotides (nts)]. The MLPA analysis of the proband's sample also showed the presence of the -(α)(5.2) deletion in heterozygous state. We report here the presence of the -(α)(5.2) deletion, for the first time in the Americas, in a Uruguayan family with Italian ancestry, detected with a previously described multiplex gap-PCR.

Demonstration of Time Domain Multiplexed Readout for Magnetically Coupled Calorimeters

NASA Technical Reports Server (NTRS)

Porst, J.-P.; Adams, J. S.; Balvin, M.; Bandler, S.; Beyer, J.; Busch, S. E.; Drung, D.; Seidel, G. M.; Smith, S. J.; Stevenson, T. R.

2012-01-01

Magnetically coupled calorimeters (MCC) have extremely high potential for x-ray applications due to the inherent high energy resolution capability and being non-dissipative. Although very high energy-resolution has been demonstrated, until now there has been no demonstration of multiplexed read-out. We report on the first realization of a time domain multiplexed (TDM) read-out. While this has many similarities with TDM of transition-edge-sensors (TES), for MGGs the energy resolution is limited by the SQUID read-out noise and requires the well established scheme to be altered in order to minimize degradation due to noise aliasing effects. In cur approach, each pixel is read out by a single first stage SQUID (SQ1) that is operated in open loop. The outputs of the SQ1 s are low-pass filtered with an array of low cross-talk inductors, then fed into a single-stage SQUID TD multiplexer. The multiplexer is addressed from room temperature and read out through a single amplifier channel. We present results achieved with a new detector platform. Noise performance is presented and compared to expectations. We have demonstrated multiplexed X-ray spectroscopy at 5.9keV with delta_FWHM=10eV. In an optimized setup, we show it is possible to multiplex 32 detectors without significantly degrading the Intrinsic detector resolution.

Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips.

PubMed

Zhong, Xiao-Bo; Reynolds, Robert; Kidd, Judith R; Kidd, Kenneth K; Jenison, Robert; Marlar, Richard A; Ward, David C

2003-09-30

Single-nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. A rapid, sensitive, and inexpensive assay is important for large-scale SNP scoring. Here we report the development of a multiplex SNP detection system using silicon chips coated to create a thin-film optical biosensor. Allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attached to a hydrazinederivatized chip surface. Target sequences (e.g., PCR amplicons) then are hybridized in the presence of a mixture of biotinylated detector probes, one for each SNP, and a thermostable DNA ligase. After a stringent wash (0.01 M NaOH), ligation of biotinylated detector probes to perfectly matched capture oligomers is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. Testing of PCR fragments is completed in 30-40 min. Up to several hundred SNPs can be assayed on a 36-mm2 chip, and SNP scoring can be done by eye or with a simple digital-camera system. This assay is extremely robust, exhibits high sensitivity and specificity, and is format-flexible and economical. In studies of mutations associated with risk for venous thrombosis and genotyping/haplotyping of African-American samples, we document high-fidelity analysis with 0 misassignments in 500 assays performed in duplicate.

Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

PubMed Central

Kabadi, Ami M.; Ousterout, David G.; Hilton, Isaac B.; Gersbach, Charles A.

2014-01-01

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746

Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization.

PubMed

Lee, Chi-Ying; Panicker, Gitika; Bej, Asim K

2003-05-01

Outbreak of diseases associated with consumption of raw shellfish especially oysters is a major concern to the seafood industry and public health agencies. A multiplex PCR amplification of targeted gene segments followed by DNA-DNA sandwich hybridization was optimized to detect the etiologic agents. First, a multiplex PCR amplification of hns, spvB, vvh, ctx and tl was developed enabling simultaneous detection of total Salmonella enterica serotype Typhimurium, Vibrio vulnificus, Vibrio cholerae and Vibrio parahaemolyticus from both pure cultures and seeded oysters. Amplicons were then subjected to a colorimetric CovaLink NH microwell plate sandwich hybridization using phosphorylated and biotinlylated oligonucleotide probes, the nucleotide sequences of which were located internal to the amplified DNA. The results from the hybridization with the multiplexed PCR amplified DNA exhibited a high signal/noise ratio ranging between 14.1 and 43.2 measured at 405 nm wavelength. The sensitivity of detection for each pathogen was 10(2) cells/g of oyster tissue homogenate. The results from this study showed that the combination of the multiplex PCR with a colorimetric microwell plate sandwich hybridization assay permits a specific, sensitive, and reproducible system for the detection of the microbial pathogens in shellfish, thereby improving the microbiological safety of shellfish to consumers.

Pico-strain multiplexed fiber optic sensor array operating down to infra-sonic frequencies.

PubMed

Littler, Ian C M; Gray, Malcolm B; Chow, Jong H; Shaddock, Daniel A; McClelland, David E

2009-06-22

An integrated sensor system is presented which displays passive long range operation to 100 km at pico-strain (pepsilon) sensitivity to low frequencies (4 Hz) in wavelength division multiplexed operation with negligible cross-talk (better than -75 dB). This has been achieved by pre-stabilizing and multiplexing all interrogation lasers for the sensor array to a single optical frequency reference. This single frequency reference allows each laser to be locked to an arbitrary wavelength and independently tuned, while maintaining suppression of laser frequency noise. With appropriate packaging, such a multiplexed strain sensing system can form the core of a low frequency accelerometer or hydrophone array.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Seven Novel Probe Systems for Real-Time PCR Provide Absolute Single-Base Discrimination, Higher Signaling, and Generic Components

PubMed Central

Murray, James L.; Hu, Peixu; Shafer, David A.

2015-01-01

We have developed novel probe systems for real-time PCR that provide higher specificity, greater sensitivity, and lower cost relative to dual-labeled probes. The seven DNA Detection Switch (DDS)-probe systems reported here employ two interacting polynucleotide components: a fluorescently labeled probe and a quencher antiprobe. High-fidelity detection is achieved with three DDS designs: two internal probes (internal DDS and Flip probes) and a primer probe (ZIPR probe), wherein each probe is combined with a carefully engineered, slightly mismatched, error-checking antiprobe. The antiprobe blocks off-target detection over a wide range of temperatures and facilitates multiplexing. Other designs (Universal probe, Half-Universal probe, and MacMan probe) use generic components that enable low-cost detection. Finally, single-molecule G-Force probes employ guanine-mediated fluorescent quenching by forming a hairpin between adjacent C-rich and G-rich sequences. Examples provided show how these probe technologies discriminate drug-resistant Mycobacterium tuberculosis mutants, Escherichia coli O157:H7, oncogenic EGFR deletion mutations, hepatitis B virus, influenza A/B strains, and single-nucleotide polymorphisms in the human VKORC1 gene. PMID:25307756

Equivalence of time-multiplexed and frequency-multiplexed signals in digital communications.

NASA Technical Reports Server (NTRS)

Timor, U.

1972-01-01

In comparing different techniques for multiplexing N binary data signals into a single channel, time-division multiplexing (TDM) is known to have a theoretic efficiency of 100 percent (neglecting sync power) and thus seems to outperform frequency-division multiplexing systems (FDM). By considering more general FDM systems, we will show that both TDM and FDM are equivalent and have an efficiency of 100 percent. The difference between the systems is in the multiplexing and demultiplexing subsystems, but not in the performance or in the generated waveforms.

Partial AZFc duplications not deletions are associated with male infertility in the Yi population of Yunnan Province, China.

PubMed

Ye, Jun-jie; Ma, Li; Yang, Li-juan; Wang, Jin-huan; Wang, Yue-li; Guo, Hai; Gong, Ning; Nie, Wen-hui; Zhao, Shu-hua

2013-09-01

There are many reports on associations between spermatogenesis and partial azoospermia factor c (AZFc) deletions as well as duplications; however, results are conflicting, possibly due to differences in methodology and ethnic background. The purpose of this study is to investigate the association of AZFc polymorphisms and male infertility in the Yi ethnic population, residents within Yunnan Province, China. A total of 224 infertile patients and 153 fertile subjects were selected in the Yi ethnic population. The study was performed by sequence-tagged site plus/minus (STS+/-) analysis followed by gene dosage and gene copy definition analysis. Y haplotypes of 215 cases and 115 controls were defined by 12 binary markers using single nucleotide polymorphism on Y chromosome (Y-SNP) multiplex assays based on single base primer extension technology. The distribution of Y haplotypes was not significantly different between the case and control groups. The frequencies of both gr/gr (7.6% vs. 8.5%) and b2/b3 (6.3% vs. 8.5%) deletions do not show significant differences. Similarly, single nucleotide variant (SNV) analysis shows no significant difference of gene copy definition between the cases and controls. However, the frequency of partial duplications in the infertile group (4.0%) is significantly higher than that in the control group (0.7%). Further, we found a case with sY1206 deletion which had two CDY1 copies but removed half of DAZ genes. Our results show that male infertility is associated with partial AZFc duplications, but neither gr/gr nor b2/b3 deletions, suggesting that partial AZFc duplications rather than deletions are risk factors for male infertility in Chinese-Yi population.

Identification and validation of single nucleotide polymorphisms in growth- and maturation-related candidate genes in sole (Solea solea L.).

PubMed

Diopere, Eveline; Hellemans, Bart; Volckaert, Filip A M; Maes, Gregory E

2013-03-01

Genomic methodologies applied in evolutionary and fisheries research have been of great benefit to understand the marine ecosystem and the management of natural resources. Although single nucleotide polymorphisms (SNPs) are attractive for the study of local adaptation, spatial stock management and traceability, and investigating the effects of fisheries-induced selection, they have rarely been exploited in non-model organisms. This is partly due to difficulties in finding and validating SNPs in species with limited or no genomic resources. Complementary to random genome-scan approaches, a targeted candidate gene approach has the potential to unveil pre-selected functional diversity and provides more in depth information on the action of selection at specific genes. For example genes can be under selective pressure due to climate change and sustained periods of heavy fishing pressure. In this study, we applied a candidate gene approach in sole (Solea solea L.), an important member of the demersal ecosystem. As consumption flatfish it is heavy exploited and has experienced associated life-history changes over the last 60years. To discover novel genetic polymorphisms in or around genes linked to important life history traits in sole, we screened a total of 76 candidate genes related to growth and maturation using a targeted resequencing approach. We identified in total 86 putative SNPs in 22 genes and validated 29 SNPs using a multiplex single-base extension genotyping assay. We found 22 informative SNPs, of which two represent non-synonymous mutations, potentially of functional relevance. These novel markers should be rapidly and broadly applicable in analyses of natural sole populations, as a measure of the evolutionary signature of overfishing and for initiatives on marker assisted selection. Copyright © 2012 Elsevier B.V. All rights reserved.

Use of a Hierarchical Oligonucleotide Primer Extension Approach for Multiplexed Relative Abundance Analysis of Methanogens in Anaerobic Digestion Systems

PubMed Central

Chuang, Hui-Ping; Hsu, Mao-Hsuan; Chen, Wei-Yu

2013-01-01

In this study, we established a rapid multiplex method to detect the relative abundances of amplified 16S rRNA genes from known cultivatable methanogens at hierarchical specificities in anaerobic digestion systems treating industrial wastewater and sewage sludge. The method was based on the hierarchical oligonucleotide primer extension (HOPE) technique and combined with a set of 27 primers designed to target the total archaeal populations and methanogens from 22 genera within 4 taxonomic orders. After optimization for their specificities and detection sensitivity under the conditions of multiple single-nucleotide primer extension reactions, the HOPE approach was applied to analyze the methanogens in 19 consortium samples from 7 anaerobic treatment systems (i.e., 513 reactions). Among the samples, the methanogen populations detected with order-level primers accounted for >77.2% of the PCR-amplified 16S rRNA genes detected using an Archaea-specific primer. The archaeal communities typically consisted of 2 to 7 known methanogen genera within the Methanobacteriales, Methanomicrobiales, and Methanosarcinales and displayed population dynamic and spatial distributions in anaerobic reactor operations. Principal component analysis of the HOPE data further showed that the methanogen communities could be clustered into 3 distinctive groups, in accordance with the distribution of the Methanosaeta, Methanolinea, and Methanomethylovorans, respectively. This finding suggested that in addition to acetotrophic and hydrogenotrophic methanogens, the methylotrophic methanogens might play a key role in the anaerobic treatment of industrial wastewater. Overall, the results demonstrated that the HOPE approach is a specific, rapid, and multiplexing platform to determine the relative abundances of targeted methanogens in PCR-amplified 16S rRNA gene products. PMID:24077716

High-Resolution Melting Analysis as a Powerful Tool to Discriminate and Genotype Pseudomonas savastanoi Pathovars and Strains

PubMed Central

Gori, Andrea; Cerboneschi, Matteo; Tegli, Stefania

2012-01-01

Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants. PMID:22295075

Measuring and modeling correlations in multiplex networks.

PubMed

Nicosia, Vincenzo; Latora, Vito

2015-09-01

The interactions among the elementary components of many complex systems can be qualitatively different. Such systems are therefore naturally described in terms of multiplex or multilayer networks, i.e., networks where each layer stands for a different type of interaction between the same set of nodes. There is today a growing interest in understanding when and why a description in terms of a multiplex network is necessary and more informative than a single-layer projection. Here we contribute to this debate by presenting a comprehensive study of correlations in multiplex networks. Correlations in node properties, especially degree-degree correlations, have been thoroughly studied in single-layer networks. Here we extend this idea to investigate and characterize correlations between the different layers of a multiplex network. Such correlations are intrinsically multiplex, and we first study them empirically by constructing and analyzing several multiplex networks from the real world. In particular, we introduce various measures to characterize correlations in the activity of the nodes and in their degree at the different layers and between activities and degrees. We show that real-world networks exhibit indeed nontrivial multiplex correlations. For instance, we find cases where two layers of the same multiplex network are positively correlated in terms of node degrees, while other two layers are negatively correlated. We then focus on constructing synthetic multiplex networks, proposing a series of models to reproduce the correlations observed empirically and/or to assess their relevance.

Single tube multiplex real-time PCR for the rapid detection of herpesvirus infections of the central nervous system.

PubMed

Sankuntaw, Nipaporn; Sukprasert, Saovaluk; Engchanil, Chulapan; Kaewkes, Wanlop; Chantratita, Wasun; Pairoj, Vantanit; Lulitanond, Viraphong

2011-01-01

Human herpesvirus infection of immunocompromised hosts may lead to central nervous system (CNS) infection and diseases. In this study, a single tube multiplex real-time PCR was developed for the detection of five herpesviruses (HSV-1, HSV-2, VZV, EBV and CMV) in clinical cerebrospinal fluid (CSF) specimens. Two primer pairs specific for the herpesvirus polymerase gene and five hybridization probe pairs for the specific identification of the herpesvirus types were used in a LightCycler multiplex real-time PCR. A singleplex real-time PCR was first optimized and then applied to the multiplex real-time PCR. The singleplex and multiplex real-time PCRs showed no cross-reactivity. The sensitivity of the singleplex real-time PCR was 1 copy per reaction for each herpesvirus, while that of the multiplex real-time PCR was 1 copy per reaction for HSV-1 and VZV and 10 copies per reaction for HSV-2, EBV and CMV. Intra and inter-assay variations of the single tube multiplex assay were in the range of 0.02%-3.67% and 0.79%-4.35%, respectively. The assay was evaluated by testing 62 clinical CSF samples and was found to have equivalent sensitivity, specificity and agreement as the routine real-time PCR, but reducing time, cost and amount of used sample. Copyright © 2011 Elsevier Ltd. All rights reserved.

Multimode fiber optic wavelength division multiplexing

NASA Technical Reports Server (NTRS)

Spencer, J. L.

1982-01-01

Optical wavelength division multiplexing (WDM) systems, with signals transmitted on different wavelengths through a single optical fiber, can have increased bandwidth and fault isolation properties over single wavelength optical systems. Two WDM system designs that might be used with multimode fibers are considered and a general description of the components which could be used to implement the system are given. The components described are sources, multiplexers, demultiplexers, and detectors. Emphasis is given to the demultiplexer technique which is the major developmental component in the WDM system.

Simple Multiplexing Hand-Held Control Unit

NASA Technical Reports Server (NTRS)

Hannaford, Blake

1989-01-01

Multiplexer consists of series of resistors, each shunted by single-pole, single-throw switch. User operates switches by pressing buttons or squeezing triggers. Prototype includes three switches operated successfully in over 200 hours of system operations. Number of switches accommodated determined by signal-to-noise ratio of current source, noise induced in control unit and cable, and number of bits in output of analog-to-digital converter. Because many computer-contolled robots have extra analog-to-digital channels, such multiplexer added at little extra cost.

Multiplexing of Radio-Frequency Single Electron Transistors

NASA Technical Reports Server (NTRS)

Stevenson, Thomas R.; Pellerano, F. A.; Stahle, C. M.; Aidala, K.; Schoelkopf, R. J.; Krebs, Carolyn (Technical Monitor)

2001-01-01

We present results on wavelength division multiplexing of radio-frequency single electron transistors. We use a network of resonant impedance matching circuits to direct applied rf carrier waves to different transistors depending on carrier frequency. A two-channel demonstration of this concept using discrete components successfully reconstructed input signals with small levels of cross coupling. A lithographic version of the rf circuits had measured parameters in agreement with electromagnetic modeling, with reduced cross capacitance and inductance, and should allow 20 to 50 channels to be multiplexed.

DNA polymerase preference determines PCR priming efficiency.

PubMed

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

2014-01-30

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

Microelectronic DNA assay for the detection of BRCA1 gene mutations

NASA Technical Reports Server (NTRS)

Chen, Hua; Han, Jie; Li, Jun; Meyyappan, Meyya

2004-01-01

Mutations in BRCA1 are characterized by predisposition to breast cancer, ovarian cancer and prostate cancer as well as colon cancer. Prognosis for this cancer survival depends upon the stage at which cancer is diagnosed. Reliable and rapid mutation detection is crucial for the early diagnosis and treatment. We developed an electronic assay for the detection of a representative single nucleotide polymorphism (SNP), deletion and insertion in BRCA1 gene by the microelectronics microarray instrumentation. The assay is rapid, and it takes 30 minutes for the immobilization of target DNA samples, hybridization, washing and readout. The assay is multiplexing since it is carried out at the same temperature and buffer conditions for each step. The assay is also highly specific, as the signal-to-noise ratio is much larger than recommended value (72.86 to 321.05 vs. 5) for homozygotes genotyping, and signal ratio close to the perfect value 1 for heterozygotes genotyping (1.04).

Fluorogenic DNA Sequencing in PDMS Microreactors

PubMed Central

Sims, Peter A.; Greenleaf, William J.; Duan, Haifeng; Xie, X. Sunney

2012-01-01

We have developed a multiplex sequencing-by-synthesis method combining terminal-phosphate labeled fluorogenic nucleotides (TPLFNs) and resealable microreactors. In the presence of phosphatase, the incorporation of a non-fluorescent TPLFN into a DNA primer by DNA polymerase results in a fluorophore. We immobilize DNA templates within polydimethylsiloxane (PDMS) microreactors, sequentially introduce one of the four identically labeled TPLFNs, seal the microreactors, allow template-directed TPLFN incorporation, and measure the signal from the fluorophores trapped in the microreactors. This workflow allows sequencing in a manner akin to pyrosequencing but without constant monitoring of each microreactor. With cycle times of <10 minutes, we demonstrate 30 base reads with ∼99% raw accuracy. “Fluorogenic pyrosequencing” combines benefits of pyrosequencing, such as rapid turn-around, native DNA generation, and single-color detection, with benefits of fluorescence-based approaches, such as highly sensitive detection and simple parallelization. PMID:21666670

Allele quantification using molecular inversion probes (MIP)

PubMed Central

Wang, Yuker; Moorhead, Martin; Karlin-Neumann, George; Falkowski, Matthew; Chen, Chunnuan; Siddiqui, Farooq; Davis, Ronald W.; Willis, Thomas D.; Faham, Malek

2005-01-01

Detection of genomic copy number changes has been an important research area, especially in cancer. Several high-throughput technologies have been developed to detect these changes. Features that are important for the utility of technologies assessing copy number changes include the ability to interrogate regions of interest at the desired density as well as the ability to differentiate the two homologs. In addition, assessing formaldehyde fixed and paraffin embedded (FFPE) samples allows the utilization of the vast majority of cancer samples. To address these points we demonstrate the use of molecular inversion probe (MIP) technology to the study of copy number. MIP is a high-throughput genotyping technology capable of interrogating >20 000 single nucleotide polymorphisms in the same tube. We have shown the ability of MIP at this multiplex level to provide copy number measurements while obtaining the allele information. In addition we have demonstrated a proof of principle for copy number analysis in FFPE samples. PMID:16314297

Wavelength meter having single mode fiber optics multiplexed inputs

DOEpatents

Hackel, R.P.; Paris, R.D.; Feldman, M.

1993-02-23

A wavelength meter having a single mode fiber optics input is disclosed. The single mode fiber enables a plurality of laser beams to be multiplexed to form a multiplexed input to the wavelength meter. The wavelength meter can provide a determination of the wavelength of any one or all of the plurality of laser beams by suitable processing. Another aspect of the present invention is that one of the laser beams could be a known reference laser having a predetermined wavelength. Hence, the improved wavelength meter can provide an on-line calibration capability with the reference laser input as one of the plurality of laser beams.

Wavelength meter having single mode fiber optics multiplexed inputs

DOEpatents

Hackel, Richard P.; Paris, Robert D.; Feldman, Mark

1993-01-01

A wavelength meter having a single mode fiber optics input is disclosed. The single mode fiber enables a plurality of laser beams to be multiplexed to form a multiplexed input to the wavelength meter. The wavelength meter can provide a determination of the wavelength of any one or all of the plurality of laser beams by suitable processing. Another aspect of the present invention is that one of the laser beams could be a known reference laser having a predetermined wavelength. Hence, the improved wavelength meter can provide an on-line calibration capability with the reference laser input as one of the plurality of laser beams.

Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay.

PubMed

Ajzenberg, Daniel; Collinet, Frédéric; Mercier, Aurélien; Vignoles, Philippe; Dardé, Marie-Laure

2010-12-01

We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers.

Magnetically multiplexed heating of single domain nanoparticles

NASA Astrophysics Data System (ADS)

Christiansen, M. G.; Senko, A. W.; Chen, R.; Romero, G.; Anikeeva, P.

2014-05-01

Selective hysteretic heating of multiple collocated types of single domain magnetic nanoparticles (SDMNPs) by alternating magnetic fields (AMFs) may offer a useful tool for biomedical applications. The possibility of "magnetothermal multiplexing" has not yet been realized, in part due to prevalent use of linear response theory to model SDMNP heating in AMFs. Dynamic hysteresis modeling suggests that specific driving conditions play an underappreciated role in determining optimal material selection strategies for high heat dissipation. Motivated by this observation, magnetothermal multiplexing is theoretically predicted and empirically demonstrated by selecting SDMNPs with properties that suggest optimal hysteretic heat dissipation at dissimilar AMF driving conditions. This form of multiplexing could effectively offer multiple channels for minimally invasive biological signaling applications.

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

Mangoni, Monica; Bisanzi, Simonetta; Carozzi, Francesca

Purpose: Clinical radiosensitivity varies considerably among patients, and radiation-induced side effects developing in normal tissue can be therapy limiting. Some single nucleotide polymorphisms (SNPs) have been shown to correlate with hypersensitivity to radiotherapy. We conducted a prospective study of 87 female patients with breast cancer who received radiotherapy after breast surgery. We evaluated the association between acute skin reaction following radiotherapy and 11 genetic polymorphisms in DNA repair genes: XRCC1 (Arg399Gln and Arg194Trp), XRCC3 (Thr241Met), XPD (Asp312Asn and Lys751Gln), MSH2 (gIVS12-6T>C), MLH1 (Ile219Val), MSH3 (Ala1045Thr), MGMT (Leu84Phe), and in damage-detoxification GSTM1 and GSTT1 genes (allele deletion). Methods and Materials: Individualmore » genetic polymorphisms were determined by polymerase chain reaction and single nucleotide primer extension for single nucleotide polymorphisms or by a multiplex polymerase chain reaction assay for deletion polymorphisms. The development of severe acute skin reaction (moist desquamation or interruption of radiotherapy due to toxicity) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for cumulative biologically effective radiation dose. Results: Radiosensitivity developed in eight patients and was increased in carriers of variants XRCC3-241Met allele (hazard ratio [HR] unquantifiably high), MSH2 gIVS12-6nt-C allele (HR = 53.36; 95% confidence intervals [95% CI], 3.56-798.98), and MSH3-1045Ala allele (HR unquantifiably high). Carriers of XRCC1-Arg194Trp variant allele in combination with XRCC1-Arg399Gln wild-type allele had a significant risk of radiosensitivity (HR = 38.26; 95% CI, 1.19-1232.52). Conclusions: To our knowledge, this is the first report to find an association between MSH2 and MSH3 genetic variants and the development of radiosensitivity in breast cancer patients. Our findings suggest the hypothesis that mismatch repair mechanisms may be involved in cellular response to radiotherapy. Genetic polymorphisms may be promising candidates for predicting acute radiosensitivity, but further studies are necessary to confirm our findings.« less

Genotyping of Toxoplasma gondii Isolates with 15 Microsatellite Markers in a Single Multiplex PCR Assay ▿

PubMed Central

Ajzenberg, Daniel; Collinet, Frédéric; Mercier, Aurélien; Vignoles, Philippe; Dardé, Marie-Laure

2010-01-01

We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers. PMID:20881166

Systematic search for single nucleotide polymorphisms in a lymphoid tyrosine phosphatase gene (PTPN22): association between a promoter polymorphism and type 1 diabetes in Asian populations.

PubMed

Kawasaki, Eiji; Awata, Takuya; Ikegami, Hiroshi; Kobayashi, Tetsuro; Maruyama, Taro; Nakanishi, Koji; Shimada, Akira; Uga, Miho; Uga, Mho; Kurihara, Susumu; Kawabata, Yumiko; Tanaka, Shoichiro; Kanazawa, Yasuhiko; Lee, Inkyu; Eguchi, Katsumi

2006-03-15

The protein tyrosine phosphatase, nonreceptor 22 gene (PTPN22) maps to human chromosome 1p13.3-p13.1 and encodes an important negative regulator of T-cell activation, lymphoid-specific phosphatase (Lyp). Recently, the minor allele of a single-nucleotide polymorphism (SNP) at nucleotide position 1858 (rs2476601, +1858C > T) was found to be associated with type 1 diabetes. However, the degree of the association is variable among ethnic populations, suggesting the presence of other disease-associated variants in PTPN22. To examine this possibility, we carried out a systemic search for PTPN22 using direct sequencing of PCR-amplified products in the Japanese population. Association and linkage studies were also conducted in 1,690 Japanese samples, 180 Korean samples, and 472 Caucasian samples from 95 nuclear families. We identified five novel SNPs, but not the +1858C > T SNP. Of these two frequent SNPs, -1123G > C, and +2740C > T were in strong linkage disequilibrium (LD), and the -1123G > C promoter SNP was associated with acute-onset but not slow-onset type 1 diabetes in the Japanese population (odds ratio [OR] = 1.42, 95% CI = 1.07-1.89, P = 0.015). This association was observed also in Korean patients with type 1 diabetes (Mantel-Haenszel chi2= 6.543, P = 0.0105, combined OR = 1.41 95% CI = 1.09-1.82). Furthermore, the affected family-based control (AFBAC) association test and the transmission disequilibrium analysis of multiplex families of European descent from the British Diabetes Association (BDA) Warren Repository indicated that the association was stronger in -1123G > C compared to +1858C > T. In conclusion, the type 1 diabetes association with PTPN22 is confirmed, but it cannot be attributed solely to the +1858C > T variant. The promoter -1123G > C SNP is a more likely causative variant in PTPN22. 2006 Wiley-Liss, Inc.

Designing robust watermark barcodes for multiplex long-read sequencing.

PubMed

Ezpeleta, Joaquín; Krsticevic, Flavia J; Bulacio, Pilar; Tapia, Elizabeth

2017-03-15

To attain acceptable sample misassignment rates, current approaches to multiplex single-molecule real-time sequencing require upstream quality improvement, which is obtained from multiple passes over the sequenced insert and significantly reduces the effective read length. In order to fully exploit the raw read length on multiplex applications, robust barcodes capable of dealing with the full single-pass error rates are needed. We present a method for designing sequencing barcodes that can withstand a large number of insertion, deletion and substitution errors and are suitable for use in multiplex single-molecule real-time sequencing. The manuscript focuses on the design of barcodes for full-length single-pass reads, impaired by challenging error rates in the order of 11%. The proposed barcodes can multiplex hundreds or thousands of samples while achieving sample misassignment probabilities as low as 10-7 under the above conditions, and are designed to be compatible with chemical constraints imposed by the sequencing process. Software tools for constructing watermark barcode sets and demultiplexing barcoded reads, together with example sets of barcodes and synthetic barcoded reads, are freely available at www.cifasis-conicet.gov.ar/ezpeleta/NS-watermark . ezpeleta@cifasis-conicet.gov.ar. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Mini-SNaPshot multiplex assays authenticate elephant ivory and simultaneously identify the species origin.

PubMed

Kitpipit, Thitika; Thongjued, Kantima; Penchart, Kitichaya; Ouithavon, Kanita; Chotigeat, Wilaiwan

2017-03-01

Illegal trading of ivory is mainly responsible for the dramatic decline in elephant populations. Thailand is one of the largest laundering hotspots for African ivory, as the domestic Asian elephant ivory can be legally traded. So, to help combat ivory poaching and smuggling, an efficient method is needed to identify the elephant species from its ivory and ivory products. In this study, a mini-SNaPshot ® multiplex assay was developed and fully validated for the identification of confiscated ivory and low DNA template ivory products. Elephantid- and elephant species-specific mitochondrial single nucleotide polymorphisms (SNPs) were identified from 207 mammalian and 1705 elephant/mammoth cytochrome b sequence alignments. Seven informative SNPs were used for assay development. The assay unambiguously and accurately identified authentic elephant ivory and its species of origin on the basis of peak size and color observed in the haplotype profile. The assay was highly efficient for analysis of confiscated ivory and low-template ivory products with a 99.29% success rate (N=140). It was highly reproducible, exhibited no cross-reaction with eight other mammalian DNA; and had 100% identification accuracy. In addition, nested and direct PCR amplification were also compatible with the developed assay. This efficient assay should benefit wildlife forensic laboratories and aid in the prosecution of elephant-related crimes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers.

PubMed

Asari, Masaru; Okuda, Katsuhiro; Hoshina, Chisato; Omura, Tomohiro; Tasaki, Yoshikazu; Shiono, Hiroshi; Matsubara, Kazuo; Shimizu, Keiko

2016-02-01

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis. Copyright © 2015 Elsevier Inc. All rights reserved.

Short tandem repeat analysis in Japanese population.

PubMed

Hashiyada, M

2000-01-01

Short tandem repeats (STRs), known as microsatellites, are one of the most informative genetic markers for characterizing biological materials. Because of the relatively small size of STR alleles (generally 100-350 nucleotides), amplification by polymerase chain reaction (PCR) is relatively easy, affording a high sensitivity of detection. In addition, STR loci can be amplified simultaneously in a multiplex PCR. Thus, substantial information can be obtained in a single analysis with the benefits of using less template DNA, reducing labor, and reducing the contamination. We investigated 14 STR loci in a Japanese population living in Sendai by three multiplex PCR kits, GenePrint PowerPlex 1.1 and 2.2. Fluorescent STR System (Promega, Madison, WI, USA) and AmpF/STR Profiler (Perkin-Elmer, Norwalk, CT, USA). Genomic DNA was extracted using sodium dodecyl sulfate (SDS) proteinase K or Chelex 100 treatment followed by the phenol/chloroform extraction. PCR was performed according to the manufacturer's protocols. Electrophoresis was carried out on an ABI 377 sequencer and the alleles were determined by GeneScan 2.0.2 software (Perkin-Elmer). In 14 STRs loci, statistical parameters indicated a relatively high rate, and no significant deviation from Hardy-Weinberg equilibrium was detected. We apply this STR system to paternity testing and forensic casework, e.g., personal identification in rape cases. This system is an effective tool in the forensic sciences to obtain information on individual identification.

Rapid multiplexed genotyping for hereditary thrombophilia by SELDI-TOF mass spectrometry.

PubMed

Yang, Shangbin; Xu, Lihui; Wu, Haifeng M

2010-03-01

Approximately 50% of patients with venous thromboembolism also present with hereditary predisposition. The most common genetic factors are single nucleotide polymorphisms (SNPs) of factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T. Genotyping these SNPs helps clinicians to correctly diagnose the disease and properly manage patients. In this study, we report a novel method using surface-enhanced laser desorption and ionization time of flight mass spectrometry to rapidly genotype, in a multiplex fashion, 3 SNPs that predispose patients to thrombosis. First, patient DNA samples were subjected to polymerase chain reaction to amplify and extend the DNA products with masses corresponding to specific genotypes. Polymerase chain reaction products were then applied to Q10 anionic protein chips, undergoing on-chip sample enrichment and clean-up. Finally, the genotypes of the SNPs were determined by surface-enhanced laser desorption and ionization time of flight mass spectrometry. This method offers a rapid turnaround time of less than 5 hours from sample collection to result reporting. The analytical accuracy of each SNP genotyping result has been confirmed by DNA sequencing. In addition, the genotype results produced by this method were validated by comparing them with results obtained by the approved method in the clinical reference laboratory. This novel method is fast, accurate, and reproducible, and thus provides an excellent platform to promote personalized medicine in the management of clotting disorders.

Analysis of chromosome 22q11 copy number variations by multiplex ligation-dependent probe amplification for prenatal diagnosis of congenital heart defect.

PubMed

Zhang, Jingjing; Ma, Dingyuan; Wang, Yan; Cao, Li; Wu, Yun; Qiao, Fengchang; Liu, An; Li, Li; Lin, Ying; Liu, Gang; Liu, Cuiyun; Hu, Ping; Xu, Zhengfeng

2015-01-01

Congenital heart defects (CHD) represent one of the most common birth defects. This study aimed to evaluate the value of multiplex ligation-dependent probe amplification (MLPA) as a tool to detect the copy number variations (CNVs) of 22q11 in fetuses with CHD. A large cohort of 225 fetuses with CHD was screened by fetal echocardiography. Once common chromosome abnormalities in 30 fetuses were screened out by conventional G-banding analysis, the CNVs of chromosome 22q11 in the remaining 195 fetuses were determined by MLPA for prenatal genetic counseling. In 195 CHD fetuses with normal karyotype, 11 cases had pathological CNVs, including 22q11.2 deletion (seven cases), the deletion of 22q11 cat eye syndrome (CES) region (one case), 22q11.2 duplication (one case), 22q13.3 deletion (one case) and 17p13.3 deletion (one case). In total, our findings from MLPA screening represented 4.9 % in our cohort. Among these, three cases were inherited CNVs, and eight cases were de novo. These CNVs were further verified by single nucleotide polymorphism (SNP)-array analysis, and their chromosomal location was refined. This study indicated that MLPA could serve as an effective test for routine prenatal diagnosis of 22q11 in fetuses with CHD.

A scoring strategy combining statistics and functional genomics supports a possible role for common polygenic variation in autism

PubMed Central

Carayol, Jérôme; Schellenberg, Gerard D.; Dombroski, Beth; Amiet, Claire; Génin, Bérengère; Fontaine, Karine; Rousseau, Francis; Vazart, Céline; Cohen, David; Frazier, Thomas W.; Hardan, Antonio Y.; Dawson, Geraldine; Rio Frio, Thomas

2014-01-01

Autism spectrum disorders (ASD) are highly heritable complex neurodevelopmental disorders with a 4:1 male: female ratio. Common genetic variation could explain 40–60% of the variance in liability to autism. Because of their small effect, genome-wide association studies (GWASs) have only identified a small number of individual single-nucleotide polymorphisms (SNPs). To increase the power of GWASs in complex disorders, methods like convergent functional genomics (CFG) have emerged to extract true association signals from noise and to identify and prioritize genes from SNPs using a scoring strategy combining statistics and functional genomics. We adapted and applied this approach to analyze data from a GWAS performed on families with multiple children affected with autism from Autism Speaks Autism Genetic Resource Exchange (AGRE). We identified a set of 133 candidate markers that were localized in or close to genes with functional relevance in ASD from a discovery population (545 multiplex families); a gender specific genetic score (GS) based on these common variants explained 1% (P = 0.01 in males) and 5% (P = 8.7 × 10−7 in females) of genetic variance in an independent sample of multiplex families. Overall, our work demonstrates that prioritization of GWAS data based on functional genomics identified common variants associated with autism and provided additional support for a common polygenic background in autism. PMID:24600472

Linkage and association studies identify a novel locus for Alzheimer disease at 7q36 in a Dutch population-based sample.

PubMed

Rademakers, Rosa; Cruts, Marc; Sleegers, Kristel; Dermaut, Bart; Theuns, Jessie; Aulchenko, Yurii; Weckx, Stefan; De Pooter, Tim; Van den Broeck, Marleen; Corsmit, Ellen; De Rijk, Peter; Del-Favero, Jurgen; van Swieten, John; van Duijn, Cornelia M; Van Broeckhoven, Christine

2005-10-01

We obtained conclusive linkage of Alzheimer disease (AD) with a candidate region of 19.7 cM at 7q36 in an extended multiplex family, family 1270, ascertained in a population-based study of early-onset AD in the northern Netherlands. Single-nucleotide polymorphism and haplotype association analyses of a Dutch patient-control sample further supported the linkage at 7q36. In addition, we identified a shared haplotype at 7q36 between family 1270 and three of six multiplex AD-affected families from the same geographical region, which is indicative of a founder effect and defines a priority region of 9.3 cM. Mutation analysis of coding exons of 29 candidate genes identified one linked synonymous mutation, g.38030G-->C in exon 10, that affected codon 626 of the PAX transactivation domain interacting protein gene (PAXIP1). It remains to be determined whether PAXIP1 has a functional role in the expression of AD in family 1270 or whether another mutation at this locus explains the observed linkage and sharing. Together, our linkage data from the informative family 1270 and the association data in the population-based early-onset AD patient-control sample strongly support the identification of a novel AD locus at 7q36 and re-emphasize the genetic heterogeneity of AD.

Screening for common copy-number variants in cancer genes.

PubMed

Tyson, Jess; Majerus, Tamsin M O; Walker, Susan; Armour, John A L

2010-12-01

For most cases of colorectal cancer that arise without a family history of the disease, it is proposed that an appreciable heritable component of predisposition is the result of contributions from many loci. Although progress has been made in identifying single nucleotide variants associated with colorectal cancer risk, the involvement of low-penetrance copy number variants is relatively unexplored. We have used multiplex amplifiable probe hybridization (MAPH) in a fourfold multiplex (QuadMAPH), positioned at an average resolution of one probe per 2 kb, to screen a total of 1.56 Mb of genomic DNA for copy number variants around the genes APC, AXIN1, BRCA1, BRCA2, CTNNB1, HRAS, MLH1, MSH2, and TP53. Two deletion events were detected, one upstream of MLH1 in a control individual and the other in APC in a colorectal cancer patient, but these do not seem to correspond to copy number polymorphisms with measurably high population frequencies. In summary, by means of our QuadMAPH assay, copy number measurement data were of sufficient resolution and accuracy to detect any copy number variants with high probability. However, this study has demonstrated a very low incidence of deletion and duplication variants within intronic and flanking regions of these nine genes, in both control individuals and colorectal cancer patients. Copyright © 2010 Elsevier Inc. All rights reserved.

Available number of multiplexed holograms based on signal-to-noise ratio analysis in reflection-type holographic memory using three-dimensional speckle-shift multiplexing.

PubMed

Nishizaki, Tatsuya; Matoba, Osamu; Nitta, Kouichi

2014-09-01

The recording properties of three-dimensional speckle-shift multiplexing in reflection-type holographic memory are analyzed numerically. Three-dimensional recording can increase the number of multiplexed holograms by suppressing the cross-talk noise from adjacent holograms by using depth-direction multiplexing rather than in-plane multiplexing. Numerical results indicate that the number of multiplexed holograms in three-layer recording can be increased by 1.44 times as large as that of a single-layer recording when an acceptable signal-to-noise ratio is set to be 2 when NA=0.43 and the thickness of the recording medium is 0.5 mm.

A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

PubMed Central

Lynd, Amy; Ranson, Hilary; McCall, P J; Randle, Nadine P; Black, William C; Walker, Edward D; Donnelly, Martin J

2005-01-01

Background A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation. Methods A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique. Results and Discussion The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous. Conclusion The method is capable of detecting both the East and West African kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries. PMID:15766386

State-of-the-art survey of multimode fiber optic wavelength division multiplexing

NASA Astrophysics Data System (ADS)

Spencer, J. L.

1983-05-01

Optical wavelength division multiplexing (WDM) systems, with signals transmitted on different wavelengths through a single fiber, can have increased information capacity and fault isolation properties over single wavelength optical systems. This paper describes a typical WDM system. Also, a state-of-the-art survey of optical multimode components which could be used to implement the system is made. The components to be surveyed are sources, multiplexers, and detectors. Emphasis is given to the demultiplexer techniques which are the major development components in the WDM system.

Wavelength Division Multiplexing Scheme for Radio-Frequency Single Electron Transistors

NASA Technical Reports Server (NTRS)

Stevenson, Thomas R.; Pellerano, F. A.; Stahle, C. M.; Aidala, K.; Schoelkopf, R. J.; Krebs, Carolyn (Technical Monitor)

2001-01-01

We describe work on a wavelength division multiplexing scheme for radio-frequency single electron transistors. We use a network of resonant impedance matching circuits to direct applied rf carrier waves to different transistors depending on carrier frequency. Using discrete components, we made a two-channel demonstration of this concept and successfully reconstructed input signals with small levels of cross coupling. A lithographic version of the rf circuits had measured parameters in agreement with electromagnetic modeling, with reduced cross capacitance and inductance, and should allow 20 to 50 channels to be multiplexed.

Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile, and accurate RNA structure analysis

PubMed Central

Smola, Matthew J.; Rice, Greggory M.; Busan, Steven; Siegfried, Nathan A.; Weeks, Kevin M.

2016-01-01

SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures. PMID:26426499

Common rs5918 (PlA1/A2) polymorphism in the ITGB3 gene and risk of coronary artery disease

PubMed Central

Heidari, Mohammad Mehdi; Soheilyfar, Sorour

2016-01-01

Introduction The T to C transition at nucleotide 1565 of the human glycoprotein IIIa (ITGB3) gene represents a genetic polymorphism (PlA1/A2) that can influence both platelet activation and aggregation and that has been associated with many types of disease. Here, we present a newly designed multiplex tetra-primer amplification refractory mutation system – polymerase chain reaction (T-ARMS-PCR) for genotyping a single nucleotide polymorphism (SNP) (dbSNP ID: rs5918) in the human ITGB3 gene. Material and methods We set up T-ARMS-PCR for the rs5918 SNP in a single-step PCR and the results were validated by the PCR-RFLP method in 132 coronary artery disease (CAD) patients and 122 unrelated healthy individuals. Results Full accordance was found for genotype determination by the PCR-RFLP method. The multiple logistic regression analysis showed a significant association of the rs5918 polymorphism and CAD according to dominant and recessive models (dominant model OR: 2.40, 95% CI: 1.33–4.35; p = 0.003, recessive model OR: 4.71, 95% CI: 1.32–16.80; p = 0.0067). Conclusions Our T-ARMS-PCR in comparison with RFLP and allele-specific PCR is more advantageous because this PCR method allows the evaluation of both the wild type and the mutant allele in the same tube. Our results suggest that the rs5918 (PlA1/A2) polymorphism in the ITGB3 gene may contribute to the susceptibility of sporadic Iranian coronary artery disease (CAD) patients. PMID:28905013

Massively parallel sequencing of 68 insertion/deletion markers identifies novel microhaplotypes for utility in human identity testing.

PubMed

Wendt, Frank R; Warshauer, David H; Zeng, Xiangpei; Churchill, Jennifer D; Novroski, Nicole M M; Song, Bing; King, Jonathan L; LaRue, Bobby L; Budowle, Bruce

2016-11-01

Short tandem repeat (STR) loci are the traditional markers used for kinship, missing persons, and direct comparison human identity testing. These markers hold considerable value due to their highly polymorphic nature, amplicon size, and ability to be multiplexed. However, many STRs are still too large for use in analysis of highly degraded DNA. Small bi-allelic polymorphisms, such as insertions/deletions (INDELs), may be better suited for analyzing compromised samples, and their allele size differences are amenable to analysis by capillary electrophoresis. The INDEL marker allelic states range in size from 2 to 6 base pairs, enabling small amplicon size. In addition, heterozygote balance may be increased by minimizing preferential amplification of the smaller allele, as is more common with STR markers. Multiplexing a large number of INDELs allows for generating panels with high discrimination power. The Nextera™ Rapid Capture Custom Enrichment Kit (Illumina, Inc., San Diego, CA) and massively parallel sequencing (MPS) on the Illumina MiSeq were used to sequence 68 well-characterized INDELs in four major US population groups. In addition, the STR Allele Identification Tool: Razor (STRait Razor) was used in a novel way to analyze INDEL sequences and detect adjacent single nucleotide polymorphisms (SNPs) and other polymorphisms. This application enabled the discovery of unique allelic variants, which increased the discrimination power and decreased the single-locus random match probabilities (RMPs) of 22 of these well-characterized INDELs which can be considered as microhaplotypes. These findings suggest that additional microhaplotypes containing human identification (HID) INDELs may exist elsewhere in the genome. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Single-band upconversion nanoprobes for multiplexed simultaneous in situ molecular mapping of cancer biomarkers.

PubMed

Zhou, Lei; Wang, Rui; Yao, Chi; Li, Xiaomin; Wang, Chengli; Zhang, Xiaoyan; Xu, Congjian; Zeng, Aijun; Zhao, Dongyuan; Zhang, Fan

2015-04-24

The identification of potential diagnostic markers and target molecules among the plethora of tumour oncoproteins for cancer diagnosis requires facile technology that is capable of quantitatively analysing multiple biomarkers in tumour cells and tissues. Diagnostic and prognostic classifications of human tumours are currently based on the western blotting and single-colour immunohistochemical methods that are not suitable for multiplexed detection. Herein, we report a general and novel method to prepare single-band upconversion nanoparticles with different colours. The expression levels of three biomarkers in breast cancer cells were determined using single-band upconversion nanoparticles, western blotting and immunohistochemical technologies with excellent correlation. Significantly, the application of antibody-conjugated single-band upconversion nanoparticle molecular profiling technology can achieve the multiplexed simultaneous in situ biodetection of biomarkers in breast cancer cells and tissue specimens and produce more accurate results for the simultaneous quantification of proteins present at low levels compared with classical immunohistochemical technology.

Quantum Point Contact Single-Nucleotide Conductance for DNA and RNA Sequence Identification.

PubMed

Afsari, Sepideh; Korshoj, Lee E; Abel, Gary R; Khan, Sajida; Chatterjee, Anushree; Nagpal, Prashant

2017-11-28

Several nanoscale electronic methods have been proposed for high-throughput single-molecule nucleic acid sequence identification. While many studies display a large ensemble of measurements as "electronic fingerprints" with some promise for distinguishing the DNA and RNA nucleobases (adenine, guanine, cytosine, thymine, and uracil), important metrics such as accuracy and confidence of base calling fall well below the current genomic methods. Issues such as unreliable metal-molecule junction formation, variation of nucleotide conformations, insufficient differences between the molecular orbitals responsible for single-nucleotide conduction, and lack of rigorous base calling algorithms lead to overlapping nanoelectronic measurements and poor nucleotide discrimination, especially at low coverage on single molecules. Here, we demonstrate a technique for reproducible conductance measurements on conformation-constrained single nucleotides and an advanced algorithmic approach for distinguishing the nucleobases. Our quantum point contact single-nucleotide conductance sequencing (QPICS) method uses combed and electrostatically bound single DNA and RNA nucleotides on a self-assembled monolayer of cysteamine molecules. We demonstrate that by varying the applied bias and pH conditions, molecular conductance can be switched ON and OFF, leading to reversible nucleotide perturbation for electronic recognition (NPER). We utilize NPER as a method to achieve >99.7% accuracy for DNA and RNA base calling at low molecular coverage (∼12×) using unbiased single measurements on DNA/RNA nucleotides, which represents a significant advance compared to existing sequencing methods. These results demonstrate the potential for utilizing simple surface modifications and existing biochemical moieties in individual nucleobases for a reliable, direct, single-molecule, nanoelectronic DNA and RNA nucleotide identification method for sequencing.

MtDNA SNP multiplexes for efficient inference of matrilineal genetic ancestry within Oceania.

PubMed

Ballantyne, Kaye N; van Oven, Mannis; Ralf, Arwin; Stoneking, Mark; Mitchell, R John; van Oorschot, Roland A H; Kayser, Manfred

2012-07-01

Human mitochondrial DNA (mtDNA) is a convenient marker for tracing matrilineal bio-geographic ancestry and is widely applied in forensic, genealogical and anthropological studies. In forensic applications, DNA-based ancestry inference can be useful for finding unknown suspects by concentrating police investigations in cases where autosomal STR profiling was unable to provide a match, or can help provide clues in missing person identification. Although multiplexed mtDNA single nucleotide polymorphism (SNP) assays to infer matrilineal ancestry at a (near) continental level are already available, such tools are lacking for the Oceania region. Here, we have developed a hierarchical system of three SNaPshot multiplexes for genotyping 26 SNPs defining all major mtDNA haplogroups for Oceania (including Australia, Near Oceania and Remote Oceania). With this system, it was possible to conclusively assign 74% of Oceanian individuals to their Oceanian matrilineal ancestry in an established literature database (after correcting for obvious external admixture). Furthermore, in a set of 161 genotyped individuals collected in Australia, Papua New Guinea and Fiji, 87.6% were conclusively assigned an Oceanian matrilineal origin. For the remaining 12.4% of the genotyped samples either a Eurasian origin was detected indicating likely European admixture (1.9%), the identified haplogroups are shared between Oceania and S/SE-Asia (5%), or the SNPs applied did not allow a geographic inference to be assigned (5.6%). Sub-regional assignment within Oceania was possible for 32.9% of the individuals genotyped: 49.5% of Australians were assigned an Australian origin and 13.7% of the Papua New Guineans were assigned a Near Oceanian origin, although none of the Fijians could be assigned a specific Remote Oceanian origin. The low assignment rates of Near and Remote Oceania are explained by recent migrations from Asia via Near Oceania into Remote Oceania. Combining the mtDNA multiplexes for Oceania introduced here with those we developed earlier for all other continental regions, global matrilineal bio-geographic ancestry assignment from DNA is now achievable in a highly efficient way that is also suitable for applications with limited material such as forensic case work. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Fiber optics for the future - wavelength division multiplexing

NASA Technical Reports Server (NTRS)

Spencer, J. L.

1982-01-01

Optical wavelength division multiplexing (WDM) systems, with signals transmitted on different wavelengths through a single fiber, can have increased information capacity and fault isolation properties over single wavelength optical systems. This paper describes a typical WDM system. The applicability of future standards to such a system are discussed. Also, a state-of-the-art survey of optical multimode components which could be used to implement the system are made. The components to be surveyed are sources, multiplexers, and detectors. Emphasis is given to the demultiplexer techniques which are the major developmental components in the WDM system.

System for producing chroma signals

NASA Technical Reports Server (NTRS)

Vorhaben, K. H.; Lipoma, P. C. (Inventor)

1977-01-01

A method for obtaining electronic chroma signals with a single scanning-type image device is described. A color multiplexed light signal is produced using an arrangement of dichroic filter stripes. In the particular system described, a two layer filter is used to color modulate external light which is then detected by an image pickup tube. The resulting time division multiplexed electronic signal from the pickup tube is converted by a decoder into a green color signal, and a single red-blue multiplexed signal, which is demultiplexed to produce red and blue color signals. The three primary color signals can be encoded as standard NTSC color signals.

Seven novel probe systems for real-time PCR provide absolute single-base discrimination, higher signaling, and generic components.

PubMed

Murray, James L; Hu, Peixu; Shafer, David A

2014-11-01

We have developed novel probe systems for real-time PCR that provide higher specificity, greater sensitivity, and lower cost relative to dual-labeled probes. The seven DNA Detection Switch (DDS)-probe systems reported here employ two interacting polynucleotide components: a fluorescently labeled probe and a quencher antiprobe. High-fidelity detection is achieved with three DDS designs: two internal probes (internal DDS and Flip probes) and a primer probe (ZIPR probe), wherein each probe is combined with a carefully engineered, slightly mismatched, error-checking antiprobe. The antiprobe blocks off-target detection over a wide range of temperatures and facilitates multiplexing. Other designs (Universal probe, Half-Universal probe, and MacMan probe) use generic components that enable low-cost detection. Finally, single-molecule G-Force probes employ guanine-mediated fluorescent quenching by forming a hairpin between adjacent C-rich and G-rich sequences. Examples provided show how these probe technologies discriminate drug-resistant Mycobacterium tuberculosis mutants, Escherichia coli O157:H7, oncogenic EGFR deletion mutations, hepatitis B virus, influenza A/B strains, and single-nucleotide polymorphisms in the human VKORC1 gene. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Aptamer-Based Multiplexed Proteomic Technology for Biomarker Discovery

PubMed Central

Gold, Larry; Ayers, Deborah; Bertino, Jennifer; Bock, Christopher; Bock, Ashley; Brody, Edward N.; Carter, Jeff; Dalby, Andrew B.; Eaton, Bruce E.; Fitzwater, Tim; Flather, Dylan; Forbes, Ashley; Foreman, Trudi; Fowler, Cate; Gawande, Bharat; Goss, Meredith; Gunn, Magda; Gupta, Shashi; Halladay, Dennis; Heil, Jim; Heilig, Joe; Hicke, Brian; Husar, Gregory; Janjic, Nebojsa; Jarvis, Thale; Jennings, Susan; Katilius, Evaldas; Keeney, Tracy R.; Kim, Nancy; Koch, Tad H.; Kraemer, Stephan; Kroiss, Luke; Le, Ngan; Levine, Daniel; Lindsey, Wes; Lollo, Bridget; Mayfield, Wes; Mehan, Mike; Mehler, Robert; Nelson, Sally K.; Nelson, Michele; Nieuwlandt, Dan; Nikrad, Malti; Ochsner, Urs; Ostroff, Rachel M.; Otis, Matt; Parker, Thomas; Pietrasiewicz, Steve; Resnicow, Daniel I.; Rohloff, John; Sanders, Glenn; Sattin, Sarah; Schneider, Daniel; Singer, Britta; Stanton, Martin; Sterkel, Alana; Stewart, Alex; Stratford, Suzanne; Vaught, Jonathan D.; Vrkljan, Mike; Walker, Jeffrey J.; Watrobka, Mike; Waugh, Sheela; Weiss, Allison; Wilcox, Sheri K.; Wolfson, Alexey; Wolk, Steven K.; Zhang, Chi; Zichi, Dom

2010-01-01

Background The interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. Methodology/Principal Findings We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. Conclusions/Significance We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine. PMID:21165148

Aptamer-based multiplexed proteomic technology for biomarker discovery.

PubMed

Gold, Larry; Ayers, Deborah; Bertino, Jennifer; Bock, Christopher; Bock, Ashley; Brody, Edward N; Carter, Jeff; Dalby, Andrew B; Eaton, Bruce E; Fitzwater, Tim; Flather, Dylan; Forbes, Ashley; Foreman, Trudi; Fowler, Cate; Gawande, Bharat; Goss, Meredith; Gunn, Magda; Gupta, Shashi; Halladay, Dennis; Heil, Jim; Heilig, Joe; Hicke, Brian; Husar, Gregory; Janjic, Nebojsa; Jarvis, Thale; Jennings, Susan; Katilius, Evaldas; Keeney, Tracy R; Kim, Nancy; Koch, Tad H; Kraemer, Stephan; Kroiss, Luke; Le, Ngan; Levine, Daniel; Lindsey, Wes; Lollo, Bridget; Mayfield, Wes; Mehan, Mike; Mehler, Robert; Nelson, Sally K; Nelson, Michele; Nieuwlandt, Dan; Nikrad, Malti; Ochsner, Urs; Ostroff, Rachel M; Otis, Matt; Parker, Thomas; Pietrasiewicz, Steve; Resnicow, Daniel I; Rohloff, John; Sanders, Glenn; Sattin, Sarah; Schneider, Daniel; Singer, Britta; Stanton, Martin; Sterkel, Alana; Stewart, Alex; Stratford, Suzanne; Vaught, Jonathan D; Vrkljan, Mike; Walker, Jeffrey J; Watrobka, Mike; Waugh, Sheela; Weiss, Allison; Wilcox, Sheri K; Wolfson, Alexey; Wolk, Steven K; Zhang, Chi; Zichi, Dom

2010-12-07

The interrogation of proteomes ("proteomics") in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (~100 fM-1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states. We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.

Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

PubMed

Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

2015-01-01

An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.

Frequency-multiplexed bias and readout of a 16-pixel superconducting nanowire single-photon detector array

NASA Astrophysics Data System (ADS)

Doerner, S.; Kuzmin, A.; Wuensch, S.; Charaev, I.; Boes, F.; Zwick, T.; Siegel, M.

2017-07-01

We demonstrate a 16-pixel array of microwave-current driven superconducting nanowire single-photon detectors with an integrated and scalable frequency-division multiplexing architecture, which reduces the required number of bias and readout lines to a single microwave feed line. The electrical behavior of the photon-sensitive nanowires, embedded in a resonant circuit, as well as the optical performance and timing jitter of the single detectors is discussed. Besides the single pixel measurements, we also demonstrate the operation of a 16-pixel array with a temporal, spatial, and photon-number resolution.

Genetic Structures of Copy Number Variants Revealed by Genotyping Single Sperm

PubMed Central

Luo, Minjie; Cui, Xiangfeng; Fredman, David; Brookes, Anthony J.; Azaro, Marco A.; Greenawalt, Danielle M.; Hu, Guohong; Wang, Hui-Yun; Tereshchenko, Irina V.; Lin, Yong; Shentu, Yue; Gao, Richeng; Shen, Li; Li, Honghua

2009-01-01

Background Copy number variants (CNVs) occupy a significant portion of the human genome and may have important roles in meiotic recombination, human genome evolution and gene expression. Many genetic diseases may be underlain by CNVs. However, because of the presence of their multiple copies, variability in copy numbers and the diploidy of the human genome, detailed genetic structure of CNVs cannot be readily studied by available techniques. Methodology/Principal Findings Single sperm samples were used as the primary subjects for the study so that CNV haplotypes in the sperm donors could be studied individually. Forty-eight CNVs characterized in a previous study were analyzed using a microarray-based high-throughput genotyping method after multiplex amplification. Seventeen single nucleotide polymorphisms (SNPs) were also included as controls. Two single-base variants, either allelic or paralogous, could be discriminated for all markers. Microarray data were used to resolve SNP alleles and CNV haplotypes, to quantitatively assess the numbers and compositions of the paralogous segments in each CNV haplotype. Conclusions/Significance This is the first study of the genetic structure of CNVs on a large scale. Resulting information may help understand evolution of the human genome, gain insight into many genetic processes, and discriminate between CNVs and SNPs. The highly sensitive high-throughput experimental system with haploid sperm samples as subjects may be used to facilitate detailed large-scale CNV analysis. PMID:19384415

Pulse patterning effect in optical pulse division multiplexing for flexible single wavelength multiple access optical network

NASA Astrophysics Data System (ADS)

Jung, Sun-Young; Kim, Chang-Hun; Han, Sang-Kook

2018-05-01

A demand for high spectral efficiency requires multiple access within a single wavelength, but the uplink signals are significantly degraded because of optical beat interference (OBI) in intensity modulation/direct detection system. An optical pulse division multiplexing (OPDM) technique was proposed that could effectively reduce the OBI via a simple method as long as near-orthogonality is satisfied, but the condition was strict, and thus, the number of multiplexing units was very limited. We propose pulse pattern enhanced OPDM (e-OPDM) to reduce the OBI and improve the flexibility in multiple access within a single wavelength. The performance of the e-OPDM and patterning effect are experimentally verified after 23-km single mode fiber transmission. By employing pulse patterning in OPDM, the tight requirement was relaxed by extending the optical delay dynamic range. This could support more number of access with reduced OBI, which could eventually enhance a multiple access function.

Optical delay encoding for fast timing and detector signal multiplexing in PET

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

Grant, Alexander M.; Levin, Craig S., E-mail: cslevin@stanford.edu; Molecular Imaging Program at Stanford

2015-08-15

Purpose: The large number of detector channels in modern positron emission tomography (PET) scanners poses a challenge in terms of readout electronics complexity. Multiplexing schemes are typically implemented to reduce the number of physical readout channels, but often result in performance degradation. Novel methods of multiplexing in PET must be developed to avoid this data degradation. The preservation of fast timing information is especially important for time-of-flight PET. Methods: A new multiplexing scheme based on encoding detector interaction events with a series of extremely fast overlapping optical pulses with precise delays is demonstrated in this work. Encoding events in thismore » way potentially allows many detector channels to be simultaneously encoded onto a single optical fiber that is then read out by a single digitizer. A two channel silicon photomultiplier-based prototype utilizing this optical delay encoding technique along with dual threshold time-over-threshold is demonstrated. Results: The optical encoding and multiplexing prototype achieves a coincidence time resolution of 160 ps full width at half maximum (FWHM) and an energy resolution of 13.1% FWHM at 511 keV with 3 × 3 × 5 mm{sup 3} LYSO crystals. All interaction information for both detectors, including timing, energy, and channel identification, is encoded onto a single optical fiber with little degradation. Conclusions: Optical delay encoding and multiplexing technology could lead to time-of-flight PET scanners with fewer readout channels and simplified data acquisition systems.« less

Ultra high speed optical transmission using subcarrier-multiplexed four-dimensional LDPC-coded modulation.

PubMed

Batshon, Hussam G; Djordjevic, Ivan; Schmidt, Ted

2010-09-13

We propose a subcarrier-multiplexed four-dimensional LDPC bit-interleaved coded modulation scheme that is capable of achieving beyond 480 Gb/s single-channel transmission rate over optical channels. Subcarrier-multiplexed four-dimensional LDPC coded modulation scheme outperforms the corresponding dual polarization schemes by up to 4.6 dB in OSNR at BER 10(-8).

Large object investigation by digital holography with effective spectrum multiplexing under single-exposure approach

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

Liu, Ning, E-mail: coolboy006@sohu.com; Zhang, Yingying; Xie, Jun

2014-10-13

We present a method to investigate large object by digital holography with effective spectrum multiplexing under single-exposure approach. This method splits the original reference beam and redirects one of its branches as a second object beam. Through the modified Mach-Zehnder interferometer, the two object beams can illuminate different parts of the large object and create a spectrum multiplexed hologram onto the focal plane array of the charge-coupled device/complementary metal oxide semiconductor camera. After correct spectrum extraction and image reconstruction, the large object can be fully observed within only one single snap-shot. The flexibility and great performance make our method amore » very attractive and promising technique for large object investigation under common 632.8 nm illumination.« less

Molecular allergy diagnostics using multiplex assays: methodological and practical considerations for use in research and clinical routine: Part 21 of the Series Molecular Allergology.

PubMed

Jakob, Thilo; Forstenlechner, Peter; Matricardi, Paolo; Kleine-Tebbe, Jörg

The availability of single allergens and their use in microarray technology enables the simultaneous determination of specific IgE (sIgE) to a multitude of different allergens (> 100) in a multiplex procedure requiring only minute amounts of serum. This allows extensive individual sensitization profiles to be determined from a single analysis. Combined with a patient's medical history, these profiles simplify identification of cross-reactivity; permit a more accurate estimation of the risk of severe reactions; and enable the indication for specific immunotherapy to be more precisely established, particularly in cases of polysensitization. Strictly speaking, a multiplex assay is not a single test, but instead more than 100 simultaneous tests. This places considerable demands on the production, quality assurance, and interpretation of data. The following chapter describes the multiplex test systems currently available and discusses their characteristics. Performance data are presented and the sIgE values obtained from multiplex and singleplex assays are compared. Finally, the advantages and limitations of molecular allergy diagnostics using multiplex assays in clinical routine are discussed, and innovative possibilities for clinical research are described. The multiplex diagnostic tests available for clinical routine have now become well established. The interpretation of test results is demanding, particularly since all individual results need to be checked for their plausibility and clinical relevance on the basis of previous history (patient history, clinical symptoms, challenge test results). There is still room for improvement in certain areas, for example with respect to the overall test sensitivity of the method, as well as the availability and quality of particular allergens. The current test systems are just the beginning of a continuous development that will influence and most likely change clinical allergology in the coming years.

Simultaneous readout of 128 X-ray and gamma-ray transition-edge microcalorimeters using microwave SQUID multiplexing

NASA Astrophysics Data System (ADS)

Mates, J. A. B.; Becker, D. T.; Bennett, D. A.; Dober, B. J.; Gard, J. D.; Hays-Wehle, J. P.; Fowler, J. W.; Hilton, G. C.; Reintsema, C. D.; Schmidt, D. R.; Swetz, D. S.; Vale, L. R.; Ullom, J. N.

2017-08-01

The number of elements in most cryogenic sensor arrays is limited by the technology available to multiplex signals from the arrays into a smaller number of wires and readout amplifiers. The largest demonstrated arrays of transition-edge sensor (TES) microcalorimeters contain roughly 250 detectors and use time-division multiplexing with Superconducting Quantum Interference Devices (SQUIDs). The bandwidth limits of this technology constrain the number of sensors per amplifier chain, a quantity known as the multiplexing factor, to several 10s. With microwave SQUID multiplexing, we can expand the readout bandwidth and enable much larger multiplexing factors. While microwave SQUID multiplexing of TES microcalorimeters has been previously demonstrated with small numbers of detectors, we now present a fully scalable demonstration in which 128 TES detectors are read out on a single pair of coaxial cables.

Percolation in multiplex networks with overlap.

PubMed

Cellai, Davide; López, Eduardo; Zhou, Jie; Gleeson, James P; Bianconi, Ginestra

2013-11-01

From transportation networks to complex infrastructures, and to social and communication networks, a large variety of systems can be described in terms of multiplexes formed by a set of nodes interacting through different networks (layers). Multiplexes may display an increased fragility with respect to the single layers that constitute them. However, so far the overlap of the links in different layers has been mostly neglected, despite the fact that it is an ubiquitous phenomenon in most multiplexes. Here, we show that the overlap among layers can improve the robustness of interdependent multiplex systems and change the critical behavior of the percolation phase transition in a complex way.

Stable Gene Targeting in Human Cells Using Single-Strand Oligonucleotides with Modified Bases

PubMed Central

Rios, Xavier; Briggs, Adrian W.; Christodoulou, Danos; Gorham, Josh M.; Seidman, Jonathan G.; Church, George M.

2012-01-01

Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells. PMID:22615794

Rapid targeted somatic mutation analysis of solid tumors in routine clinical diagnostics.

PubMed

Magliacane, Gilda; Grassini, Greta; Bartocci, Paola; Francaviglia, Ilaria; Dal Cin, Elena; Barbieri, Gianluca; Arrigoni, Gianluigi; Pecciarini, Lorenza; Doglioni, Claudio; Cangi, Maria Giulia

2015-10-13

Tumor genotyping is an essential step in routine clinical practice and pathology laboratories face a major challenge in being able to provide rapid, sensitive and updated molecular tests. We developed a novel mass spectrometry multiplexed genotyping platform named PentaPanel to concurrently assess single nucleotide polymorphisms in 56 hotspots of the 5 most clinically relevant cancer genes, KRAS, NRAS, BRAF, EGFR and PIK3CA for a total of 221 detectable mutations. To both evaluate and validate the PentaPanel performance, we investigated 1025 tumor specimens of 6 different cancer types (carcinomas of colon, lung, breast, pancreas, and biliary tract, and melanomas), systematically addressing sensitivity, specificity, and reproducibility of our platform. Sanger sequencing was also performed for all the study samples. Our data showed that PentaPanel is a high throughput and robust tool, allowing genotyping for targeted therapy selection of 10 patients in the same run, with a practical turnaround time of 2 working days. Importantly, it was successfully used to interrogate different DNAs isolated from routinely processed specimens (formalin-fixed paraffin embedded, frozen, and cytological samples), covering all the requirements of clinical tests. In conclusion, the PentaPanel platform can provide an immediate, accurate and cost effective multiplex approach for clinically relevant gene mutation analysis in many solid tumors and its utility across many diseases can be particularly relevant in multiple clinical trials, including the new basket trial approach, aiming to identify appropriate targeted drug combination strategies.

Forensic genetic informativeness of an SNP panel consisting of 19 multi-allelic SNPs.

PubMed

Gao, Zehua; Chen, Xiaogang; Zhao, Yuancun; Zhao, Xiaohong; Zhang, Shu; Yang, Yiwen; Wang, Yufang; Zhang, Ji

2018-05-01

Current research focusing on forensic personal identification, phenotype inference and ancestry information on single-nucleotide polymorphisms (SNPs) has been widely reported. In the present study, we focused on tetra-allelic SNPs in the Chinese Han population. A total of 48 tetra-allelic SNPs were screened out from the Chinese Han population of the 1000 Genomes Database, including Chinese Han in Beijing (CHB) and Chinese Han South (CHS). Considering the forensic genetic requirement for the polymorphisms, only 11 tetra-allelic SNPs with a heterozygosity >0.06 were selected for further multiplex panel construction. In order to meet the demands of personal identification and parentage identification, an additional 8 tri-allelic SNPs were combined into the final multiplex panel. To ensure application in the degraded DNA analysis, all the PCR products were designed to be 87-188 bp. Employing multiple PCR reactions and SNaPshot minisequencing, 511 unrelated Chinese Han individuals from Sichuan were genotyped. The combined match probability (CMP), combined discrimination power (CDP), and cumulative probability of exclusion (CPE) of the panel were 6.07 × 10 -11 , 0.9999999999393 and 0.996764, respectively. Based on the population data retrieved from the 1000 Genomes Project, Fst values between Chinese Han in Sichuan (SCH) and all the populations included in the 1000 Genomes Project were calculated. The results indicated that two SNPs in this panel may contain ancestry information and may be used as markers of forensic biogeographical ancestry inference. Copyright © 2018 Elsevier B.V. All rights reserved.

Causative Role of Grapevine Red Blotch Virus in Red Blotch Disease.

PubMed

Yepes, Luz Marcela; Cieniewicz, Elizabeth; Krenz, Björn; McLane, Heather; Thompson, Jeremy R; Perry, Keith Lloyd; Fuchs, Marc

2018-05-17

Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch's postulates and revealed the causative role of GRBV in red blotch disease.

Quantitation of Marek's disease and chicken anemia viruses in organs of experimentally infected chickens and commercial chickens by multiplex real-time PCR.

PubMed

Davidson, Irit; Raibshtein, I; Al-Touri, A

2013-06-01

The worldwide distribution of chicken anemia virus (CAV) and Marek's disease virus (MDV) is well documented. In addition to their economic significance in single- or dual-virus infections, the two viruses can often accompany various other pathogens and affect poultry health either directly, by causing tumors, anemia, and delayed growth, or indirectly, by aggravating other diseases, as a result of their immunosuppressive effects. After a decade of employing the molecular diagnosis of those viruses, which replaced conventional virus isolation, we present the development of a real-time multiplex PCR for the simultaneous detection of both viruses. The real-time PCRs for MDV and for CAV alone are more sensitive than the respective end-point PCRs. In addition, the multiplex real-time shows a similar sensitivity when compared to the single real-time PCR for each virus. The newly developed real-time multiplex PCR is of importance in terms of the diagnosis and detection of low copies of each virus, MDV and CAV in single- and in multiple-virus infections, and its applicability will be further evaluated.

Validation and optimization of the Ion Torrent S5 XL sequencer and Oncomine workflow for BRCA1 and BRCA2 genetic testing.

PubMed

Shin, Saeam; Kim, Yoonjung; Chul Oh, Seoung; Yu, Nae; Lee, Seung-Tae; Rak Choi, Jong; Lee, Kyung-A

2017-05-23

In this study, we validated the analytical performance of BRCA1/2 sequencing using Ion Torrent's new bench-top sequencer with amplicon panel with optimized bioinformatics pipelines. Using 43 samples that were previously validated by Illumina's MiSeq platform and/or by Sanger sequencing/multiplex ligation-dependent probe amplification, we amplified the target with the Oncomine™ BRCA Research Assay and sequenced on Ion Torrent S5 XL (Thermo Fisher Scientific, Waltham, MA, USA). We compared two bioinformatics pipelines for optimal processing of S5 XL sequence data: the Torrent Suite with a plug-in Torrent Variant Caller (Thermo Fisher Scientific), and commercial NextGENe software (Softgenetics, State College, PA, USA). All expected 681 single nucleotide variants, 15 small indels, and three copy number variants were correctly called, except one common variant adjacent to a rare variant on the primer-binding site. The sensitivity, specificity, false positive rate, and accuracy for detection of single nucleotide variant and small indels of S5 XL sequencing were 99.85%, 100%, 0%, and 99.99% for the Torrent Variant Caller and 99.85%, 99.99%, 0.14%, and 99.99% for NextGENe, respectively. The reproducibility of variant calling was 100%, and the precision of variant frequency also showed good performance with coefficients of variation between 0.32 and 5.29%. We obtained highly accurate data through uniform and sufficient coverage depth over all target regions and through optimization of the bioinformatics pipeline. We confirmed that our platform is accurate and practical for diagnostic BRCA1/2 testing in a clinical laboratory.

Differentiation of Mycobacterium tuberculosis complex from non-tubercular mycobacteria by nested multiplex PCR targeting IS6110, MTP40 and 32kD alpha antigen encoding gene fragments.

PubMed

Sinha, Pallavi; Gupta, Anamika; Prakash, Pradyot; Anupurba, Shampa; Tripathi, Rajneesh; Srivastava, G N

2016-03-12

Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragments IS6110, MTP40 and 32kD α-antigen encoding genes specific for Mycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy. The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing. Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5%) and 19 (29.2%) cases were culture positive for M. tuberculosis. Based on CRS, 450 patients had "clinical TB" (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed "non-TB" cases. For culture, the sensitivity was low, 79.3% for pulmonary and 54.3% for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1% for pulmonary and 91.4% for extra-pulmonary TB cases with specificity of 100% and 93.3% respectively. Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB.

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.

Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.

PubMed

Garst, Andrew D; Bassalo, Marcelo C; Pines, Gur; Lynch, Sean A; Halweg-Edwards, Andrea L; Liu, Rongming; Liang, Liya; Wang, Zhiwen; Zeitoun, Ramsey; Alexander, William G; Gill, Ryan T

2017-01-01

Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.

Characterization of lettuce big-vein associated virus and Mirafiori lettuce big-vein virus infecting lettuce in Saudi Arabia.

PubMed

Umar, M; Amer, M A; Al-Saleh, M A; Al-Shahwan, I M; Shakeel, M T; Zakri, A M; Katis, N I

2017-07-01

During 2014 and 2015, 97 lettuce plants that showed big-vein-disease-like symptoms and seven weed plants were collected from the Riyadh region. DAS-ELISA revealed that 25% and 9% of the lettuce plants were singly infected with LBVaV and MiLBVV, respectively, whereas 63% had a mixed infection with both viruses. The results were confirmed by multiplex reverse transcription polymerase chain reaction using primers specific for LBVaV and MiLBVV. LBVaV and MiLBVV were also detected in Sonchus oleraceus and Eruca sativa, respectively. The nucleotide sequence of LBVaV and MiLBVV Saudi isolates ranged from 94.3-100%, and their similarities to isolates with sequences in the GenBank database ranged from 93.9 to 99.6% and 93.8 to 99.3%, respectively. Olpidium sp. was present in the roots of lettuce plants with big-vein disease and it was shown to facilitate transmission of both viruses.

Noninvasive Prenatal Paternity Testing (NIPAT) through Maternal Plasma DNA Sequencing: A Pilot Study.

PubMed

Jiang, Haojun; Xie, Yifan; Li, Xuchao; Ge, Huijuan; Deng, Yongqiang; Mu, Haofang; Feng, Xiaoli; Yin, Lu; Du, Zhou; Chen, Fang; He, Nongyue

2016-01-01

Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) have been already used to perform noninvasive prenatal paternity testing from maternal plasma DNA. The frequently used technologies were PCR followed by capillary electrophoresis and SNP typing array, respectively. Here, we developed a noninvasive prenatal paternity testing (NIPAT) based on SNP typing with maternal plasma DNA sequencing. We evaluated the influence factors (minor allele frequency (MAF), the number of total SNP, fetal fraction and effective sequencing depth) and designed three different selective SNP panels in order to verify the performance in clinical cases. Combining targeted deep sequencing of selective SNP and informative bioinformatics pipeline, we calculated the combined paternity index (CPI) of 17 cases to determine paternity. Sequencing-based NIPAT results fully agreed with invasive prenatal paternity test using STR multiplex system. Our study here proved that the maternal plasma DNA sequencing-based technology is feasible and accurate in determining paternity, which may provide an alternative in forensic application in the future.

Time Division Multiplexing of Semiconductor Qubits

NASA Astrophysics Data System (ADS)

Jarratt, Marie Claire; Hornibrook, John; Croot, Xanthe; Watson, John; Gardner, Geoff; Fallahi, Saeed; Manfra, Michael; Reilly, David

Readout chains, comprising resonators, amplifiers, and demodulators, are likely to be precious resources in quantum computing architectures. The potential to share readout resources is contingent on realising efficient means of time-division multiplexing (TDM) schemes that are compatible with quantum computing. Here, we demonstrate TDM using a GaAs quantum dot device with multiple charge sensors. Our device incorporates chip-level switches that do not load the impedance matching network. When used in conjunction with frequency multiplexing, each frequency tone addresses multiple time-multiplexed qubits, vastly increasing the capacity of a single readout line.

Mass cytometry: a highly multiplexed single-cell technology for advancing drug development.

PubMed

Atkuri, Kondala R; Stevens, Jeffrey C; Neubert, Hendrik

2015-02-01

Advanced single-cell analysis technologies (e.g., mass cytometry) that help in multiplexing cellular measurements in limited-volume primary samples are critical in bridging discovery efforts to successful drug approval. Mass cytometry is the state-of-the-art technology in multiparametric single-cell analysis. Mass cytometers (also known as cytometry by time-of-flight or CyTOF) combine the cellular analysis principles of traditional fluorescence-based flow cytometry with the selectivity and quantitative power of inductively coupled plasma-mass spectrometry. Standard flow cytometry is limited in the number of parameters that can be measured owing to the overlap in signal when detecting fluorescently labeled antibodies. Mass cytometry uses antibodies tagged to stable isotopes of rare earth metals, which requires minimal signal compensation between the different metal tags. This unique feature enables researchers to seamlessly multiplex up to 40 independent measurements on single cells. In this overview we first present an overview of mass cytometry and compare it with traditional flow cytometry. We then discuss the emerging and potential applications of CyTOF technology in the pharmaceutical industry, including quantitative and qualitative deep profiling of immune cells and their applications in assessing drug immunogenicity, extensive mapping of signaling networks in single cells, cell surface receptor quantification and multiplexed internalization kinetics, multiplexing sample analysis by barcoding, and establishing cell ontologies on the basis of phenotype and/or function. We end with a discussion of the anticipated impact of this technology on drug development lifecycle with special emphasis on the utility of mass cytometry in deciphering a drug's pharmacokinetics and pharmacodynamics relationship. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Microwave SQUID Multiplexer for the Readout of Metallic Magnetic Calorimeters

NASA Astrophysics Data System (ADS)

Kempf, S.; Gastaldo, L.; Fleischmann, A.; Enss, C.

2014-06-01

We have realized a frequency-domain multiplexing technique for the readout of large metallic magnetic calorimeter detector arrays. It is based on non-hysteretic single-junction SQUIDs and allows for a simultaneous readout of hundreds or thousands of detectors by using a single cryogenic high electron mobility transistor amplifier and two coaxial cables that are routed from room-temperature to the detector array. We discuss the working principle of the multiplexer and present details about our prototype multiplexer design. We show that fabricated devices are fully operational and that characteristic SQUID parameters such as the input sensitivity of the SQUID or the resonance frequency of the readout circuit can be predicted with confidence. Our best device so far has shown a magnetic flux white noise level of 1.4 m which can in future be reduced by an optimization of the fabrication processes as well as an improved microwave readout system.

Multiplexed Five-Color Molecular Imaging of Cancer Cells and Tumor Tissues with Carbon Nanotube Raman Tags in the Near-Infrared

PubMed Central

Liu, Zhuang; Tabakman, Scott; Sherlock, Sarah; Li, Xiaolin; Chen, Zhuo; Jiang, Kaili; Fan, Shoushan; Dai, Hongjie

2011-01-01

Single-walled carbon nanotubes (SWNTs) with five different C13/C12 isotope compositions and well-separated Raman peaks have been synthesized and conjugated to five targeting ligands in order to impart molecular specificity. Multiplexed Raman imaging of live cells has been carried out by highly specific staining of cells with a five-color mixture of SWNTs. Ex vivo multiplexed Raman imaging of tumor samples uncovers a surprising up-regulation of epidermal growth factor receptor (EGFR) on LS174T colon cancer cells from cell culture to in vivo tumor growth. This is the first time five-color multiplexed molecular imaging has been performed in the near-infrared (NIR) region under a single laser excitation. Near zero interfering background of imaging is achieved due to the sharp Raman peaks unique to nanotubes over the low, smooth autofluorescence background of biological species. PMID:21442006

Energy-efficient process-stacking multiplexing access for 60-GHz mm-wave wireless personal area networks.

PubMed

Estevez, Claudio; Kailas, Aravind

2012-01-01

Millimeter-wave technology shows high potential for future wireless personal area networks, reaching over 1 Gbps transmissions using simple modulation techniques. Current specifications consider dividing the spectrum into effortlessly separable spectrum ranges. These low requirements open a research area in time and space multiplexing techniques for millimeter-waves. In this work a process-stacking multiplexing access algorithm is designed for single channel operation. The concept is intuitive, but its implementation is not trivial. The key to stacking single channel events is to operate while simultaneously obtaining and handling a-posteriori time-frame information of scheduled events. This information is used to shift a global time pointer that the wireless access point manages and uses to synchronize all serviced nodes. The performance of the proposed multiplexing access technique is lower bounded by the performance of legacy TDMA and can significantly improve the effective throughput. Work is validated by simulation results.

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

Hsia, Chu Chieh; Chizhikov, Vladimir E.; Yang, Amy X.

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminatedmore » the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.« less

Multiplexed single-molecule force spectroscopy using a centrifuge.

PubMed

Yang, Darren; Ward, Andrew; Halvorsen, Ken; Wong, Wesley P

2016-03-17

We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise.

Multiplexed single-molecule force spectroscopy using a centrifuge

PubMed Central

Yang, Darren; Ward, Andrew; Halvorsen, Ken; Wong, Wesley P.

2016-01-01

We present a miniature centrifuge force microscope (CFM) that repurposes a benchtop centrifuge for high-throughput single-molecule experiments with high-resolution particle tracking, a large force range, temperature control and simple push-button operation. Incorporating DNA nanoswitches to enable repeated interrogation by force of single molecular pairs, we demonstrate increased throughput, reliability and the ability to characterize population heterogeneity. We perform spatiotemporally multiplexed experiments to collect 1,863 bond rupture statistics from 538 traceable molecular pairs in a single experiment, and show that 2 populations of DNA zippers can be distinguished using per-molecule statistics to reduce noise. PMID:26984516

Single-exposure two-dimensional superresolution in digital holography using a vertical cavity surface-emitting laser source array.

PubMed

Granero, Luis; Zalevsky, Zeev; Micó, Vicente

2011-04-01

We present a new implementation capable of producing two-dimensional (2D) superresolution (SR) imaging in a single exposure by aperture synthesis in digital lensless Fourier holography when using angular multiplexing provided by a vertical cavity surface-emitting laser source array. The system performs the recording in a single CCD snapshot of a multiplexed hologram coming from the incoherent addition of multiple subholograms, where each contains information about a different 2D spatial frequency band of the object's spectrum. Thus, a set of nonoverlapping bandpass images of the input object can be recovered by Fourier transformation (FT) of the multiplexed hologram. The SR is obtained by coherent addition of the information contained in each bandpass image while generating an enlarged synthetic aperture. Experimental results demonstrate improvement in resolution and image quality.

[A review of mixed gas detection system based on infrared spectroscopic technique].

PubMed

Dang, Jing-Min; Fu, Li; Yan, Zi-Hui; Zheng, Chuan-Tao; Chang, Yu-Chun; Chen, Chen; Wang, Yi-Din

2014-10-01

In order to provide the experiences and references to the researchers who are working on infrared (IR) mixed gas detection field. The proposed manuscript reviews two sections of the aforementioned field, including optical multiplexing structure and detection method. At present, the coherent light sources whose representative are quantum cascade laser (QCL) and inter-band cascade laser(ICL) become the mainstream light source in IR mixed gas detection, which replace the traditional non-coherent light source, such as IR radiation source and IR light emitting diode. In addition, the photon detector which has a super high detectivity and very short response time is gradually beyond thermal infrared detector, dominant in the field of infrared detector. The optical multiplexing structure is the key factor of IR mixed gas detection system, which consists of single light source multi-plexing detection structure and multi light source multiplexing detection structure. Particularly, single light source multiplexing detection structure is advantages of small volume and high integration, which make it a plausible candidate for the portable mixed gas detection system; Meanwhile, multi light source multiplexing detection structure is embodiment of time division multiplex, frequency division multiplexing and wavelength division multiplexing, and become the leading structure of the mixed gas detection system because of its wider spectral range, higher spectral resolution, etc. The detection method applied to IR mixed gas detection includes non-dispersive infrared (NDIR) spectroscopy, wavelength and frequency-modulation spectroscopy, cavity-enhanced spectroscopy and photoacoustic spectroscopy, etc. The IR mixed gas detection system designed by researchers after recognizing the whole sections of the proposed system, which play a significant role in industrial and agricultural production, environmental monitoring, and life science, etc.

Longitudinal study of a heteroplasmic 3460 Leber hereditary optic neuropathy family by multiplexed primer-extension analysis and nucleotide sequencing

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

Ghosh, S.S.; Fahy, E.; Bodis-Wollner, I.

1996-02-01

Nucleotide-sequencing and multiplexed primer-extension assays have been used to quantitate the mutant-allele frequency in 14 maternal relatives, spanning three generations, from a family that is heteroplasmic for the primary Leber hereditary optic neuropathy (LHON) mutation at nucleotide 3460 of the mitochondrial genome. There was excellent agreement between the values that were obtained with the two different methods. The longitudinal study shows that the mutant-allele frequency was constant within individual family members over a sampling period of 3.5 years. Second, although there was an overall increase in the mutant-allele frequency in successive generations, segregation in the direction of the mutant allelemore » was not invariant, and there was one instance in which there was a significant decrease in the frequency from parent to offspring. From these two sets of results, and from previous studies of heteroplasmic LHON families, we conclude that there is no evidence for a marked selective pressure that determines the replication, segregation, or transmission of primary LHON mutations to white blood cells and platelets. Instead, the mtDNA molecules are most likely to replicate and segregate under conditions of random drift at the cellular level. Finally, the pattern of transmission in this maternal lineage is compatible with a developmental bottleneck model in which the number of mitochondrial units of segregation in the female germ line is relatively small in relation to the number of mtDNA molecules within a cell. However, this is not an invariant pattern for humans, and simple models of mitochondrial gene transmission are inappropriate at the present time. 37 refs., 4 figs., 1 tab.« less

Development of a rapid SNP-typing assay to differentiate Bifidobacterium animalis ssp. lactis strains used in probiotic-supplemented dairy products.

PubMed

Lomonaco, Sara; Furumoto, Emily J; Loquasto, Joseph R; Morra, Patrizia; Grassi, Ausilia; Roberts, Robert F

2015-02-01

Identification at the genus, species, and strain levels is desirable when a probiotic microorganism is added to foods. Strains of Bifidobacterium animalis ssp. lactis (BAL) are commonly used worldwide in dairy products supplemented with probiotic strains. However, strain discrimination is difficult because of the high degree of genome identity (99.975%) between different genomes of this subspecies. Typing of monomorphic species can be carried out efficiently by targeting informative single nucleotide polymorphisms (SNP). Findings from a previous study analyzing both reference and commercial strains of BAL identified SNP that could be used to discriminate common strains into 8 groups. This paper describes development of a minisequencing assay based on the primer extension reaction (PER) targeting multiple SNP that can allow strain differentiation of BAL. Based on previous data, 6 informative SNP were selected for further testing, and a multiplex preliminary PCR was optimized to amplify the DNA regions containing the selected SNP. Extension primers (EP) annealing immediately adjacent to the selected SNP were developed and tested in simplex and multiplex PER to evaluate their performance. Twenty-five strains belonging to 9 distinct genomic clusters of B. animalis ssp. lactis were selected and analyzed using the developed minisequencing assay, simultaneously targeting the 6 selected SNP. Fragment analysis was subsequently carried out in duplicate and demonstrated that the assay yielded 8 specific profiles separating the most commonly used commercial strains. This novel multiplex PER approach provides a simple, rapid, flexible SNP-based subtyping method for proper characterization and identification of commercial probiotic strains of BAL from fermented dairy products. To assess the usefulness of this method, DNA was extracted from yogurt manufactured with and without the addition of B. animalis ssp. lactis BB-12. Extracted DNA was then subjected to the minisequencing protocol, resulting in a SNP profile matching the profile for the strain BB-12. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Multiplex quantification of 16S rDNA of predominant bacteria group within human fecal samples by polymerase chain reaction--ligase detection reaction (PCR-LDR).

PubMed

Li, Kai; Chen, Bei; Zhou, Yuxun; Huang, Rui; Liang, Yinming; Wang, Qinxi; Xiao, Zhenxian; Xiao, Junhua

2009-03-01

A new method, based on ligase detection reaction (LDR), was developed for quantitative detection of multiplex PCR amplicons of 16S rRNA genes present in complex mixtures (specifically feces). LDR has been widely used in single nucleotide polymorphism (SNP) assay but never applied for quantification of multiplex PCR products. This method employs one pair of DNA probes, one of which is labeled with fluorescence for signal capture, complementary to the target sequence. For multiple target sequence analysis, probes were modified with different lengths of polyT at the 5' end and 3' end. Using a DNA sequencer, these ligated probes were separated and identified by size and dye color. Then, relative abundance of target DNA were normalized and quantified based on the fluorescence intensities and exterior size standards. 16S rRNA gene of three preponderant bacteria groups in human feces: Clostridium coccoides, Bacteroides and related genera, and Clostridium leptum group, were amplified and cloned into plasmid DNA so as to make standard curves. After PCR-LDR analysis, a strong linear relationship was found between the florescence intensity and the diluted plasmid DNA concentrations. Furthermore, based on this method, 100 human fecal samples were quantified for the relative abundance of the three bacterial groups. Relative abundance of C. coccoides was significantly higher in elderly people in comparison with young adults, without gender differences. Relative abundance of Bacteroides and related genera and C. leptum group were significantly higher in young and middle aged than in the elderly. Regarding the whole set of sample, C. coccoides showed the highest relative abundance, followed by decreasing groups Bacteroides and related genera, and C. leptum. These results imply that PCR-LDR can be feasible and flexible applied to large scale epidemiological studies.

Ultrafast FADC multiplexer

NASA Astrophysics Data System (ADS)

Mirzoyan, R.; Cortina, J.; Lorenz, E.; Martinez, M.; Ostankov, A.; Paneque, D.

2002-10-01

Ultrafast Flash amplitude-to-digital converters (FADCs) are still very expensive. Here we propose a multiplexing scheme allowing one in common trigger mode to read out multiple signal sources by using a single FADC channel. Usual coaxial cables can be used in the multiplexer as analog signal delay elements. The limited bandwidth of the coaxial cable, depending on its type and length will set an upper limit to the number of multiplexed channels. Better bandwidth and the correspondingly higher number of multiplexed channels one can obtain when using the technique of transmission of analog signals via optical fibers. Low-cost vertical cavity surface emitting laser (VCSEL) diodes can be used as converters of fast electrical signals into near infrared light. Multiplexing can be an economically priced solution when one needs ultrafast digitization of hundreds of fast signal channels.

Association between genetic polymorphisms in the XRCC1, XRCC3, XPD, GSTM1, GSTT1, MSH2, MLH1, MSH3, and MGMT genes and radiosensitivity in breast cancer patients.

PubMed

Mangoni, Monica; Bisanzi, Simonetta; Carozzi, Francesca; Sani, Cristina; Biti, Giampaolo; Livi, Lorenzo; Barletta, Emanuela; Costantini, Adele Seniori; Gorini, Giuseppe

2011-09-01

Clinical radiosensitivity varies considerably among patients, and radiation-induced side effects developing in normal tissue can be therapy limiting. Some single nucleotide polymorphisms (SNPs) have been shown to correlate with hypersensitivity to radiotherapy. We conducted a prospective study of 87 female patients with breast cancer who received radiotherapy after breast surgery. We evaluated the association between acute skin reaction following radiotherapy and 11 genetic polymorphisms in DNA repair genes: XRCC1 (Arg399Gln and Arg194Trp), XRCC3 (Thr241Met), XPD (Asp312Asn and Lys751Gln), MSH2 (gIVS12-6T>C), MLH1 (Ile219Val), MSH3 (Ala1045Thr), MGMT (Leu84Phe), and in damage-detoxification GSTM1 and GSTT1 genes (allele deletion). Individual genetic polymorphisms were determined by polymerase chain reaction and single nucleotide primer extension for single nucleotide polymorphisms or by a multiplex polymerase chain reaction assay for deletion polymorphisms. The development of severe acute skin reaction (moist desquamation or interruption of radiotherapy due to toxicity) associated with genetic polymorphisms was modeled using Cox proportional hazards, accounting for cumulative biologically effective radiation dose. Radiosensitivity developed in eight patients and was increased in carriers of variants XRCC3-241Met allele (hazard ratio [HR] unquantifiably high), MSH2 gIVS12-6nt-C allele (HR=53.36; 95% confidence intervals [95% CI], 3.56-798.98), and MSH3-1045Ala allele (HR unquantifiably high). Carriers of XRCC1-Arg194Trp variant allele in combination with XRCC1-Arg399Gln wild-type allele had a significant risk of radiosensitivity (HR=38.26; 95% CI, 1.19-1232.52). To our knowledge, this is the first report to find an association between MSH2 and MSH3 genetic variants and the development of radiosensitivity in breast cancer patients. Our findings suggest the hypothesis that mismatch repair mechanisms may be involved in cellular response to radiotherapy. Genetic polymorphisms may be promising candidates for predicting acute radiosensitivity, but further studies are necessary to confirm our findings. Copyright © 2011 Elsevier Inc. All rights reserved.

Wavelength-Division Multiplexing Of Bipolar Digital Signals

NASA Technical Reports Server (NTRS)

Gibbons, Ronnie D.; Ubele, John L., II

1994-01-01

In system, bipolar digital data transmitted by use of wavelength-division multiplexing on single optical fiber. Two different wavelengths used to transmit pulses signifying "positive" or "negative" bipolar digital data. Simultaneous absence of pulses at both wavelengths signifies digital "zero."

Nucleobases, nucleosides, and nucleotides: versatile biomolecules for generating functional nanomaterials.

PubMed

Pu, Fang; Ren, Jinsong; Qu, Xiaogang

2018-02-21

The incorporation of biomolecules into nanomaterials generates functional nanosystems with novel and advanced properties, presenting great potential for applications in various fields. Nucleobases, nucleosides and nucleotides, as building blocks of nucleic acids and biological coenzymes, constitute necessary components of the foundation of life. In recent years, as versatile biomolecules for the construction or regulation of functional nanomaterials, they have stimulated interest in researchers, due to their unique properties such as structural diversity, multiplex binding sites, self-assembly ability, stability, biocompatibility, and chirality. In this review, strategies for the synthesis of nanomaterials and the regulation of their morphologies and functions using nucleobases, nucleosides, and nucleotides as building blocks, templates or modulators are summarized alongside selected applications. The diverse applications range from sensing, bioimaging, and drug delivery to mimicking light-harvesting antenna, the construction of logic gates, and beyond. Furthermore, some perspectives and challenges in this emerging field are proposed. This review is directed toward the broader scientific community interested in biomolecule-based functional nanomaterials.

Wavevector multiplexed atomic quantum memory via spatially-resolved single-photon detection.

PubMed

Parniak, Michał; Dąbrowski, Michał; Mazelanik, Mateusz; Leszczyński, Adam; Lipka, Michał; Wasilewski, Wojciech

2017-12-15

Parallelized quantum information processing requires tailored quantum memories to simultaneously handle multiple photons. The spatial degree of freedom is a promising candidate to facilitate such photonic multiplexing. Using a single-photon resolving camera, we demonstrate a wavevector multiplexed quantum memory based on a cold atomic ensemble. Observation of nonclassical correlations between Raman scattered photons is confirmed by an average value of the second-order correlation function [Formula: see text] in 665 separated modes simultaneously. The proposed protocol utilizing the multimode memory along with the camera will facilitate generation of multi-photon states, which are a necessity in quantum-enhanced sensing technologies and as an input to photonic quantum circuits.

Holographically Encoded Volume Phase Masks

DTIC Science & Technology

2015-07-13

Lu et al., “Coherent beam combination of fiber laser arrays via multiplexed volume Bragg gratings,” in Conf. on Lasers and Electro- Optics: Science...combining of fiber lasers using multiplexed volume Bragg gratings,” in Conf. on Lasers and Electro- Optics: Science and Innovations, OSA Technical Digest...satisfying the Bragg condition of the hologram. Moreover, this approach enables the capability to encode and multiplex several phase masks into a single

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review.

PubMed

Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

2016-05-28

The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review

PubMed Central

Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

2016-01-01

The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process. PMID:27239109

Optical switch compatible with wavelength division multiplexing and mode division multiplexing for photonic networks-on-chip.

PubMed

Jia, Hao; Zhou, Ting; Zhang, Lei; Ding, Jianfeng; Fu, Xin; Yang, Lin

2017-08-21

We propose a 2 × 2 multimode optical switch, which is composed of two mode de-multiplexers, n 2 × 2 single-mode optical switches where n is the number of the supported spatial modes, and two mode multiplexers. As a proof of concept, asymmetric directional couplers are employed to construct the mode multiplexers and de-multiplexers, balanced Mach-Zehnder interferometer is utilized to construct the 2 × 2 single-mode optical switches. The fabricated silicon 2 × 2 multimode optical switch has a broad optical bandwidth and can support four spatial modes. The link-crosstalk for all four modes is smaller than -18.8 dB. The inter-mode crosstalk for the same optical link is less than -22.1 dB. 40 Gbps data transmission is performed for all spatial modes and all optical links. The power penalties for the error-free switching (BER<10 -9 ) at 25 Gbps are less than 1.8 dB for all channels at the wavelength of 1550 nm. The power consumption of the device is 117.9 mW in the "cross" state and 116.2 mW in the "bar" state. The switching time is about 21 μs. This work enables large-capacity multimode photonic networks-on-chip.

Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

PubMed

Ullal, Adeeti V; Weissleder, Ralph

2015-01-01

We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

Association between Single Nucleotide Polymorphisms of the Major Histocompatibility Complex Class II Gene and Newcastle Disease Virus Titre and Body Weight in Leung Hang Khao Chickens

PubMed Central

Molee, A.; Kongroi, K.; Kuadsantia, P.; Poompramun, C.; Likitdecharote, B.

2016-01-01

The aim of the present study was to investigate the effect of single nucleotide polymorphisms in the major histocompatibility complex (MHC) class II gene on resistance to Newcastle disease virus and body weight of the Thai indigenous chicken, Leung Hang Khao (Gallus gallus domesticus). Blood samples were collected for single nucleotide polymorphism analysis from 485 chickens. Polymerase chain reaction sequencing was used to classify single nucleotide polymorphisms of class II MHC. Body weights were measured at the ages of 3, 4, 5, and 7 months. Titres of Newcastle disease virus at 2 weeks to 7 months were determined and the correlation between body weight and titre was analysed. The association between single nucleotide polymorphisms and body weight and titre were analysed by a generalized linear model. Seven single nucleotide polymorphisms were identified: C125T, A126T, C209G, C242T, A243T, C244T, and A254T. Significant correlations between log titre and body weight were found at 2 and 4 weeks. Associations between single nucleotide polymorphisms and titre were found for C209G and A254T, and between all single nucleotide polymorphisms (except A243T) and body weight. The results showed that class II MHC is associated with both titre of Newcastle disease virus and body weight in Leung Hang Khao chickens. This is of concern because improved growth traits are the main goal of breeding selection. Moreover, the results suggested that MHC has a pleiotropic effect on the titre and growth performance. This mechanism should be investigated in a future study. PMID:26732325

A triplex ribozyme expression system based on a single hairpin ribozyme.

PubMed

Aquino-Jarquin, Guillermo; Benítez-Hess, María Luisa; DiPaolo, Joseph A; Alvarez-Salas, Luis M

2008-09-01

Triplex ribozyme (RZ) configurations allow for the individual activity of trans-acting RZs in multiple expression cassettes (multiplex), thereby increasing target cleavage relative to conventionally expressed RZs. Although hairpin RZs have been advantageously compared to hammerhead RZs, their longer size and structural features complicated triplex design. We present a triplex expression system based on a single hairpin RZ with transcleavage capability and simple engineering. The system was tested in vitro using cis- and trans-cleavage kinetic assays against a known target RNA from HPV-16 E6/E7 mRNA. Single and multiplex triplex RZ constructs were more efficient in cleaving the target than tandem-cloned hairpin RZs, suggesting that the release of individual RZs enhanced trans-cleavage kinetics. Multiplex systems constructed with two different hairpin RZs resulted in better trans-cleavage compared to standard double-RZ constructs. In addition, the triplex RZ performed cis- and trans-cleavage in cervical cancer cells. The use of triplex configurations with multiplex RZs permit differential targeting of the same or different RNA, thus improving potential use against unstable targets. This prototype will provide the basis for the development of future RZ-based therapies and technologies.

Integrated-Optic Wavelength Multiplexer In Glass Fabricated By A Charge Controlled Ion Exchange

NASA Astrophysics Data System (ADS)

Klein, R.; Jestel, D.; Lilienhof, H. J.; Rottman, F.; Voges, E.

1989-02-01

Integrated-optic wavelength division multiplexing (WDM) is commonly used in communication systems. These WDM-devices are also well suited to build up optical fiber networks for both intensity and interferometric sensor types. The operation principle of our wavelength division multiplexing devise is based on the wavelength dependent two-mode interference in a two-moded waveguide, which is coupled adiabatically to the single-mode input and output strip waveguides. The single-mode input and output waveguides are connected via two Y-branches ( "'kJ- 1° branching angle ) with a two-moded intersection region. The ratio of the light powers in the single-mode output waveguides depends on wavelength . The two-mode interference within the two-moded center waveguide leads to an almost wavelength periodic transmission caracteristic . Dual-channel multiplexers/demultiplexers were fabricated by a charge controlled field assisted pottasium exchange in B-270 glass (Desag). The devices have a typical channel separation of 30 - 40 nm and a far-end crosstalk attenuation of better than 16 dB. The operation wavelength regions of the fabricated devices are 0.6 - 0.8 µm and 1.3 - 1.6 µm, respectively.

Helicase dependent OnChip-amplification and its use in multiplex pathogen detection.

PubMed

Andresen, Dennie; von Nickisch-Rosenegk, Markus; Bier, Frank F

2009-05-01

The need for fast, specific and sensitive multiparametric detection methods is an ever growing demand in molecular diagnostics. Here we report on a newly developed method, the helicase dependent OnChip amplification (OnChip-HDA). This approach integrates the analysis and detection in one single reaction thus leading to time and cost savings in multiparametric analysis. HDA is an isothermal amplification method that is not depending on thermocycling as known from PCR due to the helicases' ability to unwind DNA double-strands. We have combined the HDA with microarray based detection, making it suitable for multiplex detection. As an example we used the OnChip HDA in single and multiplex amplifications for the detection of the two pathogens N. gonorrhoeae and S. aureus directly on surface bound primers. We have successfully shown the OnChip-HDA and applied it for single- and duplex-detection of the pathogens N. gonorrhoeae and S. aureus. We have developed a new method, the OnChip-HDA for the multiplex detection of pathogens. Its simplicity in reaction setup and potential for miniaturization and multiparametric analysis is advantageous for the integration in miniaturized Lab on Chip systems, e.g. needed in point of care diagnostics.

Identification of individual powdery mildew fungi infecting leaves and direct detection of gene expression by single conidium polymerase chain reaction.

PubMed

Matsuda, Yoshinori; Sameshima, Takeshi; Moriura, Nobuyuki; Inoue, Kanako; Nonomura, Teruo; Kakutani, Koji; Nishimura, Hiroaki; Kusakari, Shin-Ichi; Takamatsu, Susumu; Toyoda, Hideyoshi

2005-10-01

ABSTRACT Greenhouse-grown tomato seedlings were inoculated naturally with two genera of powdery mildew conidia forming appressorial germ tubes that could not be differentiated by length alone. For direct identification, single germinated conidia were removed from leaves by means of a glass pipette linked to the manipulator of a high-fidelity digital microscope. This microscope enabled in vivo observation of the fungi without leaf decoloration or fungal staining. The isolated conidia were subjected to PCR amplification of the 5.8S rDNA and its adjacent internal transcribed spacer sequences followed by nested PCR to attain sensitivity high enough to amplify target nucleotide sequences (PCR/nested PCR). Target sequences from the conidia were completely coincident with those of the pathogen Oidium neolycopersici or Erysiphe trifolii (syn. Microsphaera trifolii), which is nonpathogenic on tomato. Using RT-PCR/nested PCR or multiplex RT-PCR/nested PCR, it was possible to amplify transcripts expressed in single conidia. Conidia at pre- and postgermination stages were removed individually from tomato leaves, and two powdery mildew genes were monitored. The results indicated that the beta-tubulin homolog TUB2-ol was expressed at pre- and postgermination stages and the cutinase homolog CUT1-ol was only expressed postgermination. Combining digital microscopic micromanipulation and two-step PCR amplification is thus useful for investigation of individual propagules on the surface of plants.

Development of genomic microsatellite multiplex PCR using dye-labeled universal primer and its validation in pedigree analysis of Pacific oyster ( Crassostrea gigas)

NASA Astrophysics Data System (ADS)

Liu, Ting; Li, Qi; Song, Junlin; Yu, Hong

2017-02-01

There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.

Functional analysis of regulatory single-nucleotide polymorphisms.

PubMed

Pampín, Sandra; Rodríguez-Rey, José C

2007-04-01

The identification of regulatory polymorphisms has become a key problem in human genetics. In the past few years there has been a conceptual change in the way in which regulatory single-nucleotide polymorphisms are studied. We revise the new approaches and discuss how gene expression studies can contribute to a better knowledge of the genetics of common diseases. New techniques for the association of single-nucleotide polymorphisms with changes in gene expression have been recently developed. This, together with a more comprehensive use of the old in-vitro methods, has produced a great amount of genetic information. When added to current databases, it will help to design better tools for the detection of regulatory single-nucleotide polymorphisms. The identification of functional regulatory single-nucleotide polymorphisms cannot be done by the simple inspection of DNA sequence. In-vivo techniques, based on primer-extension, and the more recently developed 'haploChIP' allow the association of gene variants to changes in gene expression. Gene expression analysis by conventional in-vitro techniques is the only way to identify the functional consequences of regulatory single-nucleotide polymorphisms. The amount of information produced in the last few years will help to refine the tools for the future analysis of regulatory gene variants.

A highly addressable static droplet array enabling digital control of a single droplet at pico-volume resolution.

PubMed

Jeong, Heon-Ho; Lee, Byungjin; Jin, Si Hyung; Jeong, Seong-Geun; Lee, Chang-Soo

2016-04-26

Droplet-based microfluidics enabling exquisite liquid-handling has been developed for diagnosis, drug discovery and quantitative biology. Compartmentalization of samples into a large number of tiny droplets is a great approach to perform multiplex assays and to improve reliability and accuracy using a limited volume of samples. Despite significant advances in microfluidic technology, individual droplet handling in pico-volume resolution is still a challenge in obtaining more efficient and varying multiplex assays. We present a highly addressable static droplet array (SDA) enabling individual digital manipulation of a single droplet using a microvalve system. In a conventional single-layer microvalve system, the number of microvalves required is dictated by the number of operation objects; thus, individual trap-and-release on a large-scale 2D array format is highly challenging. By integrating double-layer microvalves, we achieve a "balloon" valve that preserves the pressure-on state under released pressure; this valve can allow the selective releasing and trapping of 7200 multiplexed pico-droplets using only 1 μL of sample without volume loss. This selectivity and addressability completely arranged only single-cell encapsulated droplets from a mixture of droplet compositions via repetitive selective trapping and releasing. Thus, it will be useful for efficient handling of miniscule volumes of rare or clinical samples in multiplex or combinatory assays, and the selective collection of samples.

Multiplexed Neurochemical Signaling by Neurons of the Ventral Tegmental Area

PubMed Central

Barker, David J.; Root, David H.; Zhang, Shiliang; Morales, Marisela

2016-01-01

The ventral tegmental area (VTA) is an evolutionarily conserved structure that has roles in reward-seeking, safety-seeking, learning, motivation, and neuropsychiatric disorders such as addiction and depression. The involvement of the VTA in these various behaviors and disorders is paralleled by its diverse signaling mechanisms. Here we review recent advances in our understanding of neuronal diversity in the VTA with a focus on cell phenotypes that participate in ‘multiplexed’ neurotransmission involving distinct signaling mechanisms. First, we describe the cellular diversity within the VTA, including neurons capable of transmitting dopamine, glutamate or GABA as well as neurons capable of multiplexing combinations of these neurotransmitters. Next, we describe the complex synaptic architecture used by VTA neurons in order to accommodate the transmission of multiple transmitters. We specifically cover recent findings showing that VTA multiplexed neurotransmission may be mediated by either the segregation of dopamine and glutamate into distinct microdomains within a single axon or by the integration of glutamate and GABA into a single axon terminal. In addition, we discuss our current understanding of the functional role that these multiplexed signaling pathways have in the lateral habenula and the nucleus accumbens. Finally, we consider the putative roles of VTA multiplexed neurotransmission in synaptic plasticity and discuss how changes in VTA multiplexed neurons may relate to various psychopathologies including drug addiction and depression. PMID:26763116

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

Highly Multiplexed, Single Cell Transcriptomic Analysis of T-Cells by Microfluidic PCR.

PubMed

Dominguez, Maria; Roederer, Mario; Chattopadhyay, Pratip K

2017-01-01

Recently, technologies have been developed to measure expression of 96 (or more) mRNA transcripts at once from a single cell. Here we describe methods and important considerations for use of Fluidigm's BioMark platform for multiplexed single cell gene expression. We describe how to qualify primer/probes, select genes to examine in 96-parameter panels, perform the reverse transcription/cDNA synthesis step, and operate the instrument. In addition, we describe data analysis considerations. This technology has enormous value for characterizing the heterogeneity of T-cells, thereby providing a useful tool for immune monitoring.

Diversity of genetic events associated with MLH1 promoter methylation in Lynch syndrome families with heritable constitutional epimutation.

PubMed

Leclerc, Julie; Flament, Cathy; Lovecchio, Tonio; Delattre, Lucie; Ait Yahya, Emilie; Baert-Desurmont, Stéphanie; Burnichon, Nelly; Bronner, Myriam; Cabaret, Odile; Lejeune, Sophie; Guimbaud, Rosine; Morin, Gilles; Mauillon, Jacques; Jonveaux, Philippe; Laurent-Puig, Pierre; Frébourg, Thierry; Porchet, Nicole; Buisine, Marie-Pierre

2018-04-12

PurposeConstitutional epimutations are an alternative to genetic mutations in the etiology of genetic diseases. Some of these epimutations, termed secondary, correspond to the epigenetic effects of cis-acting genetic defects transmitted to the offspring following a Mendelian inheritance pattern. In Lynch syndrome, a few families with such apparently heritable MLH1 epimutations have been reported so far.MethodsWe designed a long-range polymerase chain reaction next-generation sequencing strategy to screen MLH1 entire gene and applied it to 4 French families with heritable epimutations and 10 additional patients with no proven transmission of their epimutations.ResultsThis strategy successfully detected the insertion of an Alu element in MLH1 coding sequence in one family. Two previously unreported MLH1 variants were also identified in other epimutation carriers: a nucleotide substitution within intron 1 and a single-nucleotide deletion in the 5'-UTR. Detection of a partial MLH1 duplication in another family required multiplex ligation-dependent probe amplification technology. We demonstrated the segregation of these variants with MLH1 methylation and studied the functional consequences of these defects on transcription.ConclusionThis is the largest cohort of patients with MLH1 secondary epimutations associated with a broad spectrum of genetic defects. This study provides further insight into the complexity of molecular mechanisms leading to secondary epimutations.GENETICS in MEDICINE advance online publication, 12 April 2018; doi:10.1038/gim.2018.47.

Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses

PubMed Central

Waggoner, Jesse J.; Gresh, Lionel; Mohamed-Hadley, Alisha; Ballesteros, Gabriela; Davila, Maria Jose Vargas; Tellez, Yolanda; Sahoo, Malaya K.; Balmaseda, Angel; Harris, Eva

2016-01-01

Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses. PMID:27184629

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

USDA-ARS?s Scientific Manuscript database

Bead based multiplex assays (BBMA) also referred to as Luminex, MultiAnalyte Profiling or cytometric bead array (CBA) assays, are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several, up to 50-500 analytes within a single, small sample volume). Curren...

A multiplex PCR for detection of six viruses in ducks.

PubMed

Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

2017-10-01

In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10 2 copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

Multiplex Immunoassay Profiling.

PubMed

Stephen, Laurie

2017-01-01

Multiplex immunoassays allow for the rapid profiling of biomarker proteins in biological fluids, using less sample and labor than single immunoassays. This chapter details the methods to develop and manufacture multiplex assays for the Luminex ® platform. Although assay development is not included here, the same methods can be used to covalently couple antibodies to the Luminex beads and to label antibodies for the screening of sandwich pairs, if needed. The assay optimization, detection of cross-reactivity, and minimizing antibody interactions and matrix interferences will be addressed.

[Do Multiplex PCR techniques displace classical cultures in microbiology?].

PubMed

Auckenthaler, Raymond; Risch, Martin

2015-02-01

Multiplex PCR technologies progressively find their way in clinical microbiology. This technique allows the simultaneous amplification of multiple DNA targets in a single test run for the identification of pathogens up to the species level. Various pathogens of infectious diseases can be detected by a symptom-oriented approach clearly and quickly with high reliability. Essentially multiplex PCR panels are available for clarification of gastrointestinal, respiratory, sexually transmitted infections and meningitis. Today's offer from industry, university hospitals and large private laboratories of Switzerland is tabulated and commented.

An efficient multiplexing approach for adaptive aircraft communications via a relay satellite.

NASA Technical Reports Server (NTRS)

Devieux, C.; Bisaga, J. J.

1973-01-01

Description of a coherent wide-angle multiplexing approach which is 4 to 8 dB more efficient in the utilization of satellite power as compared to a multicarrier transmission accessing a single TWT amplifier transponder. The wide-angle multiplexing approach achieves this performance by efficiently trading the modulation power improvement against backoff at the satellite earth terminal phase modulator. A simple addition of an amplitude clipper at the modulator input is critical to the proper operation of the system.

Frequency-Domain Streak Camera and Tomography for Ultrafast Imaging of Evolving and Channeled Plasma Accelerator Structures

NASA Astrophysics Data System (ADS)

Li, Zhengyan; Zgadzaj, Rafal; Wang, Xiaoming; Reed, Stephen; Dong, Peng; Downer, Michael C.

2010-11-01

We demonstrate a prototype Frequency Domain Streak Camera (FDSC) that can capture the picosecond time evolution of the plasma accelerator structure in a single shot. In our prototype Frequency-Domain Streak Camera, a probe pulse propagates obliquely to a sub-picosecond pump pulse that creates an evolving nonlinear index "bubble" in fused silica glass, supplementing a conventional Frequency Domain Holographic (FDH) probe-reference pair that co-propagates with the "bubble". Frequency Domain Tomography (FDT) generalizes Frequency-Domain Streak Camera by probing the "bubble" from multiple angles and reconstructing its morphology and evolution using algorithms similar to those used in medical CAT scans. Multiplexing methods (Temporal Multiplexing and Angular Multiplexing) improve data storage and processing capability, demonstrating a compact Frequency Domain Tomography system with a single spectrometer.

Detecting associated single-nucleotide polymorphisms on the X chromosome in case control genome-wide association studies.

PubMed

Chen, Zhongxue; Ng, Hon Keung Tony; Li, Jing; Liu, Qingzhong; Huang, Hanwen

2017-04-01

In the past decade, hundreds of genome-wide association studies have been conducted to detect the significant single-nucleotide polymorphisms that are associated with certain diseases. However, most of the data from the X chromosome were not analyzed and only a few significant associated single-nucleotide polymorphisms from the X chromosome have been identified from genome-wide association studies. This is mainly due to the lack of powerful statistical tests. In this paper, we propose a novel statistical approach that combines the information of single-nucleotide polymorphisms on the X chromosome from both males and females in an efficient way. The proposed approach avoids the need of making strong assumptions about the underlying genetic models. Our proposed statistical test is a robust method that only makes the assumption that the risk allele is the same for both females and males if the single-nucleotide polymorphism is associated with the disease for both genders. Through simulation study and a real data application, we show that the proposed procedure is robust and have excellent performance compared to existing methods. We expect that many more associated single-nucleotide polymorphisms on the X chromosome will be identified if the proposed approach is applied to current available genome-wide association studies data.

Genome-wide divergence and linkage disequilibrium analyses for Capsicum baccatum revealed by genome-anchored single nucleotide polymorphisms

USDA-ARS?s Scientific Manuscript database

Principal component analysis (PCA) with 36,621 polymorphic genome-anchored single nucleotide polymorphisms (SNPs) identified collectively for Capsicum annuum and Capsicum baccatum was used to show the distribution of these 2 important incompatible cultivated pepper species. Estimated mean nucleotide...

Genetic polymorphisms in ESR1 and ESR2 genes, and risk of hypospadias in a multiethnic study population.

PubMed

Choudhry, Shweta; Baskin, Laurence S; Lammer, Edward J; Witte, John S; Dasgupta, Sudeshna; Ma, Chen; Surampalli, Abhilasha; Shen, Joel; Shaw, Gary M; Carmichael, Suzan L

2015-05-01

Estrogenic endocrine disruptors acting via estrogen receptors α (ESR1) and β (ESR2) have been implicated in the etiology of hypospadias, a common congenital malformation of the male external genitalia. We determined the association of single nucleotide polymorphisms in ESR1 and ESR2 genes with hypospadias in a racially/ethnically diverse study population of California births. We investigated the relationship between hypospadias and 108 ESR1 and 36 ESR2 single nucleotide polymorphisms in 647 cases and 877 population based nonmalformed controls among infants born in selected California counties from 1990 to 2003. Subgroup analyses were performed by race/ethnicity (nonHispanic white and Hispanic subjects) and by hypospadias severity (mild to moderate and severe). Odds ratios for 33 of the 108 ESR1 single nucleotide polymorphisms had p values less than 0.05 (p = 0.05 to 0.007) for risk of hypospadias. However, none of the 36 ESR2 single nucleotide polymorphisms was significantly associated. In stratified analyses the association results were consistent by disease severity but different sets of single nucleotide polymorphisms were significantly associated with hypospadias in nonHispanic white and Hispanic subjects. Due to high linkage disequilibrium across the single nucleotide polymorphisms, haplotype analyses were conducted and identified 6 haplotype blocks in ESR1 gene that had haplotypes significantly associated with an increased risk of hypospadias (OR 1.3 to 1.8, p = 0.04 to 0.00001). Similar to single nucleotide polymorphism analysis, different ESR1 haplotypes were associated with risk of hypospadias in nonHispanic white and Hispanic subjects. No significant haplotype association was observed for ESR2. The data provide evidence that ESR1 single nucleotide polymorphisms and haplotypes influence the risk of hypospadias in white and Hispanic subjects, and warrant further examination in other study populations. Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction.

PubMed

Song, Yunke; Zhang, Yi; Wang, Tza-Huei

2013-04-08

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combining spatial domain multiplexing and orbital angular momentum of photon-based multiplexing to increase the bandwidth of optical fiber communication systems

NASA Astrophysics Data System (ADS)

Murshid, Syed; Alanzi, Saud; Hridoy, Arnob; Lovell, Gregory L.; Parhar, Gurinder; Chakravarty, Abhijit; Chowdhury, Bilas

2016-06-01

Spatial domain multiplexing/space division multiplexing (SDM) can increase the bandwidth of existing and futuristic optical fibers by an order of magnitude or more. In the SDM technique, we launch multiple single-mode pigtail laser sources of the same wavelength into a carrier multimode fiber at different angles. The launching angles decide the output of the carrier fiber by allocating separate spatial locations for each channel. Each channel follows a helical trajectory while traversing the length of the carrier fiber, thereby allowing spatial reuse of optical frequencies. We launch light from five different single-mode pigtail laser sources (of same wavelength) at different angles (with respect to the axis of the carrier fiber) into the carrier fiber. Owing to helical propagation, five distinct concentric donut-shaped rings with negligible crosstalk at the output end of the fiber were obtained. These SDM channels also exhibit orbital angular momentum (OAM), thereby adding an extradegree of photon freedom. We present the experimental data of five spatially multiplexed channels and compare them with simulated results to show that this technique can potentially improve the data capacity of optical fibers by an order of magnitude: A factor of five using SDM and another factor of two using OAM.

An order of magnitude improvement in optical fiber bandwidth using spatial domain multiplexing/space division multiplexing (SDM) in conjunction with orbital angular momentum (OAM)

NASA Astrophysics Data System (ADS)

Murshid, Syed; Alanzi, Saud; Hridoy, Arnob; Lovell, Greg; Parhar, Gurinder; Chakravarty, Abhijit; Chowdhury, Bilas

2014-09-01

Spatial Domain Multiplexing/Space Division Multiplexing (SDM) can increase the bandwidth of existing and futuristic optical fibers by an order of magnitude or more. In the SDM technique, we launch multiple single mode pigtail laser sources of same wavelength into a carrier fiber at different angles. The launching angles decide the output of the carrier fiber by allocating separate spatial locations for each channel. Each channel follows a helical trajectory while traversing the length of the carrier fiber, thereby allowing spatial reuse of optical frequencies. In this endeavor we launch light from five different single mode pigtail laser sources at different angles (with respect to the axis of the carrier fiber) into the carrier fiber. Owing to helical propagation we get five distinct concentric donut shaped rings with negligible crosstalk at the output end of the fiber. These SDM channels also exhibit Orbital Angular Momentum (OAM), thereby adding an extra degree of photon freedom. We present the experimental data of five spatially multiplexed channels and compare them with simulated results to show that this technique can potentially improve the data capacity of optical fibers by an order of magnitude: A factor of five using SDM and another factor of two using OAM.

Space-division multiplexing in optical fibres

NASA Astrophysics Data System (ADS)

Richardson, D. J.; Fini, J. M.; Nelson, L. E.

2013-05-01

Optical communication technology has been advancing rapidly for several decades, supporting our increasingly information-driven society and economy. Much of this progress has been in finding innovative ways to increase the data-carrying capacity of a single optical fibre. To achieve this, researchers have explored and attempted to optimize multiplexing in time, wavelength, polarization and phase. Commercial systems now utilize all four dimensions to send more information through a single fibre than ever before. The spatial dimension has, however, remained untapped in single fibres, despite it being possible to manufacture fibres supporting hundreds of spatial modes or containing multiple cores, which could be exploited as parallel channels for independent signals.

Multiplexed lateral flow biosensors: Technological advances for radically improving point-of-care diagnoses.

PubMed

Li, Jia; Macdonald, Joanne

2016-09-15

Lateral flow biosensors are a leading technology in point-of-care diagnostics due to their simplicity, rapidness and low cost. Their primacy in this arena continues through technological breakthroughs such as multiplexing: the detection of more than one biomarker in a single assay. Multiplexing capacity is critical for improving diagnostic efficiency, enhancing the diagnostic precision for specific diseases and reducing diagnostic cost. Here we review, for the first time, the various types and strategies employed for creating multiplexed lateral flow biosensors. These are classified into four main categories in terms of specific application or multiplexing level, namely linear, parameter, spatial and conceptual. We describe the practical applications and implications for each approach and compare their advantages and disadvantages. Importantly, multiplexing is still subject to limitations of the traditional lateral flow biosensor, such as sensitivity and specificity. However, by pushing the limitations of the traditional medium into the multiplex arena, several technological breakthroughs are emerging with novel solutions that further expand the utility of lateral flow biosensing for point-of-care applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Compositions and methods for detecting single nucleotide polymorphisms

DOEpatents

Yeh, Hsin-Chih; Werner, James; Martinez, Jennifer S.

2016-11-22

Described herein are nucleic acid based probes and methods for discriminating and detecting single nucleotide variants in nucleic acid molecules (e.g., DNA). The methods include use of a pair of probes can be used to detect and identify polymorphisms, for example single nucleotide polymorphism in DNA. The pair of probes emit a different fluorescent wavelength of light depending on the association and alignment of the probes when hybridized to a target nucleic acid molecule. Each pair of probes is capable of discriminating at least two different nucleic acid molecules that differ by at least a single nucleotide difference. The methods can probes can be used, for example, for detection of DNA polymorphisms that are indicative of a particular disease or condition.

Integrated analyses of proteins and their glycans in a magnetic bead-based multiplex assay format.

PubMed

Li, Danni; Chiu, Hanching; Chen, Jing; Zhang, Hui; Chan, Daniel W

2013-01-01

Well-annotated clinical samples are valuable resources for biomarker discovery and validation. Multiplex and integrated methods that simultaneously measure multiple analytes and generate integrated information about these analytes from a single measurement are desirable because these methods help conserve precious samples. We developed a magnetic bead-based system for multiplex and integrated glycoprotein quantification by immunoassays and glycan detection by lectin immunosorbent assays (LISAs). Magnetic beads coupled with antibodies were used for capturing proteins of interest. Biotinylated antibodies in combination with streptavidin-labeled phycoerythrin were used for protein quantification. In the LISAs, biotinylated detection antibodies were replaced by biotinylated lectins for glycan detection. Using tissue inhibitor of metallopeptidase 1 (TIMP-1), tissue plasminogen activator, membrane metallo-endopeptidase, and dipeptidyl peptidase-IV (DPP-4) as models, we found that the multiplex integrated system was comparable to single immunoassays in protein quantification and LISAs in glycan detection. The merits of this system were demonstrated when applied to well-annotated prostate cancer tissues for validation of biomarkers in aggressive prostate cancer. Because of the system's multiplex ability, we used only 300 ng of tissue protein for the integrated detection of glycans in these proteins. Fucosylated TIMP-1 and DPP-4 offered improved performance over the proteins in distinguishing aggressive and nonaggressive prostate cancer. The multiplex and integrated system conserves samples and is a useful tool for validation of glycoproteins and their glycoforms as biomarkers. © 2012 American Association for Clinical Chemistry

Development of multiplex polymerase chain reaction for detection of Ehrlichia canis, Babesia spp and Hepatozoon canis in canine blood.

PubMed

Kledmanee, Kan; Suwanpakdee, Sarin; Krajangwong, Sakranmanee; Chatsiriwech, Jarin; Suksai, Parut; Suwannachat, Pongpun; Sariya, Ladawan; Buddhirongawatr, Ruangrat; Charoonrut, Phingphol; Chaichoun, Kridsada

2009-01-01

A multiplex polymerase chain reaction (PCR) has been developed for simultaneous detection of canine blood parasites, Ehrlichia canis, Babesia spp and Hepatozoon canis, from blood samples in a single reaction. The multiplex PCR primers were specific to E. canis VirB9, Babesia spp 16S rRNA and H. canis 16S rRNA genes. Specificity of the amplicons was confirmed by DNA sequencing. The assay was evaluated using normal canine and infected blood samples, which were detected by microscopic examination. This multiplex PCR offers scope for simultaneous detection of three important canine blood parasites and should be valuable in monitoring parasite infections in dogs and ticks.

Integrating IR detector imaging systems

NASA Technical Reports Server (NTRS)

Bailey, G. C. (Inventor)

1984-01-01

An integrating IR detector array for imaging is provided in a hybrid circuit with InSb mesa diodes in a linear array, a single J-FET preamplifier for readout, and a silicon integrated circuit multiplexer. Thin film conductors in a fan out pattern deposited on an Al2O3 substrate connect the diodes to the multiplexer, and thick film conductors also connect the reset switch and preamplifier to the multiplexer. Two phase clock pulses are applied with a logic return signal to the multiplexer through triax comprised of three thin film conductors deposited between layers. A lens focuses a scanned image onto the diode array for horizontal read out while a scanning mirror provides vertical scan.

Optimized and secure technique for multiplexing QR code images of single characters: application to noiseless messages retrieval

NASA Astrophysics Data System (ADS)

Trejos, Sorayda; Fredy Barrera, John; Torroba, Roberto

2015-08-01

We present for the first time an optical encrypting-decrypting protocol for recovering messages without speckle noise. This is a digital holographic technique using a 2f scheme to process QR codes entries. In the procedure, letters used to compose eventual messages are individually converted into a QR code, and then each QR code is divided into portions. Through a holographic technique, we store each processed portion. After filtering and repositioning, we add all processed data to create a single pack, thus simplifying the handling and recovery of multiple QR code images, representing the first multiplexing procedure applied to processed QR codes. All QR codes are recovered in a single step and in the same plane, showing neither cross-talk nor noise problems as in other methods. Experiments have been conducted using an interferometric configuration and comparisons between unprocessed and recovered QR codes have been performed, showing differences between them due to the involved processing. Recovered QR codes can be successfully scanned, thanks to their noise tolerance. Finally, the appropriate sequence in the scanning of the recovered QR codes brings a noiseless retrieved message. Additionally, to procure maximum security, the multiplexed pack could be multiplied by a digital diffuser as to encrypt it. The encrypted pack is easily decoded by multiplying the multiplexing with the complex conjugate of the diffuser. As it is a digital operation, no noise is added. Therefore, this technique is threefold robust, involving multiplexing, encryption, and the need of a sequence to retrieve the outcome.

Highly multiplexed signal readout for a time-of-flight positron emission tomography detector based on silicon photomultipliers.

PubMed

Cates, Joshua W; Bieniosek, Matthew F; Levin, Craig S

2017-01-01

Maintaining excellent timing resolution in the generation of silicon photomultiplier (SiPM)-based time-of-flight positron emission tomography (TOF-PET) systems requires a large number of high-speed, high-bandwidth electronic channels and components. To minimize the cost and complexity of a system's back-end architecture and data acquisition, many analog signals are often multiplexed to fewer channels using techniques that encode timing, energy, and position information. With progress in the development SiPMs having lower dark noise, after pulsing, and cross talk along with higher photodetection efficiency, a coincidence timing resolution (CTR) well below 200 ps FWHM is now easily achievable in single pixel, bench-top setups using 20-mm length, lutetium-based inorganic scintillators. However, multiplexing the output of many SiPMs to a single channel will significantly degrade CTR without appropriate signal processing. We test the performance of a PET detector readout concept that multiplexes 16 SiPMs to two channels. One channel provides timing information with fast comparators, and the second channel encodes both position and energy information in a time-over-threshold-based pulse sequence. This multiplexing readout concept was constructed with discrete components to process signals from a [Formula: see text] array of SensL MicroFC-30035 SiPMs coupled to [Formula: see text] Lu 1.8 Gd 0.2 SiO 5 (LGSO):Ce (0.025 mol. %) scintillators. This readout method yielded a calibrated, global energy resolution of 15.3% FWHM at 511 keV with a CTR of [Formula: see text] FWHM between the 16-pixel multiplexed detector array and a [Formula: see text] LGSO-SiPM reference detector. In summary, results indicate this multiplexing scheme is a scalable readout technique that provides excellent coincidence timing performance.

Identification of the first multi-exonic WDR72 deletion in isolated amelogenesis imperfecta, and generation of a WDR72-specific copy number screening tool.

PubMed

Hentschel, Julia; Tatun, Dana; Parkhomchuk, Dmitri; Kurth, Ingo; Schimmel, Bettina; Heinrich-Weltzien, Roswitha; Bertzbach, Sabine; Peters, Hartmut; Beetz, Christian

2016-09-15

Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous disorder of tooth development which is due to aberrant deposition or composition of enamel. Both syndromic and isolated forms exist; they may be inherited in an X-linked, autosomal recessive, or autosomal dominant manner. WDR72 is one of ten currently known genes for recessive isolated AI; nine WDR72 mutations affecting single nucleotides have been described to date. Based on whole exome sequencing in a large consanguineous AI pedigree, we obtained evidence for presence of a multi-exonic WDR72 deletion. A home-made multiplex ligation-dependent probe amplification assay was used to confirm the aberration, to narrow its extent, and to identify heterozygous carriers. Our study extends the mutational spectrum for WDR72 to include large deletions, and supports a relevance of the previously proposed loss-of-function mechanism. It also introduces an easy-to-use and highly sensitive tool for detecting WDR72 copy number alterations. Copyright © 2016. Published by Elsevier B.V.

Accurate and exact CNV identification from targeted high-throughput sequence data.

PubMed

Nord, Alex S; Lee, Ming; King, Mary-Claire; Walsh, Tom

2011-04-12

Massively parallel sequencing of barcoded DNA samples significantly increases screening efficiency for clinically important genes. Short read aligners are well suited to single nucleotide and indel detection. However, methods for CNV detection from targeted enrichment are lacking. We present a method combining coverage with map information for the identification of deletions and duplications in targeted sequence data. Sequencing data is first scanned for gains and losses using a comparison of normalized coverage data between samples. CNV calls are confirmed by testing for a signature of sequences that span the CNV breakpoint. With our method, CNVs can be identified regardless of whether breakpoints are within regions targeted for sequencing. For CNVs where at least one breakpoint is within targeted sequence, exact CNV breakpoints can be identified. In a test data set of 96 subjects sequenced across ~1 Mb genomic sequence using multiplexing technology, our method detected mutations as small as 31 bp, predicted quantitative copy count, and had a low false-positive rate. Application of this method allows for identification of gains and losses in targeted sequence data, providing comprehensive mutation screening when combined with a short read aligner.

Lack of Association Between Toll-like Receptor 2 Polymorphisms (R753Q and A-16934T) and Atopic Dermatitis in Children from Thrace Region of Turkey

PubMed Central

Can, Ceren; Yazıcıoğlu, Mehtap; Gürkan, Hakan; Tozkır, Hilmi; Görgülü, Adnan; Süt, Necdet Hilmi

2017-01-01

Background: Atopic dermatitis is the most common chronic inflammatory skin disease. A complex interaction of both genetic and environmental factors is thought to contribute to the disease. Aims: To evaluate whether single nucleotide polymorphisms in the TLR2 gene c.2258C>T (R753Q) (rs5743708) and TLR2 c.-148+1614T>A (A-16934T) (rs4696480) (NM_0032643) are associated with atopic dermatitis in Turkish children. Study Design: Case-control study. Methods: The study was conducted on 70 Turkish children with atopic dermatitis aged 0.5-18 years. The clinical severity of atopic dermatitis was evaluated by the severity scoring of atopic dermatitis index. Serum total IgE levels, specific IgE antibodies to inhalant and food allergens were measured in both atopic dermatitis patients and controls, skin prick tests were done on 70 children with atopic dermatitis. Genotyping for TLR2 (R753Q and A-16934T) single nucleotide polymorphisms was performed in both atopic dermatitis patients and controls. Results: Cytosine-cytosine and cytosin-thymine genotype frequencies of the TLR2 R753Q single nucleotide polymorphism in the atopic dermatitis group were determined as being 98.6% and 1.4%, cytosine allele frequency for TLR2 R753Q single nucleotide polymorphism was determined as 99.29% and the thymine allele frequency was 0.71%, thymine-thymine, thymine-adenine, and adenine-adenine genotype frequencies of the TLR2 A-16934T single nucleotide polymorphism were 24.3%, 44.3%, and 31.4%. The thymine allele frequency for the TLR2 A-16934T single nucleotide polymorphism in the atopic dermatitis group was 46.43%, and the adenine allele frequency was 53.57%, respectively. There was not statistically significant difference between the groups for all investigated polymorphisms (p>0.05). For all single nucleotide polymorphisms studied, allelic distribution was analogous among atopic dermatitis patients and controls, and no significant statistical difference was observed. No homozygous carriers of the TLR2 R753Q single nucleotide polymorphism were found in the atopic dermatitis and control groups. Conclusion: The TLR2 (R753Q and A-16934T) single nucleotide polymorphisms are not associated with atopic dermatitis in a group of Turkish patients. PMID:28443596

Lack of Association Between Toll-like Receptor 2 Polymorphisms (R753Q and A-16934T) and Atopic Dermatitis in Children from Thrace Region of Turkey.

PubMed

Can, Ceren; Yazıcıoğlu, Mehtap; Gürkan, Hakan; Tozkır, Hilmi; Görgülü, Adnan; Süt, Necdet Hilmi

2017-05-05

Atopic dermatitis is the most common chronic inflammatory skin disease. A complex interaction of both genetic and environmental factors is thought to contribute to the disease. To evaluate whether single nucleotide polymorphisms in the TLR2 gene c.2258C>T (R753Q) (rs5743708) and TLR2 c.-148+1614T>A (A-16934T) (rs4696480) (NM_0032643) are associated with atopic dermatitis in Turkish children. Case-control study. The study was conducted on 70 Turkish children with atopic dermatitis aged 0.5-18 years. The clinical severity of atopic dermatitis was evaluated by the severity scoring of atopic dermatitis index. Serum total IgE levels, specific IgE antibodies to inhalant and food allergens were measured in both atopic dermatitis patients and controls, skin prick tests were done on 70 children with atopic dermatitis. Genotyping for TLR2 (R753Q and A-16934T) single nucleotide polymorphisms was performed in both atopic dermatitis patients and controls. Cytosine-cytosine and cytosin-thymine genotype frequencies of the TLR2 R753Q single nucleotide polymorphism in the atopic dermatitis group were determined as being 98.6% and 1.4%, cytosine allele frequency for TLR2 R753Q single nucleotide polymorphism was determined as 99.29% and the thymine allele frequency was 0.71%, thymine-thymine, thymine-adenine, and adenine-adenine genotype frequencies of the TLR2 A-16934T single nucleotide polymorphism were 24.3%, 44.3%, and 31.4%. The thymine allele frequency for the TLR2 A-16934T single nucleotide polymorphism in the atopic dermatitis group was 46.43%, and the adenine allele frequency was 53.57%, respectively. There was not statistically significant difference between the groups for all investigated polymorphisms (p>0.05). For all single nucleotide polymorphisms studied, allelic distribution was analogous among atopic dermatitis patients and controls, and no significant statistical difference was observed. No homozygous carriers of the TLR2 R753Q single nucleotide polymorphism were found in the atopic dermatitis and control groups. The TLR2 (R753Q and A-16934T) single nucleotide polymorphisms are not associated with atopic dermatitis in a group of Turkish patients.

Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

PubMed

Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

2005-05-18

To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

Improved prediction of biochemical recurrence after radical prostatectomy by genetic polymorphisms.

PubMed

Morote, Juan; Del Amo, Jokin; Borque, Angel; Ars, Elisabet; Hernández, Carlos; Herranz, Felipe; Arruza, Antonio; Llarena, Roberto; Planas, Jacques; Viso, María J; Palou, Joan; Raventós, Carles X; Tejedor, Diego; Artieda, Marta; Simón, Laureano; Martínez, Antonio; Rioja, Luis A

2010-08-01

Single nucleotide polymorphisms are inherited genetic variations that can predispose or protect individuals against clinical events. We hypothesized that single nucleotide polymorphism profiling may improve the prediction of biochemical recurrence after radical prostatectomy. We performed a retrospective, multi-institutional study of 703 patients treated with radical prostatectomy for clinically localized prostate cancer who had at least 5 years of followup after surgery. All patients were genotyped for 83 prostate cancer related single nucleotide polymorphisms using a low density oligonucleotide microarray. Baseline clinicopathological variables and single nucleotide polymorphisms were analyzed to predict biochemical recurrence within 5 years using stepwise logistic regression. Discrimination was measured by ROC curve AUC, specificity, sensitivity, predictive values, net reclassification improvement and integrated discrimination index. The overall biochemical recurrence rate was 35%. The model with the best fit combined 8 covariates, including the 5 clinicopathological variables prostate specific antigen, Gleason score, pathological stage, lymph node involvement and margin status, and 3 single nucleotide polymorphisms at the KLK2, SULT1A1 and TLR4 genes. Model predictive power was defined by 80% positive predictive value, 74% negative predictive value and an AUC of 0.78. The model based on clinicopathological variables plus single nucleotide polymorphisms showed significant improvement over the model without single nucleotide polymorphisms, as indicated by 23.3% net reclassification improvement (p = 0.003), integrated discrimination index (p <0.001) and likelihood ratio test (p <0.001). Internal validation proved model robustness (bootstrap corrected AUC 0.78, range 0.74 to 0.82). The calibration plot showed close agreement between biochemical recurrence observed and predicted probabilities. Predicting biochemical recurrence after radical prostatectomy based on clinicopathological data can be significantly improved by including patient genetic information. Copyright (c) 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

PubMed Central

Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H.; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A.

2015-01-01

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates. PMID:26394042

Centrality in earthquake multiplex networks

NASA Astrophysics Data System (ADS)

Lotfi, Nastaran; Darooneh, Amir Hossein; Rodrigues, Francisco A.

2018-06-01

Seismic time series has been mapped as a complex network, where a geographical region is divided into square cells that represent the nodes and connections are defined according to the sequence of earthquakes. In this paper, we map a seismic time series to a temporal network, described by a multiplex network, and characterize the evolution of the network structure in terms of the eigenvector centrality measure. We generalize previous works that considered the single layer representation of earthquake networks. Our results suggest that the multiplex representation captures better earthquake activity than methods based on single layer networks. We also verify that the regions with highest seismological activities in Iran and California can be identified from the network centrality analysis. The temporal modeling of seismic data provided here may open new possibilities for a better comprehension of the physics of earthquakes.

Frequency-Domain Streak Camera and Tomography for Ultrafast Imaging of Evolving and Channeled Plasma Accelerator Structures

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

Li Zhengyan; Zgadzaj, Rafal; Wang Xiaoming

2010-11-04

We demonstrate a prototype Frequency Domain Streak Camera (FDSC) that can capture the picosecond time evolution of the plasma accelerator structure in a single shot. In our prototype Frequency-Domain Streak Camera, a probe pulse propagates obliquely to a sub-picosecond pump pulse that creates an evolving nonlinear index 'bubble' in fused silica glass, supplementing a conventional Frequency Domain Holographic (FDH) probe-reference pair that co-propagates with the 'bubble'. Frequency Domain Tomography (FDT) generalizes Frequency-Domain Streak Camera by probing the 'bubble' from multiple angles and reconstructing its morphology and evolution using algorithms similar to those used in medical CAT scans. Multiplexing methods (Temporalmore » Multiplexing and Angular Multiplexing) improve data storage and processing capability, demonstrating a compact Frequency Domain Tomography system with a single spectrometer.« less

Design, development and evaluation of a resistor-based multiplexing circuit for a 20×20 SiPM array

NASA Astrophysics Data System (ADS)

Wang, Zhonghai; Sun, Xishan; Lou, Kai; Meier, Joseph; Zhou, Rong; Yang, Chaowen; Zhu, Xiaorong; Shao, Yiping

2016-04-01

One technical challenge in developing a large-size scintillator detector with multiple Silicon Photomultiplier (SiPM) arrays is to read out a large number of detector output channels. To achieve this, different signal multiplexing circuits have been studied and applied with different performances and cost-effective tradeoffs. Resistor-based multiplexing circuits exhibit simplicity and signal integrity, but also present the disadvantage of timing shift among different channels. In this study, a resistor-based multiplexing circuit for a large-sized SiPM array readout was developed and evaluated by simulation and experimental studies. Similarly to a multiplexing circuit used for multi-anode PMT, grounding and branching resistors were connected to each SiPM output channel. The grounding resistor was used to simultaneously reduce the signal crosstalk among different channels and to improve timing performance. Both grounding and branching resistor values were optimized to maintain a balanced performance of the event energy, timing, and positioning. A multiplexing circuit was implemented on a compact PCB and applied for a flat-panel detector which consisted of a 32×32 LYSO scintillator crystals optically coupled to 5×5 SiPM arrays for a total 20×20 output channels. Test results showed excellent crystal identification for all 1024 LYSO crystals (each with 2×2×30 mm3 size) with 22Na flood-source irradiation. The measured peak-to-valley ratio from typical crystal map profile is around 3:1 to 6.6:1, an average single crystal energy resolution of about 17.3%, and an average single crystal timing resolution of about 2 ns. Timing shift among different crystals, as reported in some other resistor-based multiplexing circuit designs, was not observed. In summary, we have designed and implemented a practical resistor-based multiplexing circuit that can be readily applied for reading out a large SiPM array with good detector performance.

[Meta-analysis on relationship between single nucleotide polymorphism of rs2231142 in ABCG2 gene and gout in East Asian population].

PubMed

Wu, Lei; He, Yao; Zhang, Di

2015-11-01

To systematically evaluate the association between single nucleotide polymorphism of rs2231142 genetic susceptibility and gout in East Asian population. The literature retrieval was conducted by using English databases (Medline, EMbase), Chinese databases (CNKI, Vip, Wanfang, SinaMed) and others to collect the published papers on the association between single nucleotide polymorphism of rs2231142 genetic susceptibility and gout by the end of December 2014. Meta-analysis was performed with software Stata 12.0. Nine studies were included. There were significant associations between increased risk of gout and single nucleotide polymorphism of rs2231142, the combined OR was 2.04 (95%CI: 1.82-2.28) for A allele and C allele, 1.97 (95%CI: 1.57-2.48) for CA and CC, 3.71 (95%CI: 3.07-4.47) for AA and CC. Sex and region specific subgroup analysis showed less heterogeneity. There is significant association between gout and single nucleotide polymorphism of rs2231142 in East Asian population, and A allele is a high risk gene for gout.

CNTNAP2 Is Significantly Associated With Speech Sound Disorder in the Chinese Han Population.

PubMed

Zhao, Yun-Jing; Wang, Yue-Ping; Yang, Wen-Zhu; Sun, Hong-Wei; Ma, Hong-Wei; Zhao, Ya-Ru

2015-11-01

Speech sound disorder is the most common communication disorder. Some investigations support the possibility that the CNTNAP2 gene might be involved in the pathogenesis of speech-related diseases. To investigate single-nucleotide polymorphisms in the CNTNAP2 gene, 300 unrelated speech sound disorder patients and 200 normal controls were included in the study. Five single-nucleotide polymorphisms were amplified and directly sequenced. Significant differences were found in the genotype (P = .0003) and allele (P = .0056) frequencies of rs2538976 between patients and controls. The excess frequency of the A allele in the patient group remained significant after Bonferroni correction (P = .0280). A significant haplotype association with rs2710102T/+rs17236239A/+2538976A/+2710117A (P = 4.10e-006) was identified. A neighboring single-nucleotide polymorphism, rs10608123, was found in complete linkage disequilibrium with rs2538976, and the genotypes exactly corresponded to each other. The authors propose that these CNTNAP2 variants increase the susceptibility to speech sound disorder. The single-nucleotide polymorphisms rs10608123 and rs2538976 may merge into one single-nucleotide polymorphism. © The Author(s) 2015.

Erratum to "10 Gbit/s mode-multiplexed QPSK transmission using MDM-to-MFDM based single coherent receiver for intra- and inter data center networking" [Opt. Commun. 391 (2017) 106-110

NASA Astrophysics Data System (ADS)

Asif, Rameez; Haithem, Mustafa

2018-03-01

We revisited our previous work "10 Gbit/s mode-multiplexed QPSK transmission using MDM-to-MFDM based single coherent receiver for intraand inter data center networking" [Opt. Commun. 391 (2017) 106-110] and discover a mistake in the Appendix 'A', i.e. mode-selective coherent detection technique. In this section, the direct referencing of the previous work at appropriate points is not adequate (page no. 109).

Low-cost coherent receiver for long-reach optical access network using single-ended detection.

PubMed

Zhang, Xuebing; Li, Zhaohui; Li, Jianping; Yu, Changyuan; Lau, Alan Pak Tao; Lu, Chao

2014-09-15

A low-cost coherent receiver using two 2×3 optical hybrids and single-ended detection is proposed for long-reach optical access network. This structure can detect the two polarization components of polarization division multiplexing (PDM) signals. Polarization de-multiplexing and signal-to-signal beat interference (SSBI) cancellation are realized by using only three photodiodes. Simulation results for 40 Gb/s PDM-OFDM transmissions indicate that the low-cost coherent receiver has 3.2 dB optical signal-to-noise ratio difference compared with the theoretical value.

Cacao single-nucleotide polymorphism (SNP) markers: A discovery strategy to identify SNPs for genotyping, genetic mapping and genome wide association studies (GWAS)

USDA-ARS?s Scientific Manuscript database

Single-nucleotide polymorphisms (SNPs) are the most common genetic markers in Theobroma cacao, occurring approximately once in every 200 nucleotides. SNPs, like microsatellites, are co-dominant and PCR-based, but they have several advantages over microsatellites. They are unambiguous, so that a SN...

Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

A Single Multiplex crRNA Array for FnCpf1-Mediated Human Genome Editing.

PubMed

Sun, Huihui; Li, Fanfan; Liu, Jie; Yang, Fayu; Zeng, Zhenhai; Lv, Xiujuan; Tu, Mengjun; Liu, Yeqing; Ge, Xianglian; Liu, Changbao; Zhao, Junzhao; Zhang, Zongduan; Qu, Jia; Song, Zongming; Gu, Feng

2018-06-15

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Experimental demonstration of a frequency-domain Volterra series nonlinear equalizer in polarization-multiplexed transmission.

PubMed

Guiomar, Fernando P; Reis, Jacklyn D; Carena, Andrea; Bosco, Gabriella; Teixeira, António L; Pinto, Armando N

2013-01-14

Employing 100G polarization-multiplexed quaternary phase-shift keying (PM-QPSK) signals, we experimentally demonstrate a dual-polarization Volterra series nonlinear equalizer (VSNE) applied in frequency-domain, to mitigate intra-channel nonlinearities. The performance of the dual-polarization VSNE is assessed in both single-channel and in wavelength-division multiplexing (WDM) scenarios, providing direct comparisons with its single-polarization version and with the widely studied back-propagation split-step Fourier (SSF) approach. In single-channel transmission, the optimum power has been increased by about 1 dB, relatively to the single-polarization equalizers, and up to 3 dB over linear equalization, with a corresponding bit error rate (BER) reduction of up to 63% and 85%, respectively. Despite of the impact of inter-channel nonlinearities, we show that intra-channel nonlinear equalization is still able to provide approximately 1 dB improvement in the optimum power and a BER reduction of ~33%, considering a 66 GHz WDM grid. By means of simulation, we demonstrate that the performance of nonlinear equalization can be substantially enhanced if both optical and electrical filtering are optimized, enabling the VSNE technique to outperform its SSF counterpart at high input powers.

The association of single-nucleotide polymorphisms in the oxytocin receptor and G protein-coupled receptor kinase 6 (GRK6) genes with oxytocin dosing requirements and labor outcomes.

PubMed

Grotegut, Chad A; Ngan, Emily; Garrett, Melanie E; Miranda, Marie Lynn; Ashley-Koch, Allison E; Swamy, Geeta K

2017-09-01

Oxytocin is a potent uterotonic agent that is widely used for induction and augmentation of labor. Oxytocin has a narrow therapeutic index and the optimal dosing for any individual woman varies widely. The objective of this study was to determine whether genetic variation in the oxytocin receptor (OXTR) or in the gene encoding G protein-coupled receptor kinase 6 (GRK6), which regulates desensitization of the oxytocin receptor, could explain variation in oxytocin dosing and labor outcomes among women being induced near term. Pregnant women with a singleton gestation residing in Durham County, NC, were prospectively enrolled as part of the Healthy Pregnancy, Healthy Baby cohort study. Those women undergoing an induction of labor at 36 weeks or greater were genotyped for 18 haplotype-tagging single-nucleotide polymorphisms in OXTR and 7 haplotype-tagging single-nucleotide polymorphisms in GRK6 using TaqMan assays. Linear regression was used to examine the relationship between maternal genotype and maximal oxytocin infusion rate, total oxytocin dose received, and duration of labor. Logistic regression was used to test for the association of maternal genotype with mode of delivery. For each outcome, backward selection techniques were utilized to control for important confounding variables and additive genetic models were used. Race/ethnicity was included in all models because of differences in allele frequencies across populations, and Bonferroni correction for multiple testing was used. DNA was available from 482 women undergoing induction of labor at 36 weeks or greater. Eighteen haplotype-tagging single-nucleotide polymorphisms within OXTR and 7 haplotype-tagging single-nucleotide polymorphisms within GRK6 were examined. Five single-nucleotide polymorphisms in OXTR showed nominal significance with maximal infusion rate of oxytocin, and two single-nucleotide polymorphisms in OXTR were associated with total oxytocin dose received. One single-nucleotide polymorphism in OXTR and two single-nucleotide polymorphisms in GRK6 were associated with duration of labor, one of which met the multiple testing threshold (P = .0014, rs2731664 [GRK6], mean duration of labor, 17.7 hours vs 20.2 hours vs 23.5 hours for AA, AC, and CC genotypes, respectively). Three single-nucleotide polymorphisms, two in OXTR and one in GRK6, showed nominal significance with mode of delivery. Genetic variation in OXTR and GRK6 is associated with the amount of oxytocin required as well as the duration of labor and risk for cesarean delivery among women undergoing induction of labor near term. With further research, pharmacogenomic approaches may potentially be utilized to develop personalized treatment to improve safety and efficacy outcomes among women undergoing induction of labor. Copyright © 2017 Elsevier Inc. All rights reserved.

Immunization of Epidemics in Multiplex Networks

PubMed Central

Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

2014-01-01

Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks. PMID:25401755

Immunization of epidemics in multiplex networks.

PubMed

Zhao, Dawei; Wang, Lianhai; Li, Shudong; Wang, Zhen; Wang, Lin; Gao, Bo

2014-01-01

Up to now, immunization of disease propagation has attracted great attention in both theoretical and experimental researches. However, vast majority of existing achievements are limited to the simple assumption of single layer networked population, which seems obviously inconsistent with recent development of complex network theory: each node could possess multiple roles in different topology connections. Inspired by this fact, we here propose the immunization strategies on multiplex networks, including multiplex node-based random (targeted) immunization and layer node-based random (targeted) immunization. With the theory of generating function, theoretical analysis is developed to calculate the immunization threshold, which is regarded as the most critical index for the effectiveness of addressed immunization strategies. Interestingly, both types of random immunization strategies show more efficiency in controlling disease spreading on multiplex Erdös-Rényi (ER) random networks; while targeted immunization strategies provide better protection on multiplex scale-free (SF) networks.

Translating pharmacodynamic biomarkers from bench to bedside: analytical validation and fit-for-purpose studies to qualify multiplex immunofluorescent assays for use on clinical core biopsy specimens.

PubMed

Marrero, Allison; Lawrence, Scott; Wilsker, Deborah; Voth, Andrea Regier; Kinders, Robert J

2016-08-01

Multiplex pharmacodynamic (PD) assays have the potential to increase sensitivity of biomarker-based reporting for new targeted agents, as well as revealing significantly more information about target and pathway activation than single-biomarker PD assays. Stringent methodology is required to ensure reliable and reproducible results. Common to all PD assays is the importance of reagent validation, assay and instrument calibration, and the determination of suitable response calibrators; however, multiplex assays, particularly those performed on paraffin specimens from tissue blocks, bring format-specific challenges adding a layer of complexity to assay development. We discuss existing multiplex approaches and the development of a multiplex immunofluorescence assay measuring DNA damage and DNA repair enzymes in response to anti-cancer therapeutics and describe how our novel method addresses known issues. Copyright © 2016 Elsevier Inc. All rights reserved.

Mapping RNA Structure In Vitro with SHAPE Chemistry and Next-Generation Sequencing (SHAPE-Seq).

PubMed

Watters, Kyle E; Lucks, Julius B

2016-01-01

Mapping RNA structure with selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry has proven to be a versatile method for characterizing RNA structure in a variety of contexts. SHAPE reagents covalently modify RNAs in a structure-dependent manner to create adducts at the 2'-OH group of the ribose backbone at nucleotides that are structurally flexible. The positions of these adducts are detected using reverse transcriptase (RT) primer extension, which stops one nucleotide before the modification, to create a pool of cDNAs whose lengths reflect the location of SHAPE modification. Quantification of the cDNA pools is used to estimate the "reactivity" of each nucleotide in an RNA molecule to the SHAPE reagent. High reactivities indicate nucleotides that are structurally flexible, while low reactivities indicate nucleotides that are inflexible. These SHAPE reactivities can then be used to infer RNA structures by restraining RNA structure prediction algorithms. Here, we provide a state-of-the-art protocol describing how to perform in vitro RNA structure probing with SHAPE chemistry using next-generation sequencing to quantify cDNA pools and estimate reactivities (SHAPE-Seq). The use of next-generation sequencing allows for higher throughput, more consistent data analysis, and multiplexing capabilities. The technique described herein, SHAPE-Seq v2.0, uses a universal reverse transcription priming site that is ligated to the RNA after SHAPE modification. The introduced priming site allows for the structural analysis of an RNA independent of its sequence.

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

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

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes

DOE PAGES

Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; ...

2014-01-01

Background . Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results . A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Eachmore » group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions . This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.« less

Superconducting Digital Multiplexers for Sensor Arrays

NASA Technical Reports Server (NTRS)

Kadin, Alan M.; Brock, Darren K.; Gupta, Deepnarayan

2004-01-01

Arrays of cryogenic microbolometers and other cryogenic detectors are being developed for infrared imaging. If the signal from each sensor is amplified, multiplexed, and digitized using superconducting electronics, then this data can be efficiently read out to ambient temperature with a minimum of noise and thermal load. HYPRES is developing an integrated system based on SQUID amplifiers, a high-resolution analog-to-digital converter (ADC) based on RSFQ (rapid single flux quantum) logic, and a clocked RSFQ multiplexer. The ADC and SQUIDs have already been demonstrated for other projects, so this paper will focus on new results of a digital multiplexer. Several test circuits have been fabricated using Nb Josephson technology and are about to be tested at T = 4.2 K, with a more complete prototype in preparation.

2x2 MIMO-OFDM Gigabit fiber-wireless access system based on polarization division multiplexed WDM-PON.

PubMed

Deng, Lei; Pang, Xiaodan; Zhao, Ying; Othman, M B; Jensen, Jesper Bevensee; Zibar, Darko; Yu, Xianbin; Liu, Deming; Monroy, Idelfonso Tafur

2012-02-13

We propose a spectral efficient radio over wavelength division multiplexed passive optical network (WDM-PON) system by combining optical polarization division multiplexing (PDM) and wireless multiple input multiple output (MIMO) spatial multiplexing techniques. In our experiment, a training-based zero forcing (ZF) channel estimation algorithm is designed to compensate the polarization rotation and wireless multipath fading. A 797 Mb/s net data rate QPSK-OFDM signal with error free (<1 × 10(5)) performance and a 1.59 Gb/s net data rate 16QAM-OFDM signal with BER performance of 1.2 × 10(2) are achieved after transmission of 22.8 km single mode fiber followed by 3 m and 1 m air distances, respectively.

Convergent optical wired and wireless long-reach access network using high spectral-efficient modulation.

PubMed

Chow, C W; Lin, Y H

2012-04-09

To provide broadband services in a single and low cost perform, the convergent optical wired and wireless access network is promising. Here, we propose and demonstrate a convergent optical wired and wireless long-reach access networks based on orthogonal wavelength division multiplexing (WDM). Both the baseband signal and the radio-over-fiber (ROF) signal are multiplexed and de-multiplexed in optical domain, hence it is simple and the operation speed is not limited by the electronic bottleneck caused by the digital signal processing (DSP). Error-free de-multiplexing and down-conversion can be achieved for all the signals after 60 km (long-reach) fiber transmission. The scalability of the system for higher bit-rate (60 GHz) is also simulated and discussed.

Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

PubMed

Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

2015-12-01

Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

An integrated passive micromixer-magnetic separation-capillary electrophoresis microdevice for rapid and multiplex pathogen detection at the single-cell level.

PubMed

Jung, Jae Hwan; Kim, Gha-Young; Seo, Tae Seok

2011-10-21

Here we report an integrated microdevice consisting of an efficient passive mixer, a magnetic separation chamber, and a capillary electrophoretic microchannel in which DNA barcode assay, target pathogen separation, and barcode DNA capillary electrophoretic analysis were performed sequentially within 30 min for multiplex pathogen detection at the single-cell level. The intestine-shaped serpentine 3D micromixer provides a high mixing rate to generate magnetic particle-pathogenic bacteria-DNA barcode labelled AuNP complexes quantitatively. After magnetic separation and purification of those complexes, the barcode DNA strands were released and analyzed by the microfluidic capillary electrophoresis within 5 min. The size of the barcode DNA strand was controlled depending on the target bacteria (Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella typhimurium), and the different elution time of the barcode DNA peak in the electropherogram allows us to recognize the target pathogen with ease in the monoplex as well as in the multiplex analysis. In addition, the quantity of the DNA barcode strand (∼10(4)) per AuNP is enough to be observed in the laser-induced confocal fluorescence detector, thereby making single-cell analysis possible. This novel integrated microdevice enables us to perform rapid, sensitive, and multiplex pathogen detection with sample-in-answer-out capability to be applied for biosafety testing, environmental screening, and clinical trials.

Detection and molecular characterization of tomato yellow leaf curl virus naturally infecting Lycopersicon esculentum in Egypt.

PubMed

Rabie, M; Ratti, C; Abdel Aleem, E; Fattouh, F

Tomato yellow leaf curl virus (TYLCV) infections of tomato crops in Egypt were widely spread in 2014. Infected symptomatic tomato plants from different governorates were sampled. TYLCV strains Israel and Mild (TYLCV-IL, TYLCV-Mild) were identified by multiplex and real-time PCR. In addition, nucleotide sequence analysis of the V1 and V2 protein genes, revealed ten TYLCV Egyptian isolates (TYLCV from TY1 to 10). Phylogenetic analysis showed their high degree of relatedness with TYLCV-IL Jordan isolate (98%). Here we have showed the complete nucleotide sequence of the TYLCV Egyptian isolate TY10, sampled from El Beheira. A high degree of similarity to other previously reported Egyptian isolates and isolates from Jordan and Japan reflect the importance of phylogenetic analysis in monitoring virus genetic diversity and possibilities for divergence of more virulent strains or genotypes.

Comprehensive analysis of the T-cell receptor beta chain gene in rhesus monkey by high throughput sequencing

PubMed Central

Li, Zhoufang; Liu, Guangjie; Tong, Yin; Zhang, Meng; Xu, Ying; Qin, Li; Wang, Zhanhui; Chen, Xiaoping; He, Jiankui

2015-01-01

Profiling immune repertoires by high throughput sequencing enhances our understanding of immune system complexity and immune-related diseases in humans. Previously, cloning and Sanger sequencing identified limited numbers of T cell receptor (TCR) nucleotide sequences in rhesus monkeys, thus their full immune repertoire is unknown. We applied multiplex PCR and Illumina high throughput sequencing to study the TCRβ of rhesus monkeys. We identified 1.26 million TCRβ sequences corresponding to 643,570 unique TCRβ sequences and 270,557 unique complementarity-determining region 3 (CDR3) gene sequences. Precise measurements of CDR3 length distribution, CDR3 amino acid distribution, length distribution of N nucleotide of junctional region, and TCRV and TCRJ gene usage preferences were performed. A comprehensive profile of rhesus monkey immune repertoire might aid human infectious disease studies using rhesus monkeys. PMID:25961410

Multiplex lexical networks reveal patterns in early word acquisition in children

NASA Astrophysics Data System (ADS)

Stella, Massimo; Beckage, Nicole M.; Brede, Markus

2017-04-01

Network models of language have provided a way of linking cognitive processes to language structure. However, current approaches focus only on one linguistic relationship at a time, missing the complex multi-relational nature of language. In this work, we overcome this limitation by modelling the mental lexicon of English-speaking toddlers as a multiplex lexical network, i.e. a multi-layered network where N = 529 words/nodes are connected according to four relationship: (i) free association, (ii) feature sharing, (iii) co-occurrence, and (iv) phonological similarity. We investigate the topology of the resulting multiplex and then proceed to evaluate single layers and the full multiplex structure on their ability to predict empirically observed age of acquisition data of English speaking toddlers. We find that the multiplex topology is an important proxy of the cognitive processes of acquisition, capable of capturing emergent lexicon structure. In fact, we show that the multiplex structure is fundamentally more powerful than individual layers in predicting the ordering with which words are acquired. Furthermore, multiplex analysis allows for a quantification of distinct phases of lexical acquisition in early learners: while initially all the multiplex layers contribute to word learning, after about month 23 free associations take the lead in driving word acquisition.

Si:Bi switched photoconducttor infrared detector array

NASA Technical Reports Server (NTRS)

Eakin, C. E.

1983-01-01

A multiplexed infrared detector array is described. The small demonstration prototype consisted of two cryogenically cooled, bismuth doped silicon, extrinsic photoconductor pixels multiplexed onto a single output channel using an on focal plane switch integration sampling technique. Noise levels of the order of 400 to 600 rms electrons per sample were demonstrated for this chip and wire hybrid version.

106-17 Telemetry Standards. Annex A.4. Asynchronous Recorder Multiplexer Output Re-Constructor (ARMOR)

DTIC Science & Technology

2017-07-01

Output Re-Constructor 1. General This standard defines the recommended multiplexer format for single-channel data recording on small-format (1/2 in...which is 1-based, is determined by the position of the channel’s module in the ARMOR system . The first input channel found in the ARMOR system is

Association of GSK3B With Alzheimer Disease and Frontotemporal Dementia

PubMed Central

Schaffer, Barbara A. J.; Bertram, Lars; Miller, Bruce L.; Mullin, Kristina; Weintraub, Sandra; Johnson, Nancy; Bigio, Eileen H.; Mesulam, Marsel; Wiedau-Pazos, Martina; Jackson, George R.; Cummings, Jeffrey L.; Cantor, Rita M.; Levey, Allan I.; Tanzi, Rudolph E.; Geschwind, Daniel H.

2009-01-01

Background Deposits of abnormally hyperphosphorylated tau are a hallmark of several dementias, including Alzheimer disease (AD), and about 10% of familial frontotemporal dementia (FTD) cases are caused by mutations in the tau gene. As a known tau kinase, GSK3B is a promising candidate gene in the remaining cases of FTD and in AD, for which tau mutations have not been found. Objective To examine the promoter of GSK3B and all 12 exons, including the surrounding intronic sequence, in patients with FTD, patients with AD, and aged healthy subjects to identify single-nucleotide polymorphisms associated with disease. Design, Setting, and Participants Single-nucleotide polymorphism frequency was examined in a case-control cohort of 48 patients with probable AD, 102 patients with FTD, 38 patients with primary progressive aphasia, and 85 aged healthy subjects. Results were followed up in 2 independent AD family samples consisting of 437 multiplex families with AD (National Institute of Mental Health Genetics Initiative AD Study) or 150 sibships discordant for AD (Consortium on Alzheimer’s Genetics Study). Results Several rare sequence variants in GSK3B were identified in the case-control study. An intronic polymorphism (IVS2−68G>A) occurred at more than twice the frequency among patients with FTD (10.8%) and patients with AD (14.6%) than in aged healthy subjects (4.1%). The polymorphism showed association with disease in both follow-up samples independently, although only the Consortium on Alzheimer’s Genetics sample showed the same direction of association as the case-control sample. Conclusions To our knowledge, this is the first evidence that a gene known to be involved in tau phosphorylation, GSK3B, is associated with risk for primary neurodegenerative dementias. This supports previous work in animal models suggesting that such genes are therapeutic targets. PMID:18852354

A High Throughput Single Nucleotide Polymorphism Multiplex Assay for Parentage Assignment in New Zealand Sheep

PubMed Central

Clarke, Shannon M.; Henry, Hannah M.; Dodds, Ken G.; Jowett, Timothy W. D.; Manley, Tim R.; Anderson, Rayna M.; McEwan, John C.

2014-01-01

Accurate pedigree information is critical to animal breeding systems to ensure the highest rate of genetic gain and management of inbreeding. The abundance of available genomic data, together with development of high throughput genotyping platforms, means that single nucleotide polymorphisms (SNPs) are now the DNA marker of choice for genomic selection studies. Furthermore the superior qualities of SNPs compared to microsatellite markers allows for standardization between laboratories; a property that is crucial for developing an international set of markers for traceability studies. The objective of this study was to develop a high throughput SNP assay for use in the New Zealand sheep industry that gives accurate pedigree assignment and will allow a reduction in breeder input over lambing. This required two phases of development- firstly, a method of extracting quality DNA from ear-punch tissue performed in a high throughput cost efficient manner and secondly a SNP assay that has the ability to assign paternity to progeny resulting from mob mating. A likelihood based approach to infer paternity was used where sires with the highest LOD score (log of the ratio of the likelihood given parentage to likelihood given non-parentage) are assigned. An 84 “parentage SNP panel” was developed that assigned, on average, 99% of progeny to a sire in a problem where there were 3,000 progeny from 120 mob mated sires that included numerous half sib sires. In only 6% of those cases was there another sire with at least a 0.02 probability of paternity. Furthermore dam information (either recorded, or by genotyping possible dams) was absent, highlighting the SNP test’s suitability for paternity testing. Utilization of this parentage SNP assay will allow implementation of progeny testing into large commercial farms where the improved accuracy of sire assignment and genetic evaluations will increase genetic gain in the sheep industry. PMID:24740141

A high throughput single nucleotide polymorphism multiplex assay for parentage assignment in New Zealand sheep.

PubMed

Clarke, Shannon M; Henry, Hannah M; Dodds, Ken G; Jowett, Timothy W D; Manley, Tim R; Anderson, Rayna M; McEwan, John C

2014-01-01

Accurate pedigree information is critical to animal breeding systems to ensure the highest rate of genetic gain and management of inbreeding. The abundance of available genomic data, together with development of high throughput genotyping platforms, means that single nucleotide polymorphisms (SNPs) are now the DNA marker of choice for genomic selection studies. Furthermore the superior qualities of SNPs compared to microsatellite markers allows for standardization between laboratories; a property that is crucial for developing an international set of markers for traceability studies. The objective of this study was to develop a high throughput SNP assay for use in the New Zealand sheep industry that gives accurate pedigree assignment and will allow a reduction in breeder input over lambing. This required two phases of development--firstly, a method of extracting quality DNA from ear-punch tissue performed in a high throughput cost efficient manner and secondly a SNP assay that has the ability to assign paternity to progeny resulting from mob mating. A likelihood based approach to infer paternity was used where sires with the highest LOD score (log of the ratio of the likelihood given parentage to likelihood given non-parentage) are assigned. An 84 "parentage SNP panel" was developed that assigned, on average, 99% of progeny to a sire in a problem where there were 3,000 progeny from 120 mob mated sires that included numerous half sib sires. In only 6% of those cases was there another sire with at least a 0.02 probability of paternity. Furthermore dam information (either recorded, or by genotyping possible dams) was absent, highlighting the SNP test's suitability for paternity testing. Utilization of this parentage SNP assay will allow implementation of progeny testing into large commercial farms where the improved accuracy of sire assignment and genetic evaluations will increase genetic gain in the sheep industry.

Quantum detector tomography of a time-multiplexed superconducting nanowire single-photon detector at telecom wavelengths.

PubMed

Natarajan, Chandra M; Zhang, Lijian; Coldenstrodt-Ronge, Hendrik; Donati, Gaia; Dorenbos, Sander N; Zwiller, Val; Walmsley, Ian A; Hadfield, Robert H

2013-01-14

Superconducting nanowire single-photon detectors (SNSPDs) are widely used in telecom wavelength optical quantum information science applications. Quantum detector tomography allows the positive-operator-valued measure (POVM) of a single-photon detector to be determined. We use an all-fiber telecom wavelength detector tomography test bed to measure detector characteristics with respect to photon flux and polarization, and hence determine the POVM. We study the SNSPD both as a binary detector and in an 8-bin, fiber based, Time-Multiplexed (TM) configuration at repetition rates up to 4 MHz. The corresponding POVMs provide an accurate picture of the photon number resolving capability of the TM-SNSPD.

Sensitive and quantitative measurement of gene expression directly from a small amount of whole blood.

PubMed

Zheng, Zhi; Luo, Yuling; McMaster, Gary K

2006-07-01

Accurate and precise quantification of mRNA in whole blood is made difficult by gene expression changes during blood processing, and by variations and biases introduced by sample preparations. We sought to develop a quantitative whole-blood mRNA assay that eliminates blood purification, RNA isolation, reverse transcription, and target amplification while providing high-quality data in an easy assay format. We performed single- and multiplex gene expression analysis with multiple hybridization probes to capture mRNA directly from blood lysate and used branched DNA to amplify the signal. The 96-well plate singleplex assay uses chemiluminescence detection, and the multiplex assay combines Luminex-encoded beads with fluorescent detection. The single- and multiplex assays could quantitatively measure as few as 6000 and 24,000 mRNA target molecules (0.01 and 0.04 amoles), respectively, in up to 25 microL of whole blood. Both formats had CVs < 10% and dynamic ranges of 3-4 logs. Assay sensitivities allowed quantitative measurement of gene expression in the minority of cells in whole blood. The signals from whole-blood lysate correlated well with signals from purified RNA of the same sample, and absolute mRNA quantification results from the assay were similar to those obtained by quantitative reverse transcription-PCR. Both single- and multiplex assay formats were compatible with common anticoagulants and PAXgene-treated samples; however, PAXgene preparations induced expression of known antiapoptotic genes in whole blood. Both the singleplex and the multiplex branched DNA assays can quantitatively measure mRNA expression directly from small volumes of whole blood. The assay offers an alternative to current technologies that depend on RNA isolation and is amenable to high-throughput gene expression analysis of whole blood.

Multiplexed single-mode wavelength-to-time mapping of multimode light

PubMed Central

Chandrasekharan, Harikumar K; Izdebski, Frauke; Gris-Sánchez, Itandehui; Krstajić, Nikola; Walker, Richard; Bridle, Helen L.; Dalgarno, Paul A.; MacPherson, William N.; Henderson, Robert K.; Birks, Tim A.; Thomson, Robert R.

2017-01-01

When an optical pulse propagates along an optical fibre, different wavelengths travel at different group velocities. As a result, wavelength information is converted into arrival-time information, a process known as wavelength-to-time mapping. This phenomenon is most cleanly observed using a single-mode fibre transmission line, where spatial mode dispersion is not present, but the use of such fibres restricts possible applications. Here we demonstrate that photonic lanterns based on tapered single-mode multicore fibres provide an efficient way to couple multimode light to an array of single-photon avalanche detectors, each of which has its own time-to-digital converter for time-correlated single-photon counting. Exploiting this capability, we demonstrate the multiplexed single-mode wavelength-to-time mapping of multimode light using a multicore fibre photonic lantern with 121 single-mode cores, coupled to 121 detectors on a 32 × 32 detector array. This work paves the way to efficient multimode wavelength-to-time mapping systems with the spectral performance of single-mode systems. PMID:28120822

Flow-through PCR on a 3D qiandu-shaped polydimethylsiloxane (PDMS) microdevice employing a single heater: toward microscale multiplex PCR.

PubMed

Wu, Wenming; Loan, Kieu The Loan; Lee, Nae Yoon

2012-05-07

Consistent temperature control in an on-chip flow-through polymerase chain reaction (PCR) employing two or more heaters is one of the main obstacles for device miniaturization and integration when realizing micro total analysis systems (μTAS), and also leads to operational complexity. In this study, we propose a qiandu (right triangular prism)-shaped polydimethylsiloxane (PDMS) microdevice with serpentine microchannels fabricated on its slanted plane, and apply the device for an on-chip flow-through PCR employing a single heater. The inclined nature of the qiandu-shaped microdevice enables the formation of a surface temperature gradient along the slanted plane of the microdevice in a height-dependent manner by the use of a single heater, and enables liquid to traverse over wide ranges of temperatures, including the three temperature zones--denaturation, annealing, and extension temperatures--required in a typical PCR. The feasibility of the qiandu-shaped PDMS microdevice as a versatile platform for performing a flow-through PCR was examined by employing multiple templates and varying the inclination angle of the device. In addition, the potential of performing a multiplex PCR using a single qiandu-shaped PDMS microdevice was explored. A 409 bp long gene fragment effective as a marker for diagnosing lung cancer and a 230 bp long gene fragment from a plasmid vector were simultaneously amplified in less than 25 min on a single microdevice, paving the way for a microscale, multiplex PCR on a single device employing a single heater.

Multispectral computational ghost imaging with multiplexed illumination

NASA Astrophysics Data System (ADS)

Huang, Jian; Shi, Dongfeng

2017-07-01

Computational ghost imaging has attracted wide attention from researchers in many fields over the last two decades. Multispectral imaging as one application of computational ghost imaging possesses spatial and spectral resolving abilities, and is very useful for surveying scenes and extracting detailed information. Existing multispectral imagers mostly utilize narrow band filters or dispersive optical devices to separate light of different wavelengths, and then use multiple bucket detectors or an array detector to record them separately. Here, we propose a novel multispectral ghost imaging method that uses one single bucket detector with multiplexed illumination to produce a colored image. The multiplexed illumination patterns are produced by three binary encoded matrices (corresponding to the red, green and blue colored information, respectively) and random patterns. The results of the simulation and experiment have verified that our method can be effective in recovering the colored object. Multispectral images are produced simultaneously by one single-pixel detector, which significantly reduces the amount of data acquisition.

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

PubMed Central

Gootenberg, Jonathan S.; Abudayyeh, Omar O.; Kellner, Max J.; Joung, Julia; Collins, James J.; Zhang, Feng

2018-01-01

Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids. PMID:29449508

Multiplexed displacement fiber sensor using thin core fiber exciter.

PubMed

Chen, Zhen; Hefferman, Gerald; Wei, Tao

2015-06-01

This letter reports a multiplexed optical displacement sensor using a thin core fiber (TCF) exciter. The TCF exciter is followed by a stripped single mode optical fiber. A small section of buffer is used as the movable component along the single mode fiber. Ultra-weak cladding mode reflection (< - 75 dB) was employed to probe the refractive index discontinuity between the air and buffer coating boundary. The position change of the movable buffer segment results in a delay change of the cladding mode reflection. Thus, it is a measure of the displacement of the buffer segment with respect to the glass fiber. The insertion loss of one sensor was measured to be less than 3 dB. A linear relationship was evaluated between the measurement position and absolute position of the moving actuator. Multiplexed capability was demonstrated and no cross talk was found between the sensors.

A novel (A)γδβ(0)-thalassemia caused by DNA deletion-inversion-insertion of the β-globin gene cluster and five olfactory receptor genes: Genetic interactions, hematological phenotypes and molecular characterization.

PubMed

Singha, Kritsada; Fucharoen, Goonnapa; Hama, Abdulloh; Fucharoen, Supan

2015-07-01

To report the phenotypes and genetic basis of a novel (A)γδβ(0)-thalassemia found in Thai individuals with several forms of thalassemia. An initial study was done in an adult Thai woman who had hypochromic microcytic red cells with unusually 100% Hb F. Extended study was carried out on her parents and another 17 unrelated individuals with elevated Hb F. Hb analysis was performed by capillary electrophoresis and DNA analysis was done using PCR. A novel diagnostic method based on multiplex PCR assays was developed. DNA analysis of the proband revealed the homozygosity for a novel deletion of 118.3 kb, removing the entire (A)γ, ψβ, δ-, β-globin and five olfactory receptor (OR) genes with an insertion of a 179 bp inverted DNA sequence located behind the OR52A5 gene located downstream and an insertion of 7 orphan nucleotides. Her parents were both carriers of this mutation. Further screening in suspected cases in our series unexpectedly led to identification of an additional 17 cases with this mutation in different genotypes including plain heterozygote, homozygote, compound heterozygote with Hb E, and double heterozygote with several forms of α-thalassemia. Hematological features associated with these genetic interactions are presented. Haplotype analysis indicated a single origin of this novel deletion-inversion-insertion (A)γδβ(0)-thalassemia in the Thai population. Differentiation of this mutation and other high Hb F determinants documented previously could be done by using a developed multiplex PCR assay. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Paper-based solid-phase multiplexed nucleic acid hybridization assay with tunable dynamic range using immobilized quantum dots as donors in fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Krull, Ulrich J

2013-08-06

A multiplexed solid-phase nucleic acid hybridization assay on a paper-based platform is presented using multicolor immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). The surface of paper was modified with imidazole groups to immobilize two types of QD-probe oligonucleotide conjugates that were assembled in solution. Green-emitting QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as an internal standard, while the rQD/A647 FRET pair served as a detection channel, combining the control and analytical test zones in one physical location. Hybridization of dye-labeled oligonucleotide targets provided the proximity for FRET sensitized emission from the acceptor dyes, which served as an analytical signal. Hybridization assays in the multicolor format provided a limit of detection of 90 fmol and an upper limit of dynamic range of 3.5 pmol. The use of an array of detection zones was designed to provide improved analytical figures of merit compared to that which could be achieved on one type of array design in terms of relative concentration of multicolor QDs. The hybridization assays showed excellent resistance to nonspecific adsorption of oligonucleotides. Selectivity of the two-plex hybridization assay was demonstrated by single nucleotide polymorphism (SNP) detection at a contrast ratio of 50:1. Additionally, it is shown that the use of preformed QD-probe oligonucleotide conjugates and consideration of the relative number density of the two types of QD-probe conjugates in the two-color assay format is advantageous to maximize assay sensitivity and the upper limit of dynamic range.

Subtelomeric multiplex ligation-dependent probe amplification as a supplement for rapid prenatal detection of fetal chromosomal aberrations.

PubMed

Chen, Xiangnan; Li, Huanzheng; Mao, Yijian; Xu, Xueqin; Lv, Jiaojiao; Zhou, Lili; Lin, Xiaoling; Tang, Shaohua

2014-01-01

Pregnant women with high-risk indications are highly suspected of fetal chromosomal aberrations. To determine whether Multiplex Ligation-dependent Probe Amplification (MLPA) using subtelomeric probe mixes (P036-E2 and P070-B2) is a reliable method for rapid detection of fetal chromosomal aberrations. The subtelomeric MLPA probe mixes were used to evaluate 50 blood samples from healthy individuals. 168 amniocytes and 182 umbilical cord blood samples from high-risk fetuses were analyzed using the same subtelomeric MLPA probe sets. Karyotyping was also performed in all cases of high-risk pregnancies, and single nucleotide polymorphism array analysis was used to confirm submicroscopic and ambiguous results from MLPA/karyotyping. Subtelomeric MLPA analysis of normal samples showed normal result in all cases by use of P036-E2 probe mix, while P070-B2 probe mix gave normal results for all but one case. In one normal control case P070-B2 produced a duplicated signal of probe for 13q34. In the high-risk group, totally 44 chromosomal abnormalities were found by karyotyping and MLPA, including 23 aneuploidies and 21 rearrangements or mosaics. MLPA detected all 23 aneuploidies, 12 rearrangements and 1 mosaic. Importantly, MLPA revealed 4 chromosomal translocations, 2 small supernumerary marker chromosomes (sSMCs), and 3 subtelomeric imbalances that were not well characterized or not detectable by karyotyping. However, MLPA showed negetive results for the remaining 8 rearrangements or mosaics, including 3 low mosaic aneuploidies, 1 inherited sSMC, and 4 paracentric inversions. Results suggest that combined use of subtelomeric MLPA and karyotyping may be an alternative method for using karyotype analyses alone in rapid detection of aneuploidies, rearrangements, and sSMCs.

Infectious mononucleosis-linked HLA class I single nucleotide polymorphism is associated with multiple sclerosis.

PubMed

Jafari, Naghmeh; Broer, Linda; Hoppenbrouwers, Ilse A; van Duijn, Cornelia M; Hintzen, Rogier Q

2010-11-01

Multiple sclerosis is a presumed autoimmune disease associated with genetic and environmental risk factors such as infectious mononucleosis. Recent research has shown infectious mononucleosis to be associated with a specific HLA class I polymorphism. Our aim was to test if the infectious mononucleosis-linked HLA class I single nucleotide polymorphism (rs6457110) is also associated with multiple sclerosis. Genotyping of the HLA-A single nucleotide polymorphism rs6457110 using TaqMan was performed in 591 multiple sclerosis cases and 600 controls. The association of multiple sclerosis with the HLA-A single nucleotide polymorphism was tested using logistic regression adjusted for age, sex and HLA-DRB1*1501. HLA-A minor allele (A) is associated with multiple sclerosis (OR = 0.68; p = 4.08 × 10( -5)). After stratification for HLA-DRB1*1501 risk allele (T) carrier we showed a significant OR of 0.70 (p = 0.003) for HLA-A. HLA class I single nucleotide polymorphism rs6457110 is associated with infectious mononucleosis and multiple sclerosis, independent of the major class II allele, supporting the hypothesis that shared genetics may contribute to the association between infectious mononucleosis and multiple sclerosis.

Rapid and inexpensive analysis of genetic variability in Arapaima gigas by PCR multiplex panel of eight microsatellites.

PubMed

Hamoy, I G; Santos, E J M; Santos, S E B

2008-01-22

The aim of the present study was the development of a multiplex genotyping panel of eight microsatellite markers of Arapaima gigas, previously described. Specific primer pairs were developed, each one of them marked with either FAM-6, HEX or NED. The amplification conditions using the new primers were standardized for a single reaction. The results obtained demonstrate high heterozygosity (average of 0.69) in a Lower Amazon population. The multiplex system described can thus be considered a fast, efficient and inexpensive method for the investigation of genetic variability in Arapaima populations.

Low-latency optical parallel adder based on a binary decision diagram with wavelength division multiplexing scheme

NASA Astrophysics Data System (ADS)

Shinya, A.; Ishihara, T.; Inoue, K.; Nozaki, K.; Kita, S.; Notomi, M.

2018-02-01

We propose an optical parallel adder based on a binary decision diagram that can calculate simply by propagating light through electrically controlled optical pass gates. The CARRY and CARRY operations are multiplexed in one circuit by a wavelength division multiplexing scheme to reduce the number of optical elements, and only a single gate constitutes the critical path for one digit calculation. The processing time reaches picoseconds per digit when we use a 100-μm-long optical path gates, which is ten times faster than a CMOS circuit.

Real-time multiple-look synthetic aperture radar processor for spacecraft applications

NASA Technical Reports Server (NTRS)

Wu, C.; Tyree, V. C. (Inventor)

1981-01-01

A spaceborne synthetic aperture radar (SAR) having pipeline multiple-look data processing is described which makes use of excessive azimuth bandwidth in radar echo signals to produce multiple-looking images. Time multiplexed single-look image lines from an azimuth correlator go through an energy analyzer which analyzes the mean energy in each separate look to determine the radar antenna electric boresight for use in generating the correct reference functions for the production of high quality SAR images. The multiplexed single look image lines also go through a registration delay to produce multi-look images.

Analysis of hybrid subcarrier multiplexing of OCDMA based on single photodiode detection

NASA Astrophysics Data System (ADS)

Ahmad, N. A. A.; Junita, M. N.; Aljunid, S. A.; Rashidi, C. B. M.; Endut, R.

2017-11-01

This paper analyzes the performance of subcarrier multiplexing (SCM) of spectral amplitude coding optical code multiple access (SAC-OCDMA) by applying Recursive Combinatorial (RC) code based on single photodiode detection (SPD). SPD is used in the receiver part to reduce the effect of multiple access interference (MAI) which contributes as a dominant noise in incoherent SAC-OCDMA systems. Results indicate that the SCM OCDMA network performance could be improved by using lower data rates and higher number of weight. Total number of users can also be enhanced by adding lower data rates and higher number of subcarriers.

Electrical detection and quantification of single and mixed DNA nucleotides in suspension

NASA Astrophysics Data System (ADS)

Ahmad, Mahmoud Al; Panicker, Neena G.; Rizvi, Tahir A.; Mustafa, Farah

2016-09-01

High speed sequential identification of the building blocks of DNA, (deoxyribonucleotides or nucleotides for short) without labeling or processing in long reads of DNA is the need of the hour. This can be accomplished through exploiting their unique electrical properties. In this study, the four different types of nucleotides that constitute a DNA molecule were suspended in a buffer followed by performing several types of electrical measurements. These electrical parameters were then used to quantify the suspended DNA nucleotides. Thus, we present a purely electrical counting scheme based on the semiconductor theory that allows one to determine the number of nucleotides in a solution by measuring their capacitance-voltage dependency. The nucleotide count was observed to be similar to the multiplication of the corresponding dopant concentration and debye volume after de-embedding the buffer contribution. The presented approach allows for a fast and label-free quantification of single and mixed nucleotides in a solution.

Time domain multiplexed spatial division multiplexing receiver.

PubMed

van Uden, Roy G H; Okonkwo, Chigo M; Chen, Haoshuo; de Waardt, Hugo; Koonen, Antonius M J

2014-05-19

A novel time domain multiplexed (TDM) spatial division multiplexing (SDM) receiver which allows for the reception of >1 dual polarization mode with a single coherent receiver, and corresponding 4-port oscilloscope, is experimentally demonstrated. Received by two coherent receivers and respective 4-port oscilloscopes, a 3 mode transmission of 28GBaud QPSK, 8, 16, and 32QAM over 41.7km of few-mode fiber demonstrates the performance of the TDM-SDM receiver with respect to back-to-back. In addition, by using carrier phase estimation employing one digital phase locked loop per output, the frequency offset between the transmitter laser and local oscillator is shown to perform similar to previous work which employs 3 coherent receivers and 4-port oscilloscopes which are dedicated to the reception of each the three modes.

Homo-FRET Based Biosensors and Their Application to Multiplexed Imaging of Signalling Events in Live Cells

PubMed Central

Warren, Sean C.; Margineanu, Anca; Katan, Matilda; Dunsby, Chris; French, Paul M. W.

2015-01-01

Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3′-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3′-phosphoinositide accumulation. PMID:26133241

Highly efficient volume hologram multiplexing in thick dye-doped jelly-like gelatin.

PubMed

Katarkevich, Vasili M; Rubinov, Anatoli N; Efendiev, Terlan Sh

2014-08-01

Dye-doped jelly-like gelatin is a thick-layer self-developing photosensitive medium that allows single and multiplexed volume phase holograms to be successfully recorded using pulsed laser radiation. In this Letter, we present a method for multiplexed recording of volume holograms in a dye-doped jelly-like gelatin, which provides significant increase in their diffraction efficiency. The method is based on the recovery of the photobleached dye molecule concentration in the hologram recording zone of gel, thanks to molecule diffusion from other unexposed gel areas. As an example, an optical recording of a multiplexed hologram consisting of three superimposed Bragg gratings with mean values of the diffraction efficiency and angular selectivity of ∼75% and ∼21^', respectively, is demonstrated by using the proposed method.

Final Report Nucleic Acid System - Hybrid PCR and Multiplex Assay Project Phase 2

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

Koopman, R P; Langlois, R G; Nasarabadi, S

2002-04-17

This report covers phase 2 (year 2) of the Nucleic Acid System--Hybrid PCR and Multiplex Assay project. The objective of the project is to reduce to practice the detection and identification of biological warfare pathogens by the nucleic acid recognition technique of PCR (polymerase chain reaction) in a multiplex mode using flow cytometry. The Hybrid instrument consists of a flow-through PCR module capable of handling a multiplexed PCR assay, a hybridizing module capable of hybridizing multiplexed PCR amplicons and beads, and a flow cytometer module for bead-based identification, all controlled by a single computer. Multiplex immunoassay using bead-based Luminex flowmore » cytometry is available, allowing rapid screening for many agents. PCR is highly specific and complements and verifies immunoassay. It can also be multiplexed and detection provided using the bead-based Luminex flow cytometer. This approach allows full access to the speed and 100-fold multiplex capability of flow cytometry for rapid screening as well as the accuracy and specificity of PCR. This project has two principal activities: (1) Design, build and test a prototype hybrid PCR/flow cytometer with the basic capabilities for rapid, broad spectrum detection and identification, and (2) Develop and evaluate multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products. This project requires not only building operationally functional instrumentation but also developing the chemical assays for detection of priority pathogens. This involves development and evaluation of multiplex flow analysis assay protocols and reagents for the simultaneous detection of PCR products.« less

First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome

PubMed Central

Beygo, J.; Buiting, K.; Seland, S.; Lüdecke, H.-J.; Hehr, U.; Lich, C.; Prager, B.; Lohmann, D.R.; Wieczorek, D.

2012-01-01

Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1. PMID:22712005

First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome.

PubMed

Beygo, J; Buiting, K; Seland, S; Lüdecke, H-J; Hehr, U; Lich, C; Prager, B; Lohmann, D R; Wieczorek, D

2012-01-01

Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1.

A comprehensive screen for SNP associations on chromosome region 5q31-33 in Swedish/Norwegian celiac disease families.

PubMed

Amundsen, Silja Svanstrøm; Adamovic, Svetlana; Hellqvist, Asa; Nilsson, Staffan; Gudjónsdóttir, Audur H; Ascher, Henry; Ek, Johan; Larsson, Kristina; Wahlström, Jan; Lie, Benedicte A; Sollid, Ludvig M; Naluai, Asa Torinsson

2007-09-01

Celiac disease (CD) is a gluten-induced enteropathy, which results from the interplay between environmental and genetic factors. There is a strong human leukocyte antigen (HLA) association with the disease, and HLA-DQ alleles represent a major genetic risk factor. In addition to HLA-DQ, non-HLA genes appear to be crucial for CD development. Chromosomal region 5q31-33 has demonstrated linkage with CD in several genome-wide studies, including in our Swedish/Norwegian cohort. In a European meta-analysis 5q31-33 was the only region that reached a genome-wide level of significance except for the HLA region. To identify the genetic variant(s) responsible for this linkage signal, we performed a comprehensive single nucleotide polymorphism (SNP) association screen in 97 Swedish/Norwegian multiplex families who demonstrate linkage to the region. We selected tag SNPs from a 16 Mb region representing the 95% confidence interval of the linkage peak. A total of 1,404 SNPs were used for the association analysis. We identified several regions with SNPs demonstrating moderate single- or multipoint associations. However, the isolated association signals appeared insufficient to account for the linkage signal seen in our cohort. Collective effects of multiple risk genes within the region, incomplete genetic coverage or effects related to copy number variation are possible explanations for our findings.

Differentiation of African components of ancestry to stratify groups in a case-control study of a Brazilian urban population.

PubMed

Silbiger, Vivian N; Hirata, Mario H; Luchessi, Andre D; Genvigir, Fabiana D V; Cerda, Alvaro; Rodrigues, Alice C; Willrich, Maria A V; Arazi, Simone S; Dorea, Egidio L; Bernik, Marcia M S; Faludi, Andre A; Bertolami, Marcelo C; Santos, Carla; Carracedo, Angel; Salas, Antonio; Freire, Ana; Lareu, Maria Victoria; Phillips, Christopher; Porras-Hurtado, Liliana; Fondevila, Manuel; Hirata, Rosario D C

2012-06-01

Balancing the subject composition of case and control groups to create homogenous ancestries between each group is essential for medical association studies. We explored the applicability of single-tube 34-plex ancestry informative markers (AIM) single nucleotide polymorphisms (SNPs) to estimate the African Component of Ancestry (ACA) to design a future case-control association study of a Brazilian urban sample. One hundred eighty individuals (107 case group; 73 control group) self-described as white, brown-intermediate or black were selected. The proportions of the relative contribution of a variable number of ancestral population components were similar between case and control groups. Moreover, the case and control groups demonstrated similar distributions for ACA <0.25 and >0.50 categories. Notably a high number of outlier values (23 samples) were observed among individuals with ACA <0.25. These individuals presented a high probability of Native American and East Asian ancestral components; however, no individuals originally giving these self-described ancestries were observed in this study. The strategy proposed for the assessment of ancestry and adjustment of case and control groups for an association study is an important step for the proper construction of the study, particularly when subjects are taken from a complex urban population. This can be achieved using a straight forward multiplexed AIM-SNPs assay of highly discriminatory ancestry markers.

Y-chromosomal testing of brown bears (Ursus arctos): Validation of a multiplex PCR-approach for nine STRs suitable for fecal and hair samples.

PubMed

Aarnes, Siv Grethe; Hagen, Snorre B; Andreassen, Rune; Schregel, Julia; Knappskog, Per M; Hailer, Frank; Stenhouse, Gordon; Janke, Axel; Eiken, Hans Geir

2015-11-01

High-resolution Y-chromosomal markers have been applied to humans and other primates to study population genetics, migration, social structures and reproduction. Y-linked markers allow the direct assessment of the genetic structure and gene flow of uniquely male inherited lineages and may also be useful for wildlife conservation and forensics, but have so far been available only for few wild species. Thus, we have developed two multiplex PCR reactions encompassing nine Y-STR markers identified from the brown bear (Ursus arctos) and tested them on hair, fecal and tissue samples. The multiplex PCR approach was optimized and analyzed for species specificity, sensitivity and stutter-peak ratios. The nine Y-STRs also showed specific STR-fragments for male black bears and male polar bears, while none of the nine markers produced any PCR products when using DNA from female bears or males from 12 other mammals. The multiplex PCR approach in two PCR reactions could be amplified with as low as 0.2 ng template input. Precision was high in DNA templates from hairs, fecal scats and tissues, with standard deviations less than 0.14 and median stutter ratios from 0.04 to 0.63. Among the eight di- and one tetra-nucleotide repeat markers, we detected simple repeat structures in seven of the nine markers with 9-25 repeat units. Allelic variation was found for eight of the nine Y-STRs, with 2-9 alleles for each marker and a total of 36 alleles among 453 male brown bears sampled mainly from Northern Europe. We conclude that the multiplex PCR approach with these nine Y-STRs would provide male bear Y-chromosomal specificity and evidence suited for samples from conservation and wildlife forensics. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Highly multiplexed signal readout for a time-of-flight positron emission tomography detector based on silicon photomultipliers

PubMed Central

Cates, Joshua W.; Bieniosek, Matthew F.; Levin, Craig S.

2017-01-01

Abstract. Maintaining excellent timing resolution in the generation of silicon photomultiplier (SiPM)-based time-of-flight positron emission tomography (TOF-PET) systems requires a large number of high-speed, high-bandwidth electronic channels and components. To minimize the cost and complexity of a system’s back-end architecture and data acquisition, many analog signals are often multiplexed to fewer channels using techniques that encode timing, energy, and position information. With progress in the development SiPMs having lower dark noise, after pulsing, and cross talk along with higher photodetection efficiency, a coincidence timing resolution (CTR) well below 200 ps FWHM is now easily achievable in single pixel, bench-top setups using 20-mm length, lutetium-based inorganic scintillators. However, multiplexing the output of many SiPMs to a single channel will significantly degrade CTR without appropriate signal processing. We test the performance of a PET detector readout concept that multiplexes 16 SiPMs to two channels. One channel provides timing information with fast comparators, and the second channel encodes both position and energy information in a time-over-threshold-based pulse sequence. This multiplexing readout concept was constructed with discrete components to process signals from a 4×4 array of SensL MicroFC-30035 SiPMs coupled to 2.9×2.9×20  mm3 Lu1.8Gd0.2SiO5 (LGSO):Ce (0.025 mol. %) scintillators. This readout method yielded a calibrated, global energy resolution of 15.3% FWHM at 511 keV with a CTR of 198±2  ps FWHM between the 16-pixel multiplexed detector array and a 2.9×2.9×20  mm3 LGSO-SiPM reference detector. In summary, results indicate this multiplexing scheme is a scalable readout technique that provides excellent coincidence timing performance. PMID:28382312

Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis

PubMed Central

Carver-Brown, Rachel K.; Reis, Arthur H.; Rice, Lisa M.; Czajka, John W.; Wangh, Lawrence J.

2012-01-01

Aims. The goal of this study was to construct a single tube molecular diagnostic multiplex assay for the detection of microbial pathogens commonly associated with septicemia, using LATE-PCR and Lights-On/Lights-Off probe technology. Methods and Results. The assay described here identified pathogens associated with sepsis by amplification and analysis of the 16S ribosomal DNA gene sequence for bacteria and specific gene sequences for fungi. A sequence from an unidentified gene in Lactococcus lactis subsp. cremoris served as a positive control for assay function. LATE-PCR was used to generate single-stranded amplicons that were then analyzed at endpoint over a wide temperature range in a specific fluorescent color. Each bacterial target was identified by its pattern of hybridization to Lights-On/Lights-Off probes derived from molecular beacons. Complex mixtures of targets were also detected. Conclusions. All microbial targets were identified in samples containing low starting copy numbers of pathogen genomic DNA, both as individual targets and in complex mixtures. Significance and Impact of the Study. This assay uses new technology to achieve an advance in the field of molecular diagnostics: a single-tube multiplex assay for identification of pathogens commonly associated with sepsis. PMID:23326668

On-chip single photon filtering and multiplexing in hybrid quantum photonic circuits.

PubMed

Elshaari, Ali W; Zadeh, Iman Esmaeil; Fognini, Andreas; Reimer, Michael E; Dalacu, Dan; Poole, Philip J; Zwiller, Val; Jöns, Klaus D

2017-08-30

Quantum light plays a pivotal role in modern science and future photonic applications. Since the advent of integrated quantum nanophotonics different material platforms based on III-V nanostructures-, colour centers-, and nonlinear waveguides as on-chip light sources have been investigated. Each platform has unique advantages and limitations; however, all implementations face major challenges with filtering of individual quantum states, scalable integration, deterministic multiplexing of selected quantum emitters, and on-chip excitation suppression. Here we overcome all of these challenges with a hybrid and scalable approach, where single III-V quantum emitters are positioned and deterministically integrated in a complementary metal-oxide-semiconductor-compatible photonic circuit. We demonstrate reconfigurable on-chip single-photon filtering and wavelength division multiplexing with a foot print one million times smaller than similar table-top approaches, while offering excitation suppression of more than 95 dB and efficient routing of single photons over a bandwidth of 40 nm. Our work marks an important step to harvest quantum optical technologies' full potential.Combining different integration platforms on the same chip is currently one of the main challenges for quantum technologies. Here, Elshaari et al. show III-V Quantum Dots embedded in nanowires operating in a CMOS compatible circuit, with controlled on-chip filtering and tunable routing.

On-chip multiplexed solid-phase nucleic acid hybridization assay using spatial profiles of immobilized quantum dots and fluorescence resonance energy transfer.

PubMed

Noor, M Omair; Tavares, Anthony J; Krull, Ulrich J

2013-07-25

A microfluidic based solid-phase assay for the multiplexed detection of nucleic acid hybridization using quantum dot (QD) mediated fluorescence resonance energy transfer (FRET) is described herein. The glass surface of hybrid glass-polydimethylsiloxane (PDMS) microfluidic channels was chemically modified to assemble the biorecognition interface. Multiplexing was demonstrated using a detection system that was comprised of two colors of immobilized semi-conductor QDs and two different oligonucleotide probe sequences. Green-emitting and red-emitting QDs were paired with Cy3 and Alexa Fluor 647 (A647) labeled oligonucleotides, respectively. The QDs served as energy donors for the transduction of dye labeled oligonucleotide targets. The in-channel assembly of the biorecognition interface and the subsequent introduction of oligonucleotide targets was accomplished within minutes using a combination of electroosmotic flow and electrophoretic force. The concurrent quantification of femtomole quantities of two target sequences was possible by measuring the spatial coverage of FRET sensitized emission along the length of the channel. In previous reports, multiplexed QD-FRET hybridization assays that employed a ratiometric method for quantification had challenges associated with lower analytical sensitivity arising from both donor and acceptor dilution that resulted in reduced energy transfer pathways as compared to single-color hybridization assays. Herein, a spatial method for quantification that is based on in-channel QD-FRET profiles provided higher analytical sensitivity in the multiplexed assay format as compared to single-color hybridization assays. The selectivity of the multiplexed hybridization assays was demonstrated by discrimination between a fully-complementary sequence and a 3 base pair sequence at a contrast ratio of 8 to 1. Copyright © 2013 Elsevier B.V. All rights reserved.

Multiplex real-time PCR for the detection and quantification of latent and persistent viral genomes in cellular or plasma blood fractions.

PubMed

Compston, Lara Isobel; Sarkobie, Francis; Li, Chengyao; Candotti, Daniel; Opare-Sem, Ohene; Allain, Jean-Pierre

2008-07-01

In common with latent viruses such as herpesviruses, parvovirus B19, HBV and GBV-C are contained successfully by the immune response and persist in the host. When immune control breaks down, reactivation of both latent and persistent viruses occurs. Two multiplex assays were developed (B19, HBV, HHV-8), (EBV, CMV, VZV) for blood screening, and tested on blood donor samples from Ghana to determine baseline prevalence of viraemia in immunocompetent persons. Single-virus real-time quantitative PCR (qPCR) assays were optimised for viral load determination of positive initial screening. The qPCR method utilised was absolute quantification with external standards. Multiplex and single-virus qPCR assays had similar sensitivity, except for the B19 assay in which sensitivity was 100-fold lower. Assays were optimised for reproducibility and repeatability, with R(2) of 0.9 being obtained for most assays. With the exception of B19 and CMV, assays had 100% detection limit ranging between 10(1) and 10(2) copies, IU or arbitrary units under single-virus and multiplex assay conditions. The prevalence of viraemia was 1.6% HBV (0.8% DNA+/HBsAg-, 0.8% DNA+/HBsAg+), 0.8% parvovirus B19, and 3.3% GBV-C viraemia in the plasma fraction. The prevalence of four herpesviruses was 1.0% HHV-8, 0.85% CMV, and 8.3% EBV, and no detectable VZV viraemia.

All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.

PubMed

Sakuma, Tetsushi; Sakamoto, Takuya; Yamamoto, Takashi

2017-01-01

CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.

Aqueous two-phase systems enable multiplexing of homogeneous immunoassays

PubMed Central

Simon, Arlyne B.; Frampton, John P.; Huang, Nien-Tsu; Kurabayashi, Katsuo; Paczesny, Sophie; Takayama, Shuichi

2014-01-01

Quantitative measurement of protein biomarkers is critical for biomarker validation and early disease detection. Current multiplex immunoassays are time consuming costly and can suffer from low accuracy. For example, multiplex ELISAs require multiple, tedious, washing and blocking steps. Moreover, they suffer from nonspecific antibody cross-reactions, leading to high background and false-positive signals. Here, we show that co-localizing antibody-bead pairs in an aqueous two-phase system (ATPS) enables multiplexing of sensitive, no-wash, homogeneous assays, while preventing nonspecific antibody cross-reactions. Our cross-reaction-free, multiplex assay can simultaneously detect picomolar concentrations of four protein biomarkers ((C-X-C motif) ligand 10 (CXCL10), CXCL9, interleukin (IL)-8 and IL-6) in cell supernatants using a single assay well. The potential clinical utility of the assay is demonstrated by detecting diagnostic biomarkers (CXCL10 and CXCL9) in plasma from 88 patients at the onset of the clinical symptoms of chronic graft-versus-host disease (GVHD). PMID:25083509

Detection of α-thalassemia-1 Southeast Asian and Thai Type Deletions and β-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis

PubMed Central

Wiengkum, Thanatcha; Srithep, Sarinee; Chainoi, Isarapong; Singboottra, Panthong; Wongwiwatthananukit, Sanchai

2011-01-01

Background Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. Methods Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and β-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous β-thalassemia 3.5-kb gene deletion. Results HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, β-thalassemia 3.5-kb gene deletion, and the wild-type β-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. Conclusions Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and β-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate. PMID:21779184

Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection.

PubMed

Kacmarczyk, Thadeous J; Bourque, Caitlin; Zhang, Xihui; Jiang, Yanwen; Houvras, Yariv; Alonso, Alicia; Betel, Doron

2015-01-01

Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal.

Multiplexing of ChIP-Seq Samples in an Optimized Experimental Condition Has Minimal Impact on Peak Detection

PubMed Central

Kacmarczyk, Thadeous J.; Bourque, Caitlin; Zhang, Xihui; Jiang, Yanwen; Houvras, Yariv; Alonso, Alicia; Betel, Doron

2015-01-01

Multiplexing samples in sequencing experiments is a common approach to maximize information yield while minimizing cost. In most cases the number of samples that are multiplexed is determined by financial consideration or experimental convenience, with limited understanding on the effects on the experimental results. Here we set to examine the impact of multiplexing ChIP-seq experiments on the ability to identify a specific epigenetic modification. We performed peak detection analyses to determine the effects of multiplexing. These include false discovery rates, size, position and statistical significance of peak detection, and changes in gene annotation. We found that, for histone marker H3K4me3, one can multiplex up to 8 samples (7 IP + 1 input) at ~21 million single-end reads each and still detect over 90% of all peaks found when using a full lane for sample (~181 million reads). Furthermore, there are no variations introduced by indexing or lane batch effects and importantly there is no significant reduction in the number of genes with neighboring H3K4me3 peaks. We conclude that, for a well characterized antibody and, therefore, model IP condition, multiplexing 8 samples per lane is sufficient to capture most of the biological signal. PMID:26066343

Single-Cell, Multiplexed Protein Detection of Rare Tumor Cells Based on a Beads-on-Barcode Antibody Microarray.

PubMed

Yang, Liu; Wang, Zhihua; Deng, Yuliang; Li, Yan; Wei, Wei; Shi, Qihui

2016-11-15

Circulating tumor cells (CTCs) shed from tumor sites and represent the molecular characteristics of the tumor. Besides genetic and transcriptional characterization, it is important to profile a panel of proteins with single-cell precision for resolving CTCs' phenotype, organ-of-origin, and drug targets. We describe a new technology that enables profiling multiple protein markers of extraordinarily rare tumor cells at the single-cell level. This technology integrates a microchip consisting of 15000 60 pL-sized microwells and a novel beads-on-barcode antibody microarray (BOBarray). The BOBarray allows for multiplexed protein detection by assigning two independent identifiers (bead size and fluorescent color) of the beads to each protein. Four bead sizes (1.75, 3, 4.5, and 6 μm) and three colors (blue, green, and yellow) are utilized to encode up to 12 different proteins. The miniaturized BOBarray can fit an array of 60 pL-sized microwells that isolate single cells for cell lysis and the subsequent detection of protein markers. An enclosed 60 pL-sized microchamber defines a high concentration of proteins released from lysed single cells, leading to single-cell resolution of protein detection. The protein markers assayed in this study include organ-specific markers and drug targets that help to characterize the organ-of-origin and drug targets of isolated rare tumor cells from blood samples. This new approach enables handling a very small number of cells and achieves single-cell, multiplexed protein detection without loss of rare but clinically important tumor cells.

Microgels for multiplex and direct fluorescence detection

NASA Astrophysics Data System (ADS)

Causa, Filippo; Aliberti, Anna; Cusano, Angela M.; Battista, Edmondo; Netti, Paolo A.

2015-05-01

Blood borne oligonucleotides fragments contain useful clinical information whose detection and monitoring represent the new frontier in liquid biopsy as they can transform the current diagnosis procedure. For instance, recent studies have identified a new class of circulating biomarkers such as s miRNAs, and demonstrated that changes in their concentration are closely associated with the development of cancer and other pathologies. However, direct detection of miRNAs in body fluids is particularly challenging and demands high sensitivity -concentration range between atto to femtomolarspecificity, and multiplexing Here we report on engineered multifunctional microgels and innovative probe design for a direct and multiplex detection of relevant clinical miRNAs in fluorescence by single particle assay. Polyethyleneglycol-based microgels have a coreshell architecture with two spectrally encoded fluorescent dyes for multiplex analyses and are endowed with fluorescent probes for miRNA detection. Encoding and detection fluorescence signals are distinguishable by not overlapping emission spectra. Tuneable fluorescence probe conjugation and corresponding emission confinement on single microgel allows for enhanced target detection. Such suspension array has indeed high selectivity and sensitivity with a detection limit of 10-15 M and a dynamic range from 10-9 to 10-15 M. We believe that sensitivity in the fM concentration range, signal background minimization, multiplexed capability and direct measurement of such microgels will translate into diagnostic benefits opening up new roots toward liquid biopsy in the context of point-of-care testing through an easy and fast detection of sensitive diagnostic biomarkers directly in serum.

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.

PCR/LDR/capillary electrophoresis for detection of single-nucleotide differences between fetal and maternal DNA in maternal plasma.

PubMed

Yi, Ping; Chen, Zhuqin; Zhao, Yan; Guo, Jianxin; Fu, Huabin; Zhou, Yuanguo; Yu, Lili; Li, Li

2009-03-01

The discovery of fetal DNA in maternal plasma has opened up an approach for noninvasive diagnosis. We have now assessed the possibility of detecting single-nucleotide differences between fetal and maternal DNA in maternal plasma by polymerase chain reaction (PCR)/ligase detection reaction((LDR)/capillary electrophoresis. PCR/LDR/capillary electrophoresis was applied to detect the genotype of c.454-397T>gene (ESR1) from experimental DNA models of maternal plasma at different sensitivity levels and 13 maternal plasma samples.alphaC in estrogen receptor. (1) Our results demonstrated that the technique could discriminate low abundance single-nucleotide mutation with a mutant/normal allele ratio up to 1:10 000. (2) Examination of ESR1 c.454-397T>C genotypes by using the method of restriction fragment length analysis was performed in 25 pregnant women, of whom 13 pregnant women had homozygous genotypes. The c.454-397T>C genotypes of paternally inherited fetal DNA in maternal plasma of these 13 women were detected by PCR/LDR/capillary electrophoresis, which were accordant with the results of umbilical cord blood. PCR/LDR/capillary electrophoresis has very high sensitivity to distinguish low abundance single nucleotide differences and can discriminate point mutations and single-nucleotide polymorphisms(SNPs) of paternally inherited fetal DNA in maternal plasma.

Multiplex PageRank.

PubMed

Halu, Arda; Mondragón, Raúl J; Panzarasa, Pietro; Bianconi, Ginestra

2013-01-01

Many complex systems can be described as multiplex networks in which the same nodes can interact with one another in different layers, thus forming a set of interacting and co-evolving networks. Examples of such multiplex systems are social networks where people are involved in different types of relationships and interact through various forms of communication media. The ranking of nodes in multiplex networks is one of the most pressing and challenging tasks that research on complex networks is currently facing. When pairs of nodes can be connected through multiple links and in multiple layers, the ranking of nodes should necessarily reflect the importance of nodes in one layer as well as their importance in other interdependent layers. In this paper, we draw on the idea of biased random walks to define the Multiplex PageRank centrality measure in which the effects of the interplay between networks on the centrality of nodes are directly taken into account. In particular, depending on the intensity of the interaction between layers, we define the Additive, Multiplicative, Combined, and Neutral versions of Multiplex PageRank, and show how each version reflects the extent to which the importance of a node in one layer affects the importance the node can gain in another layer. We discuss these measures and apply them to an online multiplex social network. Findings indicate that taking the multiplex nature of the network into account helps uncover the emergence of rankings of nodes that differ from the rankings obtained from one single layer. Results provide support in favor of the salience of multiplex centrality measures, like Multiplex PageRank, for assessing the prominence of nodes embedded in multiple interacting networks, and for shedding a new light on structural properties that would otherwise remain undetected if each of the interacting networks were analyzed in isolation.

Fiber optic multiplexed optical transmission systems for space vehicle launch facilities

NASA Technical Reports Server (NTRS)

Bell, C. H.

1975-01-01

Low loss Fiber Optic Cable is being evaluated as a potential future replacement for Kennedy Space Center's 13,000 mile Wideband cable system. In order to make economical use of the wide bandwidth characteristic of glass fibers, a Frequency Division Multiplexing (FDM) scheme has been devised to stack many analog and digital data channels on a single fiber. The Multiplexed Optical Transmission System (MOTS) will offer a unique flexibility of plug-in modularity to meet changing data and bandwidth requirements in addition to the standard 'goodies' of immunity to lightning and other EMI, RFI type interferences, and of smaller size and lighter weight.

The Application of Fiber Optic Wavelength Division Multiplexing in RF Avionics

NASA Technical Reports Server (NTRS)

Ngo, Duc; Nguyen, Hung; Atiquzzaman, Mohammed; Sluss, James J., Jr.; Refai, Hakki H.

2004-01-01

This paper demonstrates a successful application of wavelength division multiplexing (WDM) to the avionics environment to support analog RF signal transmission. We investigate the simultaneous transmission of four RF signals (channels) over a single optical fiber. These four analog channels are sequentially multiplexed and demultiplexed at different points along a fiber optic backbone to more closely emulate the conditions found onboard aircraft. We present data from measurements of signal-to-noise ratio (SNR), transmission response (loss and gain), group delay that defines phase distortion, and dynamic range that defines nonlinear distortion. The data indicate that WDM is well-suited for avionics applications.

A polarization-division multiplexing SSB-OFDM system with beat interference cancellation receivers

NASA Astrophysics Data System (ADS)

Yang, Peiling; Ma, Jianxin; Zhang, Junyi

2018-06-01

In this paper, we have proposed a polarization-division multiplexing (PDM) single-sideband optical orthogonal frequency division multiplexing (SSB-OOFDM) scheme with signal-signal beat interference cancellation receivers with balanced detection (ICRBD). This system can double channel capacity and improve spectrum efficiency (SE) with the reduced guard band (GB) due to the PDM. Multiple input multiple output (MIMO) technique is used to solve polarization mode dispersion (PMD) associated with channel estimation and equalization. By simulation, we demonstrate the efficacy of the proposed technique for a 2 ×40 Gbit/s 16-QAM SSB-PDM-OOFDM system according to the error vector magnitude (EVM) and the constellation diagrams.

xMAP Technology: Applications in Detection of Pathogens

PubMed Central

Reslova, Nikol; Michna, Veronika; Kasny, Martin; Mikel, Pavel; Kralik, Petr

2017-01-01

xMAP technology is applicable for high-throughput, multiplex and simultaneous detection of different analytes within a single complex sample. xMAP multiplex assays are currently available in various nucleic acid and immunoassay formats, enabling simultaneous detection and typing of pathogenic viruses, bacteria, parasites and fungi and also antigen or antibody interception. As an open architecture platform, the xMAP technology is beneficial to end users and therefore it is used in various pharmaceutical, clinical and research laboratories. The main aim of this review is to summarize the latest findings and applications in the field of pathogen detection using microsphere-based multiplex assays. PMID:28179899

Multiplex mass spectrometry imaging for latent fingerprints.

PubMed

Yagnik, Gargey B; Korte, Andrew R; Lee, Young Jin

2013-01-01

We have previously developed in-parallel data acquisition of orbitrap mass spectrometry (MS) and ion trap MS and/or MS/MS scans for matrix-assisted laser desorption/ionization MS imaging (MSI) to obtain rich chemical information in less data acquisition time. In the present study, we demonstrate a novel application of this multiplex MSI methodology for latent fingerprints. In a single imaging experiment, we could obtain chemical images of various endogenous and exogenous compounds, along with simultaneous MS/MS images of a few selected compounds. This work confirms the usefulness of multiplex MSI to explore chemical markers when the sample specimen is very limited. Copyright © 2013 John Wiley & Sons, Ltd.

High performance architecture design for large scale fibre-optic sensor arrays using distributed EDFAs and hybrid TDM/DWDM

NASA Astrophysics Data System (ADS)

Liao, Yi; Austin, Ed; Nash, Philip J.; Kingsley, Stuart A.; Richardson, David J.

2013-09-01

A distributed amplified dense wavelength division multiplexing (DWDM) array architecture is presented for interferometric fibre-optic sensor array systems. This architecture employs a distributed erbium-doped fibre amplifier (EDFA) scheme to decrease the array insertion loss, and employs time division multiplexing (TDM) at each wavelength to increase the number of sensors that can be supported. The first experimental demonstration of this system is reported including results which show the potential for multiplexing and interrogating up to 4096 sensors using a single telemetry fibre pair with good system performance. The number can be increased to 8192 by using dual pump sources.

Simultaneous Genotyping of the rs4762 and rs699 Polymorphisms in Angiotensinogen Gene and Correlation with Iranian CAD Patients with Novel Hexa-primer ARMS-PCR

PubMed Central

KHATAMI, Mehri; HEIDARI, Mohammad Mehdi; HADADZADEH, Mehdi; SCHEIBER-MOJDEHKAR, Barbara; BITARAF SANI, Morteza; HOUSHMAND, Massoud

2017-01-01

Background: A significant role of Renin-angiotensin system (RAS) genetic variants in the pathogenesis of essential hypertension and cardiovascular diseases has been proved. This study aimed to develop a new, fast and cheap method for the simultaneous detection of two missense single nucleotide polymorphisms (T207M or rs4762 and M268T orrs699) of angiotensinogen (AGT) in single-step Multiplex Hexa-Primer Amplification Refractory Mutation System - polymerase chain reaction (H-ARMS-PCR). Methods: In this case-control study, 148 patients with coronary artery disease (CAD) and 135 controls were included. The patients were referred to cardiac centers in Afshar Hospital (Yazd, Iran) from 2012 to 2015. Two sets of inner primer (for each SNP) and one set outer primer pairs were designed for genotyping of rs4762 and rs699 in single tube H-ARMS-PCR. Direct sequencing of all samples was also performed to assess the accuracy of this method. DNA sequencing method validated the results of single tube H-ARMS-PCR. Results: We found full accordance for genotype adscription by sequencing method. The frequency of the AGT T521 and C702 alleles was significantly higher in CAD patients than in the control group (OR: 0.551, 95% CI: 0.359–0.846, P=0.008 and OR: 0.629, 95% CI: 0.422–0.936, P=0.028, respectively). Conclusion: This is the first work describing a rapid, low-cost, high-throughput simultaneous detection of rs4762 and rs699 polymorphisms in AGT gene, used in large clinical studies. PMID:28828324

Multiplexing Superconducting Qubit Circuit for Single Microwave Photon Generation

NASA Astrophysics Data System (ADS)

George, R. E.; Senior, J.; Saira, O.-P.; Pekola, J. P.; de Graaf, S. E.; Lindström, T.; Pashkin, Yu A.

2017-10-01

We report on a device that integrates eight superconducting transmon qubits in λ /4 superconducting coplanar waveguide resonators fed from a common feedline. Using this multiplexing architecture, each resonator and qubit can be addressed individually, thus reducing the required hardware resources and allowing their individual characterisation by spectroscopic methods. The measured device parameters agree with the designed values, and the resonators and qubits exhibit excellent coherence properties and strong coupling, with the qubit relaxation rate dominated by the Purcell effect when brought in resonance with the resonator. Our analysis shows that the circuit is suitable for generation of single microwave photons on demand with an efficiency exceeding 80%.

Self-homodyne optical OFDM for broadband WDM-PONs with crosstalk-free remodulation and enhanced tolerance to Rayleigh noise

NASA Astrophysics Data System (ADS)

Lyu, WeiChao; Wang, Andong; Xie, Dequan; Zhu, Long; Guan, Xun; Wang, Jian; Xu, Jing

2018-05-01

We propose a novel architecture for wavelength-division-multiplexed passive optical network (WDM-PON) that can simultaneously circumvent both remodulation crosstalk and Rayleigh noise, based on self-homodyne detection and optical orthogonal frequency-division multiplexing (OFDM) remodulation. The proposed self-homodyne detection at optical network unit (ONU) requires neither frequency offset compensation nor phase noise compensation, and thus can significantly reduce system complexity and power consumption. Bidirectional transmission of 12.5 Gb/s down- and up-stream signals, via single 25 km single-mode fiber without dispersion compensation, is demonstrated in a proof-of-concept experiment.

Bidirectional and simultaneous FTTX/Ethernet services using RSOA based remodulation and polarization multiplexing technique

NASA Astrophysics Data System (ADS)

Das, Anindya S.; Patra, Ardhendu S.

2015-08-01

A bidirectional and simultaneous transmission of Ethernet, FTTX services through single optical carrier wavelength employing polarization multiplexing technique in the transmitter end and the user end. 10 Gbps and 2.5 Gbps datarates are transmitted over 50 km single mode fiber employing POLMUX technique at OLT and ONU to provide Ethernet and FTTX services concurrently to the user. Reflective semiconductor optical amplifier is used to reuse and remodulate the downlink signal to uplink transmission. The upstream and the downstream transmission performances are observed by the bit error rate values and the eye diagrams obtained by the BER analyzer.

Single-sideband modulator for frequency domain multiplexing of superconducting qubit readout

NASA Astrophysics Data System (ADS)

Chapman, Benjamin J.; Rosenthal, Eric I.; Kerckhoff, Joseph; Vale, Leila R.; Hilton, Gene C.; Lehnert, K. W.

2017-04-01

We introduce and experimentally characterize a superconducting single-sideband modulator compatible with cryogenic microwave circuits and propose its use for frequency domain multiplexing of superconducting qubit readout. The monolithic double-balanced modulators that comprise the device are formed with purely reactive elements (capacitors and Josephson junction inductors) and require no microwave-frequency control tones. Microwave signals in the 4 to 8 GHz band, with power up to -85 dBm, are converted up or down in frequency by as much as 120 MHz. Spurious harmonics in the device can be suppressed by up to 25 dB for select probe and modulation frequencies.

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser-induced fluorescence detection.

PubMed

Nikcevic, Irena; Piruska, Aigars; Wehmeyer, Kenneth R; Seliskar, Carl J; Limbach, Patrick A; Heineman, William R

2010-08-01

Parallel separations using CE on a multilane microchip with multiplexed LIF detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be determined in parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pK(a) determination of small molecule analytes is demonstrated with the multilane microchip.

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser induced fluorescence detection

PubMed Central

Nikcevic, Irena; Piruska, Aigars; Wehmeyer, Kenneth R.; Seliskar, Carl J.; Limbach, Patrick A.; Heineman, William R.

2010-01-01

Parallel separations using capillary electrophoresis on a multilane microchip with multiplexed laser induced fluorescence detection is demonstrated. The detection system was developed to simultaneously record data on all channels using an expanded laser beam for excitation, a camera lens to capture emission, and a CCD camera for detection. The detection system enables monitoring of each channel continuously and distinguishing individual lanes without significant crosstalk between adjacent lanes. Multiple analytes can be analyzed on parallel lanes within a single microchip in a single run, leading to increased sample throughput. The pKa determination of small molecule analytes is demonstrated with the multilane microchip. PMID:20737446

Ultrasensitive NIR-SERRS Probes with Multiplexed Ratiometric Quantification for In Vivo Antibody Leads Validation.

PubMed

Kang, Homan; Jeong, Sinyoung; Jo, Ahla; Chang, Hyejin; Yang, Jin-Kyoung; Jeong, Cheolhwan; Kyeong, San; Lee, Youn Woo; Samanta, Animesh; Maiti, Kaustabh Kumar; Cha, Myeong Geun; Kim, Taek-Keun; Lee, Sukmook; Jun, Bong-Hyun; Chang, Young-Tae; Chung, Junho; Lee, Ho-Young; Jeong, Dae Hong; Lee, Yoon-Sik

2018-02-01

Immunotargeting ability of antibodies may show significant difference between in vitro and in vivo. To select antibody leads with high affinity and specificity, it is necessary to perform in vivo validation of antibody candidates following in vitro antibody screening. Herein, a robust in vivo validation of anti-tetraspanin-8 antibody candidates against human colon cancer using ratiometric quantification method is reported. The validation is performed on a single mouse and analyzed by multiplexed surface-enhanced Raman scattering using ultrasensitive and near infrared (NIR)-active surface-enhanced resonance Raman scattering nanoprobes (NIR-SERRS dots). The NIR-SERRS dots are composed of NIR-active labels and Au/Ag hollow-shell assembled silica nanospheres. A 93% of NIR-SERRS dots is detectable at a single-particle level and signal intensity is 100-fold stronger than that from nonresonant molecule-labeled spherical Au NPs (80 nm). The result of SERRS-based antibody validation is comparable to that of the conventional method using single-photon-emission computed tomography. The NIR-SERRS-based strategy is an alternate validation method which provides cost-effective and accurate multiplexing measurements for antibody-based drug development. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Aspergillus and Penicillium identification using DNA sequences: Barcode or MLST?

USDA-ARS?s Scientific Manuscript database

Current methods in DNA technology can detect single nucleotide polymorphisms with measurable accuracy using several different approaches appropriate for different uses. If there are even single nucleotide differences that are invariant markers of the species, we can accomplish identification through...

A new OTDR based on probe frequency multiplexing

NASA Astrophysics Data System (ADS)

Lu, Lidong; Liang, Yun; Li, Binglin; Guo, Jinghong; Zhang, Xuping

2013-12-01

Two signal multiplexing methods are proposed and experimentally demonstrated in optical time domain reflectometry (OTDR) for fault location of optical fiber transmission line to obtain high measurement efficiency. Probe signal multiplexing is individually obtained by phase modulation for generation of multi-frequency and time sequential frequency probe pulses. The backscattered Rayleigh light of the multiplexing probe signals is transferred to corresponding heterodyne intermediate frequency (IF) through heterodyning with the single frequency local oscillator (LO). Then the IFs are simultaneously acquired by use of a data acquisition card (DAQ) with sampling rate of 100Msps, and the obtained data are processed by digital band pass filtering (BPF), digital down conversion (DDC) and digital low pass filtering (BPF) procedure. For each probe frequency of the detected signals, the extraction of the time domain reflecting signal power is performed by parallel computing method. For a comprehensive performance comparison with conventional coherent OTDR on the probe frequency multiplexing methods, the potential for enhancement of dynamic range, spatial resolution and measurement time are analyzed and discussed. Experimental results show that by use of the probe frequency multiplexing method, the measurement efficiency of coherent OTDR can be enhanced by nearly 40 times.

Bitterness of the Non-nutritive Sweetener Acesulfame Potassium Varies With Polymorphisms in TAS2R9 and TAS2R31

PubMed Central

2013-01-01

Demand for nonnutritive sweeteners continues to increase due to their ability to provide desirable sweetness with minimal calories. Acesulfame potassium and saccharin are well-studied nonnutritive sweeteners commonly found in food products. Some individuals report aversive sensations from these sweeteners, such as bitter and metallic side tastes. Recent advances in molecular genetics have provided insight into the cause of perceptual differences across people. For example, common alleles for the genes TAS2R9 and TAS2R38 explain variable response to the bitter drugs ofloxacin in vitro and propylthiouracil in vivo. Here, we wanted to determine whether differences in the bitterness of acesulfame potassium could be predicted by common polymorphisms (genetic variants) in bitter taste receptor genes (TAS2Rs). We genotyped participants (n = 108) for putatively functional single nucleotide polymorphisms in 5 TAS2Rs and asked them to rate the bitterness of 25 mM acesulfame potassium on a general labeled magnitude scale. Consistent with prior reports, we found 2 single nucleotide polymorphisms in TAS2R31 were associated with acesulfame potassium bitterness. However, TAS2R9 alleles also predicted additional variation in acesulfame potassium bitterness. Conversely, single nucleotide polymorphisms in TAS2R4, TAS2R38, and near TAS2R16 were not significant predictors. Using 1 single nucleotide polymorphism each from TAS2R9 and TAS2R31, we modeled the simultaneous influence of these single nucleotide polymorphisms on acesulfame potassium bitterness; together, these 2 single nucleotide polymorphisms explained 13.4% of the variance in perceived bitterness. These data suggest multiple polymorphisms within TAS2Rs contribute to the ability to perceive the bitterness from acesulfame potassium. PMID:23599216

Nucleotide cleaving agents and method

DOEpatents

Que, Jr., Lawrence; Hanson, Richard S.; Schnaith, Leah M. T.

2000-01-01

The present invention provides a unique series of nucleotide cleaving agents and a method for cleaving a nucleotide sequence, whether single-stranded or double-stranded DNA or RNA, using and a cationic metal complex having at least one polydentate ligand to cleave the nucleotide sequence phosphate backbone to yield a hydroxyl end and a phosphate end.

Integrated cladding-pumped multicore few-mode erbium-doped fibre amplifier for space-division-multiplexed communications

NASA Astrophysics Data System (ADS)

Chen, H.; Jin, C.; Huang, B.; Fontaine, N. K.; Ryf, R.; Shang, K.; Grégoire, N.; Morency, S.; Essiambre, R.-J.; Li, G.; Messaddeq, Y.; Larochelle, S.

2016-08-01

Space-division multiplexing (SDM), whereby multiple spatial channels in multimode and multicore optical fibres are used to increase the total transmission capacity per fibre, is being investigated to avert a data capacity crunch and reduce the cost per transmitted bit. With the number of channels employed in SDM transmission experiments continuing to rise, there is a requirement for integrated SDM components that are scalable. Here, we demonstrate a cladding-pumped SDM erbium-doped fibre amplifier (EDFA) that consists of six uncoupled multimode erbium-doped cores. Each core supports three spatial modes, which enables the EDFA to amplify a total of 18 spatial channels (six cores × three modes) simultaneously with a single pump diode and a complexity similar to a single-mode EDFA. The amplifier delivers >20 dBm total output power per core and <7 dB noise figure over the C-band. This cladding-pumped EDFA enables combined space-division and wavelength-division multiplexed transmission over multiple multimode fibre spans.

Multiplexed, High Density Electrophysiology with Nanofabricated Neural Probes

PubMed Central

Du, Jiangang; Blanche, Timothy J.; Harrison, Reid R.; Lester, Henry A.; Masmanidis, Sotiris C.

2011-01-01

Extracellular electrode arrays can reveal the neuronal network correlates of behavior with single-cell, single-spike, and sub-millisecond resolution. However, implantable electrodes are inherently invasive, and efforts to scale up the number and density of recording sites must compromise on device size in order to connect the electrodes. Here, we report on silicon-based neural probes employing nanofabricated, high-density electrical leads. Furthermore, we address the challenge of reading out multichannel data with an application-specific integrated circuit (ASIC) performing signal amplification, band-pass filtering, and multiplexing functions. We demonstrate high spatial resolution extracellular measurements with a fully integrated, low noise 64-channel system weighing just 330 mg. The on-chip multiplexers make possible recordings with substantially fewer external wires than the number of input channels. By combining nanofabricated probes with ASICs we have implemented a system for performing large-scale, high-density electrophysiology in small, freely behaving animals that is both minimally invasive and highly scalable. PMID:22022568

Preparation of armored RNA as a control for multiplex real-time reverse transcription-PCR detection of influenza virus and severe acute respiratory syndrome coronavirus.

PubMed

Yu, Xin-Fen; Pan, Jing-Cao; Ye, Rong; Xiang, Hai-Qing; Kou, Yu; Huang, Zhi-Cheng

2008-03-01

The common respiratory viruses, including influenza A, influenza B, and newly emerging severe acute respiratory syndrome (SARS) viruses, may cause similar clinical symptoms. Therefore, differential diagnosis of these virus pathogens is frequently required for single clinical samples. In addition, there is an urgent need for noninfectious and stable RNA standards and controls for multivirus detection. In this study, reverse transcription-PCR (RT-PCR) targeting of the RNAs of influenza A and influenza B viruses and SARS coronavirus was performed, and the resulting products were spliced into a fragment which was packaged into armored RNA for use as a noninfectious, quantifiable synthetic substitute. Furthermore, in the present study we developed a multiplex real-time RT-PCR assay in which the armored RNA was used as an external positive control and the three RNA viruses could be detected simultaneously in a single reaction mix. The detection limit of the multiplex real-time PCR was 10 copies/microl of armored RNA.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC).

PubMed

Phillips, Zachary F; Chen, Michael; Waller, Laura

2017-01-01

We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial brightfield microscope with one hardware modification-an inexpensive 3D printed condenser insert. The method, color-multiplexed Differential Phase Contrast (cDPC), is a single-shot variant of Differential Phase Contrast (DPC), which recovers the phase of a sample from images with asymmetric illumination. We employ partially coherent illumination to achieve resolution corresponding to 2× the objective NA. Quantitative phase can then be used to synthesize DIC and phase contrast images or extract shape and density. We demonstrate amplitude and phase recovery at camera-limited frame rates (50 fps) for various in vitro cell samples and c. elegans in a micro-fluidic channel.

Transmission and full-band coherent detection of polarization-multiplexed all-optical Nyquist signals generated by Sinc-shaped Nyquist pulses

PubMed Central

Zhang, Junwen; Yu, Jianjun; Chi, Nan

2015-01-01

All optical method is considered as a promising technique for high symbol rate Nyquist signal generation, which has attracted a lot of research interests for high spectral-efficiency and high-capacity optical communication system. In this paper, we extend our previous work and report the fully experimental demonstration of polarization-division multiplexed (PDM) all-optical Nyquist signal generation based on Sinc-shaped Nyquist pulse with advanced modulation formats, fiber-transmission and single-receiver full-band coherent detection. Using this scheme, we have successfully demonstrated the generation, fiber transmission and single-receiver full-band coherent detection of all-optical Nyquist PDM-QPSK and PDM-16QAM signals up to 125-GBaud. 1-Tb/s single-carrier PDM-16QAM signal generation and full-band coherent detection is realized, which shows the advantage and feasibility of the single-carrier all-optical Nyquist signals. PMID:26323238

Single-exposure super-resolved interferometric microscopy by RGB multiplexing in lensless configuration

NASA Astrophysics Data System (ADS)

Granero, Luis; Ferreira, Carlos; Zalevsky, Zeev; García, Javier; Micó, Vicente

2016-07-01

Single-Exposure Super-Resolved Interferometric Microscopy (SESRIM) reports on a way to achieve one-dimensional (1-D) superresolved imaging in digital holographic microscopy (DHM) by a single illumination shot and digital recording. SESRIM provides color-coded angular multiplexing of the accessible sample's range of spatial frequencies and it allows their recording in a single CCD (color or monochrome) snapshot by adding 3 RGB coherent reference beams at the output plane. In this manuscript, we extend the applicability of SESRIM to the field of digital in-line holographic microscopy (DIHM), that is, working without lenses. As consequence of the in-line configuration, an additional restriction concerning the object field of view (FOV) must be imposed to the technique. Experimental results are reported for both a synthetic object (USAF resolution test target) and a biological sample (swine sperm sample) validating this new kind of superresolution imaging method named as lensless SESRIM (L-SESRIM).

An Engineered Kinetic Amplification Mechanism for Single Nucleotide Variant Discrimination by DNA Hybridization Probes.

PubMed

Chen, Sherry Xi; Seelig, Georg

2016-04-20

Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology.

Nonlinear interaction in differential mode delay managed mode-division multiplexed transmission systems.

PubMed

Rademacher, Georg; Warm, Stefan; Petermann, Klaus

2015-01-12

We analyze the impact of Differential Mode Delay (DMD) Management on the nonlinear impairments in mode-division multiplexed transmission systems. It is found out that DMD Management can lead to a degraded performance, due to enhanced intermodal nonlinear interaction. This can be attributed to an increased correlation of co-propagating channels, similar to the effects that show up in dispersion managed single-mode systems.

Time-division multiplexing of polarization-insensitive fiber-optic Michelson interferometric sensors

NASA Astrophysics Data System (ADS)

Huang, S. C.; Lin, W. W.; Chen, M. H.

1995-06-01

A system of time-division multiplexing of polarization-insensitive fiber-optic Michelson interferometric sensors that uses Faraday rotator mirror elements is demonstrated. This system is constructed with conventional low-birefringence single-mode fiber and is able to solve the polarization-fading problem by a combination of Faraday rotator mirrors with unbalanced Michelson interferometers. The system is lead-fiber insensitive and has potentials for practical field applications.

Association of rs662799 in APOA5 with CAD in Chinese Han population.

PubMed

Chen, Hua; Ding, Shifang; Zhou, Mi; Wu, Xiayin; Liu, Xi; Wu, Yun; Liu, Dechao

2018-01-08

CAD (Coronary Artery Disease) is a complex disease that influenced by various environmental and genetic factors. Previous studies have found many single nucleotide polymorphisms (SNPs) associated with the risk of CAD occurrence. However, the results are inconsistent. In this study, we aim to investigate genetic etiology in Chinese Han population by analysis of 7 SNPs in lipid metabolism pathway that previously has been reported to be associated with CAD. A total of 631 samples were used in this study, including 435 CAD cases and 196 normal healthy controls. SNP genotyping were conducted via multiplex PCR amplifying followed by NGS (next-generation sequencing). Rs662799 in APOA5 (Apolipoprotein A5) gene was associated with CAD in Chinese Han population (Odds-ratio = 1.374, P-value = 0.03). No significant association was observed between the rest of SNPs and CAD. Stratified association analysis revealed rs5882 was associated with CAD in non-hypertension group (Odds-ratio = 1.593, P-value = 0.023). Rs1800588 was associated with CAD in smoking group (Odds-ratio = 1.603, P-value = 0.035). The minor allele of rs662799 was the risk factor of CAD occurrences in Chinese Han population.

Optimization of the genotyping-by-sequencing strategy for population genomic analysis in conifers.

PubMed

Pan, Jin; Wang, Baosheng; Pei, Zhi-Yong; Zhao, Wei; Gao, Jie; Mao, Jian-Feng; Wang, Xiao-Ru

2015-07-01

Flexibility and low cost make genotyping-by-sequencing (GBS) an ideal tool for population genomic studies of nonmodel species. However, to utilize the potential of the method fully, many parameters affecting library quality and single nucleotide polymorphism (SNP) discovery require optimization, especially for conifer genomes with a high repetitive DNA content. In this study, we explored strategies for effective GBS analysis in pine species. We constructed GBS libraries using HpaII, PstI and EcoRI-MseI digestions with different multiplexing levels and examined the effect of restriction enzymes on library complexity and the impact of sequencing depth and size selection of restriction fragments on sequence coverage bias. We tested and compared UNEAK, Stacks and GATK pipelines for the GBS data, and then developed a reference-free SNP calling strategy for haploid pine genomes. Our GBS procedure proved to be effective in SNP discovery, producing 7000-11 000 and 14 751 SNPs within and among three pine species, respectively, from a PstI library. This investigation provides guidance for the design and analysis of GBS experiments, particularly for organisms for which genomic information is lacking. © 2014 John Wiley & Sons Ltd.

Analysis of Multiplexed Nanosensor Arrays Based on Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

PubMed

Dong, Juyao; Salem, Daniel P; Sun, Jessica H; Strano, Michael S

2018-04-24

The high-throughput, label-free detection of biomolecules remains an important challenge in analytical chemistry with the potential of nanosensors to significantly increase the ability to multiplex such assays. In this work, we develop an optical sensor array, printable from a single-walled carbon nanotube/chitosan ink and functionalized to enable a divalent ion-based proximity quenching mechanism for transducing binding between a capture protein or an antibody with the target analyte. Arrays of 5 × 6, 200 μm near-infrared (nIR) spots at a density of ≈300 spots/cm 2 are conjugated with immunoglobulin-binding proteins (proteins A, G, and L) for the detection of human IgG, mouse IgM, rat IgG2a, and human IgD. Binding kinetics are measured in a parallel, multiplexed fashion from each sensor spot using a custom laser scanning imaging configuration with an nIR photomultiplier tube detector. These arrays are used to examine cross-reactivity, competitive and nonspecific binding of analyte mixtures. We find that protein G and protein L functionalized sensors report selective responses to mouse IgM on the latter, as anticipated. Optically addressable platforms such as the one examined in this work have potential to significantly advance the real-time, multiplexed biomolecular detection of complex mixtures.

A large capacity time division multiplexed (TDM) laser beam combining technique enabled by nanosecond speed KTN deflector

NASA Astrophysics Data System (ADS)

Yin, Stuart (Shizhuo); Chao, Ju-Hung; Zhu, Wenbin; Chen, Chang-Jiang; Campbell, Adrian; Henry, Michael; Dubinskiy, Mark; Hoffman, Robert C.

2017-08-01

In this paper, we present a novel large capacity (a 1000+ channel) time division multiplexing (TDM) laser beam combining technique by harnessing a state-of-the-art nanosecond speed potassium tantalate niobate (KTN) electro-optic (EO) beam deflector as the time division multiplexer. The major advantages of TDM approach are: (1) large multiplexing capability (over 1000 channels), (2) high spatial beam quality (the combined beam has the same spatial profile as the individual beam), (3) high spectral beam quality (the combined beam has the same spectral width as the individual beam, and (4) insensitive to the phase fluctuation of individual laser because of the nature of the incoherent beam combining. The quantitative analyses show that it is possible to achieve over one hundred kW average power, single aperture, single transverse mode solid state and/or fiber laser by pursuing this innovative beam combining method, which represents a major technical advance in the field of high energy lasers. Such kind of 100+ kW average power diffraction limited beam quality lasers can play an important role in a variety of applications such as laser directed energy weapons (DEW) and large-capacity high-speed laser manufacturing, including cutting, welding, and printing.

A Multiplexed Assay That Monitors Effects of Multiple Compound Treatment Times Reveals Candidate Immune-Enhancing Compounds.

PubMed

Zhao, Ziyan; Henowitz, Liza; Zweifach, Adam

2018-05-01

We previously developed a flow cytometry assay that monitored lytic granule exocytosis in cytotoxic T lymphocytes stimulated by contacting beads coated with activating anti-CD3 antibodies. That assay was multiplexed in that responses of cells that did or did not receive the activating stimulus were distinguished via changes in light scatter accompanying binding of cells to beads, allowing us to discriminate compounds that activate responses on their own from compounds that enhance responses in cells that received the activating stimulus, all within a single sample. Here we add a second dimension of multiplexing by developing means to assess in a single sample the effects of treating cells with test compounds for different times. Bar-coding cells before adding them to test wells lets us determine compound treatment time while also monitoring activation status and response amplitude at the point of interrogation. This multiplexed assay is suitable for screening 96-well plates. We used it to screen compounds from the National Cancer Institute, identifying several compounds that enhance anti-LAMP1 responses. Multiple-treatment-time (MTT) screening enabled by bar-coding and read via high-throughput flow cytometry may be a generally useful method for facilitating the discovery of compounds of interest.

Identification of spider-mite species and their endosymbionts using multiplex PCR.

PubMed

Zélé, Flore; Weill, Mylène; Magalhães, Sara

2018-02-01

Spider mites of the genus Tetranychidae are severe crop pests. In the Mediterranean a few species coexist, but they are difficult to identify based on morphological characters. Additionally, spider mites often harbour several species of endosymbiotic bacteria, which may affect the biology of their hosts. Here, we propose novel, cost-effective, multiplex diagnostic methods allowing a quick identification of spider-mite species as well as of the endosymbionts they carry. First, we developed, and successfully multiplexed in a single PCR, primers to identify Tetranychus urticae, T. evansi and T. ludeni, some of the most common tetranychids found in southwest Europe. Moreover, we demonstrated that this method allows detecting multiple species in a single pool, even at low frequencies (up to 1/100), and can be used on entire mites without DNA extraction. Second, we developed another set of primers to detect spider-mite endosymbionts, namely Wolbachia, Cardinium and Rickettsia in a multiplex PCR, along with a generalist spider-mite primer to control for potential failure of DNA amplification in each PCR. Overall, our method represents a simple, cost-effective and reliable method to identify spider-mite species and their symbionts in natural field populations, as well as to detect contaminations in laboratory rearings. This method may easily be extended to other species.

Association of single nucleotide polymorphism in CD28(C/T-I3 + 17) and CD40 (C/T-1) genes with the Graves' disease.

PubMed

Mustafa, Saima; Fatima, Hira; Fatima, Sadia; Khosa, Tafheem; Akbar, Atif; Shaikh, Rehan Sadiq; Iqbal, Furhan

2018-01-01

To find out a correlation between the single nucleotide polymorphisms in cluster of differentiation 28 and cluster of differentiation 40 genes with Graves' disease, if any. This case-control study was conducted at the Multan Institute of Nuclear Medicine and Radiotherapy, Multan, Pakistan, and comprised blood samples of Graves' disease patients and controls. Various risk factors were also correlated either with the genotype at each single-nucleotide polymorphism or with various combinations of genotypes studied during present investigation. Of the 160 samples, there were 80(50%) each from patients and controls. Risk factor analysis revealed that gender (p=0.008), marital status (p<0.001), education (p<0.001), smoking (p<0.001), tri-iodothyronine (P <0.001), thyroxin (p<0.001) and thyroid-stimulating hormone (p<0.000) levels in blood were associated with Graves' disease. Both single-nucleotide polymorphisms in both genes were not associated with Graves' disease, either individually or in any combined form.

High-performance single cell genetic analysis using microfluidic emulsion generator arrays.

PubMed

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T; Mathies, Richard A

2010-04-15

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex polymerase chain reaction (PCR). Microfabricated emulsion generator array (MEGA) devices containing 4, 32, and 96 channels are developed to confer a flexible capability of generating up to 3.4 x 10(6) nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed and the beads are pooled and rapidly analyzed by multicolor flow cytometry. Using Escherichia coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1/10(5). This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations.

High-Performance Single Cell Genetic Analysis Using Microfluidic Emulsion Generator Arrays

PubMed Central

Zeng, Yong; Novak, Richard; Shuga, Joe; Smith, Martyn T.; Mathies, Richard A.

2010-01-01

High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex PCR. Microfabricated emulsion generator array (MEGA) devices containing 4, 32 and 96 channels are developed to confer a flexible capability of generating up to 3.4 × 106 nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed, the beads are pooled and rapidly analyzed by multi-color flow cytometry. Using E. coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1:105. This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations. PMID:20192178

Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease.

PubMed

Stockley, Jacqueline; Nisar, Shaista P; Leo, Vincenzo C; Sabi, Essa; Cunningham, Margaret R; Eikenboom, Jeroen C; Lethagen, Stefan; Schneppenheim, Reinhard; Goodeve, Anne C; Watson, Steve P; Mundell, Stuart J; Daly, Martina E

2015-01-01

The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.

Detecting Single-Nucleotides by Tunneling Current Measurements at Sub-MHz Temporal Resolution.

PubMed

Morikawa, Takanori; Yokota, Kazumichi; Tanimoto, Sachie; Tsutsui, Makusu; Taniguchi, Masateru

2017-04-18

Label-free detection of single-nucleotides was performed by fast tunneling current measurements in a polar solvent at 1 MHz sampling rate using SiO₂-protected Au nanoprobes. Short current spikes were observed, suggestive of trapping/detrapping of individual nucleotides between the nanoelectrodes. The fall and rise features of the electrical signatures indicated signal retardation by capacitance effects with a time constant of about 10 microseconds. The high temporal resolution revealed current fluctuations, reflecting the molecular conformation degrees of freedom in the electrode gap. The method presented in this work may enable direct characterizations of dynamic changes in single-molecule conformations in an electrode gap in liquid.

[Single nucleotide polymorphism and its application in allogeneic hematopoietic stem cell transplantation--review].

PubMed

Li, Su-Xia

2004-12-01

Single nucleotide polymorphism (SNP) is the third genetic marker after restriction fragment length polymorphism (RFLP) and short tandem repeat. It represents the most density genetic variability in the human genome and has been widely used in gene location, cloning, and research of heredity variation, as well as parenthood identification in forensic medicine. As steady heredity polymorphism, single nucleotide polymorphism is becoming the focus of attention in monitoring chimerism and minimal residual disease in the patients after allogeneic hematopoietic stem cell transplantation. The article reviews SNP heredity characterization, analysis techniques and its applications in allogeneic stem cell transplantation and other fields.

Xylella taiwanensis sp. nov., causing pear leaf scorch disease.

PubMed

Su, C-C; Deng, W-L; Jan, F-J; Chang, C-J; Huang, H; Shih, H-T; Chen, J

2016-11-01

A Gram-stain-negative, nutritionally fastidious bacterium (PLS229T) causing pear leaf scorch was identified in Taiwan and previously grouped into Xylella fastidiosa. Yet, significant variations between PLS229T and Xylellafastidiosa were noted. In this study, PLS229T was evaluated phenotypically and genotypically against representative strains of Xylellafastidiosa, including strains of the currently known subspecies of Xylellafastidiosa, Xylella fastidiosa subsp. multiplex and 'Xylella fastidiosasubsp.pauca'. Because of the difficulty of in vitro culture characterization, emphases were made to utilize the available whole-genome sequence information. The average nucleotide identity (ANI) values, an alternative for DNA-DNA hybridization relatedness, between PLS229T and Xylellafastidiosa were 83.4-83.9 %, significantly lower than the bacterial species threshold of 95 %. In contrast, sequence similarity of 16S rRNA genes was greater than 98 %, higher than the 97 % threshold to justify if two bacterial strains belong to different species. The uniqueness of PLS229T was also evident by observing only about 87 % similarity in the sequence of the 16S-23S internal transcribed spacer (ITS) between PLS229T and strains of Xylellafastidiosa, discovering significant single nucleotide polymorphisms at 18 randomly selected housekeeping gene loci, observing a distinct fatty acid profile for PLS229T compared with Xylellafastidiosa, and PLS229T having different observable phenotypes, such as different susceptibility to antibiotics. A phylogenetic tree derived from 16S rRNA gene sequences showed a distinct PLS229T phyletic lineage positioning it between Xylellafastidiosa and members of the genus Xanthomonas. On the basis of these data, a novel species, Xylella taiwanensis sp. nov. is proposed. The type strain is PLS229T (=BCRC 80915T=JCM 31187T).

Development and validation of a clinical trial patient stratification assay that interrogates 27 mutation sites in MAPK pathway genes.

PubMed

Chang, Ken C N; Galuska, Stefan; Weiner, Russell; Marton, Matthew J

2013-01-01

Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE) multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.

Genomic profiling of plastid DNA variation in the Mediterranean olive tree

PubMed Central

2011-01-01

Background Characterisation of plastid genome (or cpDNA) polymorphisms is commonly used for phylogeographic, population genetic and forensic analyses in plants, but detecting cpDNA variation is sometimes challenging, limiting the applications of such an approach. In the present study, we screened cpDNA polymorphism in the olive tree (Olea europaea L.) by sequencing the complete plastid genome of trees with a distinct cpDNA lineage. Our objective was to develop new markers for a rapid genomic profiling (by Multiplex PCRs) of cpDNA haplotypes in the Mediterranean olive tree. Results Eight complete cpDNA genomes of Olea were sequenced de novo. The nucleotide divergence between olive cpDNA lineages was low and not exceeding 0.07%. Based on these sequences, markers were developed for studying two single nucleotide substitutions and length polymorphism of 62 regions (with variable microsatellite motifs or other indels). They were then used to genotype the cpDNA variation in cultivated and wild Mediterranean olive trees (315 individuals). Forty polymorphic loci were detected on this sample, allowing the distinction of 22 haplotypes belonging to the three Mediterranean cpDNA lineages known as E1, E2 and E3. The discriminating power of cpDNA variation was particularly low for the cultivated olive tree with one predominating haplotype, but more diversity was detected in wild populations. Conclusions We propose a method for a rapid characterisation of the Mediterranean olive germplasm. The low variation in the cultivated olive tree indicated that the utility of cpDNA variation for forensic analyses is limited to rare haplotypes. In contrast, the high cpDNA variation in wild populations demonstrated that our markers may be useful for phylogeographic and populations genetic studies in O. europaea. PMID:21569271

Low-Cutoff, High-Pass Digital Filtering of Neural Signals

NASA Technical Reports Server (NTRS)

Mojarradi,Mohammad; Johnson, Travis; Ortiz, Monico; Cunningham, Thomas; Andersen, Richard

2004-01-01

The figure depicts the major functional blocks of a system, now undergoing development, for conditioning neural signals acquired by electrodes implanted in a brain. The overall functions to be performed by this system can be summarized as preamplification, multiplexing, digitization, and high-pass filtering. Other systems under development for recording neural signals typically contain resistor-capacitor analog low-pass filters characterized by cutoff frequencies in the vicinity of 100 Hz. In the application for which this system is being developed, there is a requirement for a cutoff frequency of 5 Hz. Because the resistors needed to obtain such a low cutoff frequency would be impractically large, it was decided to perform low-pass filtering by use of digital rather than analog circuitry. In addition, it was decided to timemultiplex the digitized signals from the multiple input channels into a single stream of data in a single output channel. The signal in each input channel is first processed by a preamplifier having a voltage gain of approximately 50. Embedded in each preamplifier is a low-pass anti-aliasing filter having a cutoff frequency of approximately 10 kHz. The anti-aliasing filters make it possible to couple the outputs of the preamplifiers to the input ports of a multiplexer. The output of the multiplexer is a single stream of time-multiplexed samples of analog signals. This stream is processed by a main differential amplifier, the output of which is sent to an analog-to-digital converter (ADC). The output of the ADC is sent to a digital signal processor (DSP).

A new single-nucleotide polymorphism database for rainbow trout generated through whole genome re-sequencing

USDA-ARS?s Scientific Manuscript database

Single-nucleotide polymorphisms (SNPs) are highly abundant markers, which are broadly distributed in animal genomes. For rainbow trout, SNP discovery has been done through sequencing of restriction-site associated DNA (RAD) libraries, reduced representation libraries (RRL), RNA sequencing, and whole...

Characterization of polyploid wheat genomic diversity using a high-density 90 000 single nucleotide polymorphism array

USDA-ARS?s Scientific Manuscript database

High-density single nucleotide polymorphism (SNP) genotyping chips are a powerful tool for studying genomic patterns of diversity, inferring ancestral relationships among individuals in populations and studying marker-trait associations in mapping experiments. We developed a genotyping array includ...

High Resolution Imaging with MUSTANG-2 on the GBT

NASA Astrophysics Data System (ADS)

Stanchfield, Sara; Ade, Peter; Aguirre, James; Brevik, Justus A.; Cho, Hsiao-Mei; Datta, Rahul; Devlin, Mark; Dicker, Simon R.; Dober, Bradley; Duff, Shannon M.; Egan, Dennis; Ford, Pam; Hilton, Gene; Hubmayr, Johannes; Irwin, Kent; Knowles, Kenda; Marganian, Paul; Mason, Brian Scott; Mates, John A. B.; McMahon, Jeff; Mello, Melinda; Mroczkowski, Tony; Romero, Charles; Sievers, Jonathon; Tucker, Carole; Vale, Leila R.; Vissers, Michael; White, Steven; Whitehead, Mark; Ullom, Joel; Young, Alexander

2018-01-01

We present early science results from MUSTANG-2, a 90 GHz feedhorn-coupled, microwave SQUID-multiplexed TES bolometer array operating on the Robert C. Byrd Green Bank Telescope (GBT). The feedhorn and waveguide-probe-coupled detector technology is a mature technology, which has been used on instruments such as the South Pole Telescope, the Atacama Cosmology Telescope, and the Atacama B-mode Search telescope. The microwave SQUID multiplexer-based readout system developed for MUSTANG-2 currently reads out 66 detectors with a single coaxial cable and will eventually allow thousands of detectors to be multiplexed. This microwave SQUID multiplexer combines the proven abilities of millimeter wave TES detectors with the multiplexing capabilities of KIDs with no degradation in noise performance of the detectors. Each multiplexing device is read out using warm electronics consisting of a commercially available ROACH board, a DAC/ADC card, and an Intermediate Frequency mixer circuit. The hardware was originally developed by the Collaboration for Astronomy Signal Processing and Electronic Research (CASPER) group, whose primary goal is to develop scalable FPGA-based hardware with the flexibility to be used in a wide range of radio signal processing applications. MUSTANG-2 is the first on-sky instrument to use microwave SQUID multiplexing and is available as a shared-risk/PI instrument on the GBT. In MUSTANG-2’s first season 7 separate proposals were awarded a total of 230 hours of telescope time.

Topology-optimized silicon photonic wire mode (de)multiplexer

NASA Astrophysics Data System (ADS)

Frellsen, Louise F.; Frandsen, Lars H.; Ding, Yunhong; Elesin, Yuriy; Sigmund, Ole; Yvind, Kresten

2015-02-01

We have designed and for the first time experimentally verified a topology optimized mode (de)multiplexer, which demultiplexes the fundamental and the first order mode of a double mode photonic wire to two separate single mode waveguides (and multiplexes vice versa). The device has a footprint of ~4.4 μm x ~2.8 μm and was fabricated for different design resolutions and design threshold values to verify the robustness of the structure to fabrication tolerances. The multiplexing functionality was confirmed by recording mode profiles using an infrared camera and vertical grating couplers. All structures were experimentally found to maintain functionality throughout a 100 nm wavelength range limited by available laser sources and insertion losses were generally lower than 1.3 dB. The cross talk was around -12 dB and the extinction ratio was measured to be better than 8 dB.

Development of a multiplexing fingerprint and high wavenumber Raman spectroscopy technique for real-time in vivo tissue Raman measurements at endoscopy

NASA Astrophysics Data System (ADS)

Bergholt, Mads Sylvest; Zheng, Wei; Huang, Zhiwei

2013-03-01

We report on the development of a novel multiplexing Raman spectroscopy technique using a single laser light together with a volume phase holographic (VPH) grating that simultaneously acquires both fingerprint (FP) and high wavenumber (HW) tissue Raman spectra at endoscopy. We utilize a customized VPH dual-transmission grating, which disperses the incident Raman scattered light vertically onto two separate segments (i.e., -150 to 1950 cm-1 1750 to 3600 cm-1) of a charge-coupled device camera. We demonstrate that the multiplexing Raman technique can acquire high quality in vivo tissue Raman spectra ranging from 800 to 3600 cm-1 within 1.0 s with a spectral resolution of 3 to 6 cm-1 during clinical endoscopy. The rapid multiplexing Raman spectroscopy technique covering both FP and HW ranges developed in this work has potential for improving in vivo tissue diagnosis and characterization at endoscopy.

Switchable in-line monitor for multi-dimensional multiplexed photonic integrated circuit.

PubMed

Chen, Guanyu; Yu, Yu; Ye, Mengyuan; Zhang, Xinliang

2016-06-27

A flexible monitor suitable for the discrimination of on-chip transmitted mode division multiplexed (MDM) and wavelength division multiplexed (WDM) signals is proposed and fabricated. By selectively extracting part of the incoming signals through the tunable wavelength and mode dependent drop filter, the in-line and switchable monitor can discriminate the wavelength, mode and power information of the transmitted signals. Being different from a conventional mode and wavelength demultiplexer, the monitor is specifically designed to ensure a flexible in-line monitoring. For demonstration, three mode and three wavelength multiplexed signals are successfully processed. Assisted by the integrated photodetectors (PDs), both the measured photo currents and eye diagrams validate the performance of the proposed device. The bit error ratio (BER) measurement results show less than 0.4 dB power penalty between different modes and ~2 dB power penalty for single wavelength and WDM cases under 10^-9 BER level.

Multiplex amplification of large sets of human exons.

PubMed

Porreca, Gregory J; Zhang, Kun; Li, Jin Billy; Xie, Bin; Austin, Derek; Vassallo, Sara L; LeProust, Emily M; Peck, Bill J; Emig, Christopher J; Dahl, Fredrik; Gao, Yuan; Church, George M; Shendure, Jay

2007-11-01

A new generation of technologies is poised to reduce DNA sequencing costs by several orders of magnitude. But our ability to fully leverage the power of these technologies is crippled by the absence of suitable 'front-end' methods for isolating complex subsets of a mammalian genome at a scale that matches the throughput at which these platforms will routinely operate. We show that targeting oligonucleotides released from programmable microarrays can be used to capture and amplify approximately 10,000 human exons in a single multiplex reaction. Additionally, we show integration of this protocol with ultra-high-throughput sequencing for targeted variation discovery. Although the multiplex capture reaction is highly specific, we found that nonuniform capture is a key issue that will need to be resolved by additional optimization. We anticipate that highly multiplexed methods for targeted amplification will enable the comprehensive resequencing of human exons at a fraction of the cost of whole-genome resequencing.

Four-channel magnetic resonance imaging receiver using frequency domain multiplexing.

PubMed

He, Wang; Qin, Xu; Jiejing, Ren; Gengying, Li

2007-01-01

An alternative technique that uses frequency domain multiplexing to acquire phased array magnetic resonance images is discussed in detail. The proposed method has advantages over traditional independent receiver chains in that it utilizes an analog-to-digital converter and a single-chip multicarrier receiver with high performance to reduce the size and cost of the phased array receiver system. A practical four-channel digital receiver using frequency domain multiplexing was implemented and verified on a home-built 0.3 T magnetic resonance imaging system. The experimental results confirmed that the cross talk between each channel was below -60 dB, the phase fluctuations were about 1 degrees , and there was no obvious signal-to-noise ratio degradation. It is demonstrated that the frequency domain multiplexing is a valuable and economical technique, particularly for array coil systems where the multichannel receiver is indispensable and dynamic range is not a critical problem.

Doubling transmission capacity in optical wireless system by antenna horizontal- and vertical-polarization multiplexing.

PubMed

Li, Xinying; Yu, Jianjun; Zhang, Junwen; Dong, Ze; Chi, Nan

2013-06-15

We experimentally demonstrate 2×56 Gb/s two-channel polarization-division-multiplexing quadrature-phase-shift-keying signal delivery over 80 km single-mode fiber-28 and 2 m Q-band (33-50 GHz) wireless link, adopting antenna horizontal- (H-) and vertical-polarization (V-polarization) multiplexing. At the wireless receiver, classic constant-modulus-algorithm equalization based on digital signal processing can realize polarization demultiplexing and remove the crosstalk at the same antenna polarization. By adopting antenna polarization multiplexing, the signal baud rate and performance requirements for optical and wireless devices can be reduced but at the cost of double antennas and devices, while wireless transmission capacity can also be increased but at the cost of stricter requirements for V-polarization. The isolation is only about 19 dB when V-polarization deviation approaches 10°, which will affect high-speed (>50 Gb/s) wireless delivery.

Multiplex PCR for Rapid Detection of Genes Encoding Class A Carbapenemases

PubMed Central

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo

2012-01-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers. PMID:22950072

Multiplex PCR for rapid detection of genes encoding class A carbapenemases.

PubMed

Hong, Sang Sook; Kim, Kyeongmi; Huh, Ji Young; Jung, Bochan; Kang, Myung Seo; Hong, Seong Geun

2012-09-01

In recent years, there have been increasing reports of KPC-producing Klebsiella pneumoniae in Korea. The modified Hodge test can be used as a phenotypic screening test for class A carbapenamase (CAC)-producing clinical isolates; however, it does not distinguish between carbapenemase types. The confirmation of type of CAC is important to ensure optimal therapy and to prevent transmission. This study applied a novel multiplex PCR assay to detect and differentiate CAC genes in a single reaction. Four primer pairs were designed to amplify fragments encoding 4 CAC families (SME, IMI/NMC-A, KPC, and GES). The multiplex PCR detected all genes tested for 4 CAC families that could be differentiated by fragment size according to gene type. This multiplex PCR offers a simple and useful approach for detecting and distinguishing CAC genes in carbapenem-resistant strains that are metallo-β-lactamase nonproducers.

Genome-wide comparison and taxonomic relatedness of multiple Xylella fastidiosa strains reveal the occurrence of three subspecies and a new Xylella species.

PubMed

Marcelletti, Simone; Scortichini, Marco

2016-10-01

A total of 21 Xylella fastidiosa strains were assessed by comparing their genomes to infer their taxonomic relationships. The whole-genome-based average nucleotide identity and tetranucleotide frequency correlation coefficient analyses were performed. In addition, a consensus tree based on comparisons of 956 core gene families, and a genome-wide phylogenetic tree and a Neighbor-net network were constructed with 820,088 nucleotides (i.e., approximately 30-33 % of the entire X. fastidiosa genome). All approaches revealed the occurrence of three well-demarcated genetic clusters that represent X. fastidiosa subspecies fastidiosa, multiplex and pauca, with the latter appeared to diverge. We suggest that the proposed but never formally described subspecies 'sandyi' and 'morus' are instead members of the subspecies fastidiosa. These analyses support the view that the Xylella strain isolated from Pyrus pyrifolia in Taiwan is likely to be a new species. A widely used multilocus sequence typing analysis yielded conflicting results.

Differentiation of closely related but biologically distinct cherry isolates of Prunus necrotic ringspot virus by polymerase chain reaction.

PubMed

Hammond, R W; Crosslin, J M; Pasini, R; Howell, W E; Mink, G I

1999-07-01

Prunus necrotic ringspot ilarvirus (PNRSV) exists as a number of biologically distinct variants which differ in host specificity, serology, and pathology. Previous nucleotide sequence alignment and phylogenetic analysis of cloned reverse transcription-polymerase chain reaction (RT-PCR) products of several biologically distinct sweet cherry isolates revealed correlations between symptom type and the nucleotide and amino acid sequences of the 3a (putative movement protein) and 3b (coat protein) open reading frames. Based upon this analysis, RT-PCR assays have been developed that can identify isolates displaying different symptoms and serotypes. The incorporation of primers in a multiplex PCR protocol permits rapid detection and discrimination among the strains. The results of PCR amplification using type-specific primers that amplify a portion of the coat protein gene demonstrate that the primer-selection procedure developed for PNRSV constitutes a reliable method of viral strain discrimination in cherry for disease control and will also be useful for examining biological diversity within the PNRSV virus group.

Novel high-speed droplet-allele specific-polymerase chain reaction: application in the rapid genotyping of single nucleotide polymorphisms.

PubMed

Taira, Chiaki; Matsuda, Kazuyuki; Yamaguchi, Akemi; Sueki, Akane; Koeda, Hiroshi; Takagi, Fumio; Kobayashi, Yukihiro; Sugano, Mitsutoshi; Honda, Takayuki

2013-09-23

Single nucleotide alterations such as single nucleotide polymorphisms (SNP) and single nucleotide mutations are associated with responses to drugs and predisposition to several diseases, and they contribute to the pathogenesis of malignancies. We developed a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) with our droplet-PCR machine (droplet-AS-PCR). Using 8 SNP loci, we evaluated the specificity and sensitivity of droplet-AS-PCR. Buccal cells were pretreated with proteinase K and subjected directly to the droplet-AS-PCR without DNA extraction. The genotypes determined using the droplet-AS-PCR were then compared with those obtained by direct sequencing. Specific PCR amplifications for the 8 SNP loci were detected, and the detection limit of the droplet-AS-PCR was found to be 0.1-5.0% by dilution experiments. Droplet-AS-PCR provided specific amplification when using buccal cells, and all the genotypes determined within 9 min were consistent with those obtained by direct sequencing. Our novel droplet-AS-PCR assay enabled high-speed amplification retaining specificity and sensitivity and provided ultra-rapid genotyping. Crude samples such as buccal cells were available for the droplet-AS-PCR assay, resulting in the reduction of the total analysis time. Droplet-AS-PCR may therefore be useful for genotyping or the detection of single nucleotide alterations. Copyright © 2013 Elsevier B.V. All rights reserved.

Copy number variation in 19 Italian multiplex families with autism spectrum disorder: Importance of synaptic and neurite elongation genes.

PubMed

Lintas, Carla; Picinelli, Chiara; Piras, Ignazio Stefano; Sacco, Roberto; Brogna, Claudia; Persico, Antonio M

2017-03-17

Autism Spectrum Disorder (ASD) is endowed with impressive heritability estimates and high recurrence rates. Its genetic underpinnings are nonetheless very heterogeneous, with common, and rare contributing variants located in hundreds of different loci, each characterized by variable levels of penetrance. Multiplex families from single ethnic groups represent a useful means to reduce heterogeneity and enhance genetic load. We screened 19 Italian ASD multiplex families (3 triplets and 16 duplets, total N = 41 ASD subjects), using array-CGH (Agilent 180 K). Causal or ASD-relevant CNVs were detected in 36.6% (15/41) of ASD probands, corresponding to 36.8% (7/19) multiplex families with at least one affected sibling genetically positive. However, only in less than half (3/7) of positive families, affected siblings share the same causal or ASD-relevant CNV. Even in these three families, additional potentially relevant CNVs not shared by affected sib pairs were also detected. These results provide further evidence of genetic heterogeneity in ASD even within multiplex families belonging to a single ethnic group. Differences in CNV burden may likely contribute to the substantial clinical heterogeneity observed between affected siblings. In addition, Gene Ontology enrichment analysis indicates that most potentially causal or relevant ASD genes detected in our cohort belong to nervous system-specific categories, especially involved in neurite elongation and synaptic structure/function. These findings point toward the existence of genomic instability in these families, whose underlying genetic and epigenetic mechanisms deserve further scrutiny. © 2017 Wiley Periodicals, Inc.

Optofluidic devices for biomolecule sensing and multiplexing

NASA Astrophysics Data System (ADS)

Ozcelik, Damla

Optofluidics which integrates photonics and microfluidics, has led to highly compact, sensitive and adaptable biomedical sensors. Optofluidic biosensors based on liquid-core anti-resonant reflecting optical waveguides (LC-ARROWs), have proven to be a highly sensitive, portable, and reconfigurable platform for fluorescence spectroscopy and detection of single biomolecules such as proteins, nucleic acids, and virus particles. However, continued improvements in sensitivity remain a major goal as we approach the ultimate limit of detecting individual bio-particles labeled by single or few fluorophores. Additionally, the ability to simultaneously detect and identify multiple biological particles or biomarkers is one of the key requirements for molecular diagnostic tests. The compactness and adaptability of these platforms can further be advanced by introducing tunability, integrating off-chip components, designing reconfigurable and customizable devices, which makes these platforms very good candidates for many different applications. The goal of this thesis was to introduce new elements in these LC-ARROW optofluidics platforms that provide major enhancements in their functionality, making them more sensitive, compact, customizable and multiplexed. First, a novel integrated tunable spectral filter that achieves effective elimination of background noise on the ARROW platform was demonstrated. A unique dual liquid-core design enabled the independent multi-wavelength tuning of the spectral filter by adjusting the refractive index and chemical properties of the liquid. In order to enhance the detection sensitivity of the platform, Y-splitter waveguides were integrated to create multiple excitation spots for each target molecule. A powerful signal processing algorithm was used to analyze the data to improve the signal-to-noise ratio (SNR) of the collected data. Next, the design, optimization and characterization of the Y-splitter waveguides are presented; and single influenza virus detection with an improved SNR was demonstrated using this platform. Finally, multiplexing capacity is introduced to the ARROW detection platform by integrating multi-mode interference (MMI) waveguides. MMI waveguides create wavelength dependent multiple excitation spots at the excitation region, allowing the spectral multiplexed detection of multiple different target molecules based on the excitation pattern, without the need for additional spectral filters. Successful spectral multiplexed detection of three different types of influenza viruses is achieved by using separate wavelengths and combination of wavelengths. This multiplexing capacity is further enhanced by taking advantage of the spatial properties of the MMI pattern, designing triple liquid-core waveguides that intersect the MMI waveguide in different locations. Furthermore, the spectral and spatial multiplexing capacities are combined in these triple liquid-core MMI platforms, allowing these devices to distinguish multiple different targets and samples simultaneously.

Identification of Critical Residues for the Tight Binding of Both Correct and Incorrect Nucleotides to Human DNA Polymerase λ

PubMed Central

Brown, Jessica A.; Pack, Lindsey R.; Sherrer, Shanen M.; Kshetry, Ajay K.; Newmister, Sean A.; Fowler, Jason D.; Taylor, John-Stephen; Suo, Zucai

2010-01-01

DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase β (Pol β). Pre-steady state kinetic studies have shown that the Pol λ•DNA complex binds both correct and incorrect nucleotides 130-fold tighter on average than the Pol β•DNA complex, although, the base substitution fidelity of both polymerases is 10−4 to 10−5. To better understand Pol λ’s tight nucleotide binding affinity, we created single- and double-substitution mutants of Pol λ to disrupt interactions between active site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding with each of the common structural moieties in the following order: triphosphate ≫ base > ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and template base led to a moderate increase in the Kd. The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template. PMID:20851705

A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis

PubMed Central

Huang, Kunlun; Zhang, Nan; Yuan, Yanfang; Shang, Ying; Luo, Yunbo

2012-01-01

In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on. PMID:22272223

Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events.

PubMed

Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Štebih, Dejan; Morisset, Dany; Holst-Jensen, Arne; Žel, Jana

2018-01-01

The standard-curve based simplex quantitative polymerase chain reaction (qPCR) has been the gold standard for DNA target quantification for more than a decade. The large and growing number of individual analyses needed to test for genetically modified organisms (GMOs) is reducing the cost-effectiveness of qPCR. Droplet digital PCR (ddPCR) enables absolute quantification without standard curves, avoids the amplification efficiency bias observed with qPCR, allows more accurate estimations at low target copy numbers and, in combination with multiplexing, significantly improves cost efficiency. Here we describe two protocols for multiplex quantification of GM maize events: (1) nondiscriminating, with multiplex quantification of targets as a group (12 GM maize lines) and (2) discriminating, with multiplex quantification of individual targets (events). The first enables the quantification of twelve European Union authorized GM maize events as a group with only two assays, but does not permit determination of the individual events present. The second protocol enables the quantification of four individual targets (three GM events and one endogene) in a single reaction. Both protocols can be modified for quantification of any other DNA target.

Mapping a multiplexed zoo of mRNA expression.

PubMed

Choi, Harry M T; Calvert, Colby R; Husain, Naeem; Huss, David; Barsi, Julius C; Deverman, Benjamin E; Hunter, Ryan C; Kato, Mihoko; Lee, S Melanie; Abelin, Anna C T; Rosenthal, Adam Z; Akbari, Omar S; Li, Yuwei; Hay, Bruce A; Sternberg, Paul W; Patterson, Paul H; Davidson, Eric H; Mazmanian, Sarkis K; Prober, David A; van de Rijn, Matt; Leadbetter, Jared R; Newman, Dianne K; Readhead, Carol; Bronner, Marianne E; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E; Pierce, Niles A

2016-10-01

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. © 2016. Published by The Company of Biologists Ltd.

Mapping a multiplexed zoo of mRNA expression

PubMed Central

Choi, Harry M. T.; Calvert, Colby R.; Husain, Naeem; Huss, David; Barsi, Julius C.; Deverman, Benjamin E.; Hunter, Ryan C.; Kato, Mihoko; Lee, S. Melanie; Abelin, Anna C. T.; Rosenthal, Adam Z.; Akbari, Omar S.; Li, Yuwei; Hay, Bruce A.; Sternberg, Paul W.; Patterson, Paul H.; Davidson, Eric H.; Mazmanian, Sarkis K.; Prober, David A.; van de Rijn, Matt; Leadbetter, Jared R.; Newman, Dianne K.; Readhead, Carol; Bronner, Marianne E.; Wold, Barbara; Lansford, Rusty; Sauka-Spengler, Tatjana; Fraser, Scott E.

2016-01-01

In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences. PMID:27702788

Weak percolation on multiplex networks

NASA Astrophysics Data System (ADS)

Baxter, Gareth J.; Dorogovtsev, Sergey N.; Mendes, José F. F.; Cellai, Davide

2014-04-01

Bootstrap percolation is a simple but nontrivial model. It has applications in many areas of science and has been explored on random networks for several decades. In single-layer (simplex) networks, it has been recently observed that bootstrap percolation, which is defined as an incremental process, can be seen as the opposite of pruning percolation, where nodes are removed according to a connectivity rule. Here we propose models of both bootstrap and pruning percolation for multiplex networks. We collectively refer to these two models with the concept of "weak" percolation, to distinguish them from the somewhat classical concept of ordinary ("strong") percolation. While the two models coincide in simplex networks, we show that they decouple when considering multiplexes, giving rise to a wealth of critical phenomena. Our bootstrap model constitutes the simplest example of a contagion process on a multiplex network and has potential applications in critical infrastructure recovery and information security. Moreover, we show that our pruning percolation model may provide a way to diagnose missing layers in a multiplex network. Finally, our analytical approach allows us to calculate critical behavior and characterize critical clusters.

4 × 20 Gbit/s mode division multiplexing over free space using vector modes and a q-plate mode (de)multiplexer

NASA Astrophysics Data System (ADS)

Milione, Giovanni; Lavery, Martin P. J.; Huang, Hao; Ren, Yongxiong; Xie, Guodong; Nguyen, Thien An; Karimi, Ebrahim; Marrucci, Lorenzo; Nolan, Daniel A.; Alfano, Robert R.; Willner, Alan E.

2015-05-01

Vector modes are spatial modes that have spatially inhomogeneous states of polarization, such as, radial and azimuthal polarization. They can produce smaller spot sizes and stronger longitudinal polarization components upon focusing. As a result, they are used for many applications, including optical trapping and nanoscale imaging. In this work, vector modes are used to increase the information capacity of free space optical communication via the method of optical communication referred to as mode division multiplexing. A mode (de)multiplexer for vector modes based on a liquid crystal technology referred to as a q-plate is introduced. As a proof of principle, using the mode (de)multiplexer four vector modes each carrying a 20 Gbit/s quadrature phase shift keying signal on a single wavelength channel (~1550nm), comprising an aggregate 80 Gbit/s, were transmitted ~1m over the lab table with <-16.4 dB (<2%) mode crosstalk. Bit error rates for all vector modes were measured at the forward error correction threshold with power penalties < 3.41dB.

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

PubMed Central

Alam, Mohammad J.; Tisdel, Naradah L.; Shah, Dhara N.; Yapar, Mehmet; Lasco, Todd M.; Garey, Kevin W.

2015-01-01

Background The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. Methods The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. Results A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. Conclusions The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run. PMID:25932438

Genome-wide association study of fertility traits in dairy cattle using high-density single nucleotide polymorphism marker panels

USDA-ARS?s Scientific Manuscript database

Unfavorable genetic correlations between production and fertility traits are well documented. Genetic selection for fertility traits is slow, however, due to low heritabilities. Identification of single nucleotide polymorphisms (SNP) involved in reproduction could improve reliability of genomic esti...

Discovery, Validation and Characterization of 1039 Cattle Single Nucleotide Polymorphisms

USDA-ARS?s Scientific Manuscript database

We identified approximately 13000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat-masked BAC-end sequences from the cattle RPCI-42 BAC library with whole-genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel ...

Developing Single Nucleotide Polymorphism (SNP) markers from transcriptome sequences for the identification of longan (Dimocarpus longan) germplasm

USDA-ARS?s Scientific Manuscript database

Longan (Dimocarpus longan Lour.) is an important tropical fruit tree crop. Accurate varietal identification is essential for germplasm management and breeding. Using longan transcriptome sequences from public databases, we developed single nucleotide polymorphism (SNP) markers; validated 60 SNPs in...

Informativeness of single nucleotide polymorphisms and relationships among onion populations from important world production regions

USDA-ARS?s Scientific Manuscript database

Single nucleotide polymorphisms (SNPs) were genotyped using a high-density array and DNAs from individual plants from important onion populations from major production regions world-wide and the likely progenitor of onion, Allium vavilovii. Genotypes at 1226 SNPs were used to estimate genetic relati...

Relationships among calpastatin single nucleotide polymorphisms, calpastatin expression and tenderness in pork longissimus

USDA-ARS?s Scientific Manuscript database

Genome scans in the pig have identified a region on chromosome 2 (SSC2) associated with tenderness. Calpastatin is a likely positional candidate gene in this region because of its inhibitory role in the calpain system that is involved in postmortem tenderization. Novel single nucleotide polymorphism...

Lineage and genogroup-defining single nucleotide polymorphisms of Escherichia coli 0157:H7

USDA-ARS?s Scientific Manuscript database

Escherichia coli O157:H7 is a zoonotic human pathogen for which cattle are an important reservoir host. Using both previously published and new sequencing data, a 48-locus single nucleotide polymorphism (SNP) based typing panel was developed that redundantly identified eleven genogroups that span ...

A new single-nucleotide polymorphisms database for rainbow trout generated through whole genome resequencing of selected samples

USDA-ARS?s Scientific Manuscript database

Single-nucleotide polymorphisms (SNPs) are highly abundant markers, which are broadly distributed in animal genomes. For rainbow trout, SNP discovery has been done through sequencing of restriction-site associated DNA (RAD) libraries, reduced representation libraries (RRL), RNA sequencing, and whole...

Development a of multiplex TaqMan real-time RT-PCR assay for simultaneous detection of Asian prunus viruses, plum bark necrosis stem pitting associated virus, and peach latent mosaic virus

USDA-ARS?s Scientific Manuscript database

Asian prunus viruses (APV 1, APV 2 and APV 3) and Plum bark necrosis stem pitting associated virus (PBNSPaV) are two recently described viruses infecting Prunus spp., and Peach latent mosaic viroid (PLMVd) is a viroid that infects the same species. A single-tube multiplex, TaqMan real-time RT-PCR as...

Topological charge number multiplexing for JTC multiple-image encryption

NASA Astrophysics Data System (ADS)

Chen, Qi; Shen, Xueju; Dou, Shuaifeng; Lin, Chao; Wang, Long

2018-04-01

We propose a method of topological charge number multiplexing based on the JTC encryption system to achieve multiple-image encryption. Using this method, multi-image can be encrypted into single ciphertext, and the original images can be recovered according to the authority level. The number of encrypted images is increased, moreover, the quality of decrypted images is improved. Results of computer simulation and initial experiment identify the validity of our proposed method.

The quest for rare variants: pooled multiplexed next generation sequencing in plants.

PubMed

Marroni, Fabio; Pinosio, Sara; Morgante, Michele

2012-01-01

Next generation sequencing (NGS) instruments produce an unprecedented amount of sequence data at contained costs. This gives researchers the possibility of designing studies with adequate power to identify rare variants at a fraction of the economic and labor resources required by individual Sanger sequencing. As of today, few research groups working in plant sciences have exploited this potentiality, showing that pooled NGS provides results in excellent agreement with those obtained by individual Sanger sequencing. The aim of this review is to convey to the reader the general ideas underlying the use of pooled NGS for the identification of rare variants. To facilitate a thorough understanding of the possibilities of the method, we will explain in detail the possible experimental and analytical approaches and discuss their advantages and disadvantages. We will show that information on allele frequency obtained by pooled NGS can be used to accurately compute basic population genetics indexes such as allele frequency, nucleotide diversity, and Tajima's D. Finally, we will discuss applications and future perspectives of the multiplexed NGS approach.

A multiplex PCR method discriminating between the TYLCV and TYLCV-Mld clades of tomato yellow leaf curl virus.

PubMed

Lefeuvre, P; Hoareau, M; Delatte, H; Reynaud, B; Lett, J-M

2007-09-01

Tomato yellow leaf curl virus (TYLCV) is one of the causal agents of tomato yellow leaf curl disease (TYLCD) and can cause up to 100% yield losses in tomato fields. As TYLCV continues to spread, many isolates have been described in different parts of the world. Recently two closely related but distinct TYLCV clades, called TYLCV and TYLCV-Mld, have been identified. Isolates from those two clades differ mainly in the nucleotide sequences of their replication associated protein genes but do not display significantly different symptomatology. In order to improve monitoring of the rapidly expanding worldwide TYLCD epidemic, a multiplex polymerase chain reaction assay (mPCR) was developed. A set of three primers were designed to detect and characterize the TYLCV and TYLCV-Mld clade isolates. The specificity and sensitivity of the mPCR were validated on TYLCV infected tomato plants and Bemisia tabaci whiteflies. Being cheap, fast and highly sensitive this new diagnostic tool should greatly simplify efforts to trace the global spread of TYLCV.

Comparison of DFT-S-orthogonal frequency division multiplexing and single-carrier in a radio-over-fiber system

NASA Astrophysics Data System (ADS)

Wang, Kaihui; Li, Xinying; Yu, Jianjun

2017-09-01

DFT-S-orthogonal frequency division multiplexing (OFDM) and single-carrier (SC) modulation are two typical modulation formats in radio-over-fiber (RoF) systems. They may have respective advantages and disadvantages in different scenarios. Therefore, bit error ratio comparison results of these two modulation formats will be useful for designing and optimizing the practical RoF system. We experimentally compare these two modulation formats in a long wireless distance RoF system at W-band. It can be concluded that DFT-S-OFDM and SC modulation have similar performances in a RoF system with transmission distance over 80-km fiber and 224-m wireless link.

Single-shot quantitative phase microscopy with color-multiplexed differential phase contrast (cDPC)

PubMed Central

2017-01-01

We present a new technique for quantitative phase and amplitude microscopy from a single color image with coded illumination. Our system consists of a commercial brightfield microscope with one hardware modification—an inexpensive 3D printed condenser insert. The method, color-multiplexed Differential Phase Contrast (cDPC), is a single-shot variant of Differential Phase Contrast (DPC), which recovers the phase of a sample from images with asymmetric illumination. We employ partially coherent illumination to achieve resolution corresponding to 2× the objective NA. Quantitative phase can then be used to synthesize DIC and phase contrast images or extract shape and density. We demonstrate amplitude and phase recovery at camera-limited frame rates (50 fps) for various in vitro cell samples and c. elegans in a micro-fluidic channel. PMID:28152023

Single line-of-sight dual energy backlighter for mix width experiments

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

Baker, K. L., E-mail: baker7@llnl.gov; Glendinning, S. G.; Martinez, D.

2014-11-15

We present a diagnostic technique used to spatially multiplex two x-ray radiographs of an object onto a detector along a single line-of-sight. This technique uses a thin, <2 μm, cosputtered backlighter target to simultaneously produce both Ni and Zn He{sub α} emission. A Ni picket fence filter, 500 μm wide bars and troughs, is then placed in front of the detector to pass only the Ni He{sub α} emission in the bar region and both energies in the trough region thereby spatially multiplexing the two radiographs on a single image. Initial experimental results testing the backlighter spectrum are presented alongmore » with simulated images showing the calculated radiographic images though the nickel picket fence filter which are used to measure the mix width in an accelerated nickel foam.« less

A novel MALDI–TOF based methodology for genotyping single nucleotide polymorphisms

PubMed Central

Blondal, Thorarinn; Waage, Benedikt G.; Smarason, Sigurdur V.; Jonsson, Frosti; Fjalldal, Sigridur B.; Stefansson, Kari; Gulcher, Jeffery; Smith, Albert V.

2003-01-01

A new MALDI–TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3′-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis. PMID:14654708

Thermally multiplexed polymerase chain reaction.

PubMed

Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

2015-07-01

Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel.

Prevalence of Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus, and Salmonella enterica Typhimurium in meat and meat products using multiplex polymerase chain reaction

PubMed Central

Latha, C.; Anu, C. J.; Ajaykumar, V. J.; Sunil, B.

2017-01-01

Aim: The objective of the study was to investigate the occurrence of Listeria monocytogenes, Yersinia enterocolitica, Staphylococcus aureus, and Salmonella enterica Typhimurium in meat and meat products using the multiplex polymerase chain reaction (PCR) method. Materials and Methods: The assay combined an enrichment step in tryptic soy broth with yeast extract formulated for the simultaneous growth of target pathogens, DNA isolation and multiplex PCR. A total of 1134 samples including beef (n=349), chicken (n=325), pork (n=310), chevon (n=50), and meat products (n=100) were collected from different parts of Kerala, India. All the samples were subjected to multiplex PCR analysis and culture-based detection for the four pathogens in parallel. Results: Overall occurrence of L. monocytogenes was 0.08 % by cultural method. However, no L. monocytogenes was obtained by multiplex PCR method. Yersinia enterocolitica was obtained from beef and pork samples. A high prevalence of S. aureus (46.7%) was found in all types of meat samples tested. None of the samples was positive for S. Typhimurium. Conclusion: Multiplex PCR assay used in this study can detect more than one pathogen simultaneously by amplifying more than one target gene in a single reaction, which can save time and labor cost. PMID:28919685

Biological nanopore MspA for DNA sequencing

NASA Astrophysics Data System (ADS)

Manrao, Elizabeth A.

Unlocking the information hidden in the human genome provides insight into the inner workings of complex biological systems and can be used to greatly improve health-care. In order to allow for widespread sequencing, new technologies are required that provide fast and inexpensive readings of DNA. Nanopore sequencing is a third generation DNA sequencing technology that is currently being developed to fulfill this need. In nanopore sequencing, a voltage is applied across a small pore in an electrolyte solution and the resulting ionic current is recorded. When DNA passes through the channel, the ionic current is partially blocked. If the DNA bases uniquely modulate the ionic current flowing through the channel, the time trace of the current can be related to the sequence of DNA passing through the pore. There are two main challenges to realizing nanopore sequencing: identifying a pore with sensitivity to single nucleotides and controlling the translocation of DNA through the pore so that the small single nucleotide current signatures are distinguishable from background noise. In this dissertation, I explore the use of Mycobacterium smegmatis porin A (MspA) for nanopore sequencing. In order to determine MspA's sensitivity to single nucleotides, DNA strands of various compositions are held in the pore as the resulting ionic current is measured. DNA is immobilized in MspA by attaching it to a large molecule which acts as an anchor. This technique confirms the single nucleotide resolution of the pore and additionally shows that MspA is sensitive to epigenetic modifications and single nucleotide polymorphisms. The forces from the electric field within MspA, the effective charge of nucleotides, and elasticity of DNA are estimated using a Freely Jointed Chain model of single stranded DNA. These results offer insight into the interactions of DNA within the pore. With the nucleotide sensitivity of MspA confirmed, a method is introduced to controllably pass DNA through the pore. Using a DNA polymerase, DNA strands are stepped through MspA one nucleotide at a time. The steps are observable as distinct levels on the ionic-current time-trace and are related to the DNA sequence. These experiments overcome the two fundamental challenges to realizing MspA nanopore sequencing and pave the way to the development of a commercial technology.

Optically-synchronized encoder and multiplexer scheme for interleaved photonics analog-to-digital conversion

NASA Astrophysics Data System (ADS)

Villa, Carlos; Kumavor, Patrick; Donkor, Eric

2008-04-01

Photonics Analog-to-Digital Converters (ADCs) utilize a train of optical pulses to sample an electrical input waveform applied to an electrooptic modulator or a reverse biased photodiode. In the former, the resulting train of amplitude-modulated optical pulses is detected (converter to electrical) and quantized using a conversional electronics ADC- as at present there are no practical, cost-effective optical quantizers available with performance that rival electronic quantizers. In the latter, the electrical samples are directly quantized by the electronics ADC. In both cases however, the sampling rate is limited by the speed with which the electronics ADC can quantize the electrical samples. One way to increase the sampling rate by a factor N is by using the time-interleaved technique which consists of a parallel array of N electrical ADC converters, which have the same sampling rate but different sampling phase. Each operating at a quantization rate of fs/N where fs is the aggregated sampling rate. In a system with no real-time operation, the N channels digital outputs are stored in memory, and then aggregated (multiplexed) to obtain the digital representation of the analog input waveform. Alternatively, for real-time operation systems the reduction of storing time in the multiplexing process is desired to improve the time response of the ADC. The complete elimination of memories come expenses of concurrent timing and synchronization in the aggregation of the digital signal that became critical for a good digital representation of the analog signal waveform. In this paper we propose and demonstrate a novel optically synchronized encoder and multiplexer scheme for interleaved photonics ADCs that utilize the N optical signals used to sample different phases of an analog input signal to synchronize the multiplexing of the resulting N digital output channels in a single digital output port. As a proof of concept, four 320 Megasamples/sec 12-bit of resolution digital signals were multiplexed to form an aggregated 1.28 Gigasamples/sec single digital output signal.

a Study of Multiplexing Schemes for Voice and Data.

NASA Astrophysics Data System (ADS)

Sriram, Kotikalapudi

Voice traffic variations are characterized by on/off transitions of voice calls, and talkspurt/silence transitions of speakers in conversations. A speaker is known to be in silence for more than half the time during a telephone conversation. In this dissertation, we study some schemes which exploit speaker silences for an efficient utilization of the transmission capacity in integrated voice/data multiplexing and in digital speech interpolation. We study two voice/data multiplexing schemes. In each scheme, any time slots momentarily unutilized by the voice traffic are made available to data. In the first scheme, the multiplexer does not use speech activity detectors (SAD), and hence the voice traffic variations are due to call on/off only. In the second scheme, the multiplexer detects speaker silences using SAD and transmits voice only during talkspurts. The multiplexer with SAD performs digital speech interpolation (DSI) as well as dynamic channel allocation to voice and data. The performance of the two schemes is evaluated using discrete-time modeling and analysis. The data delay performance for the case of English speech is compared with that for the case of Japanese speech. A closed form expression for the mean data message delay is derived for the single-channel single-talker case. In a DSI system, occasional speech losses occur whenever the number of speakers in simultaneous talkspurt exceeds the number of TDM voice channels. In a buffered DSI system, speech loss is further reduced at the cost of delay. We propose a novel fixed-delay buffered DSI scheme. In this scheme, speech fill-in/hangover is not required because there are no variable delays. Hence, all silences that naturally occur in speech are fully utilized. Consequently, a substantial improvement in the DSI performance is made possible. The scheme is modeled and analyzed in discrete -time. Its performance is evaluated in terms of the probability of speech clipping, packet rejection ratio, DSI advantage, and the delay.

Variation of Cats under Domestication: Genetic Assignment of Domestic Cats to Breeds and Worldwide Random Bred Populations

PubMed Central

Kurushima, J. D.; Lipinski, M. J.; Gandolfi, B.; Froenicke, L.; Grahn, J. C.; Grahn, R. A.; Lyons, L. A.

2012-01-01

Summary Both cat breeders and the lay public have interests in the origins of their pets, not only in the genetic identity of the purebred individuals, but also the historical origins of common household cats. The cat fancy is a relatively new institution with over 85% of its 40–50 breeds arising only in the past 75 years, primarily through selection on single-gene aesthetic traits. The short, yet intense cat breed history poses a significant challenge to the development of a genetic marker-based breed identification strategy. Using different breed assignment strategies and methods, 477 cats representing 29 fancy breeds were analysed with 38 short tandem repeats, 148 intergenic and five phenotypic single nucleotide polymorphisms. Results suggest the frequentist method of Paetkau (accuracy single nucleotide polymorphisms = 0.78, short tandem repeats = 0.88) surpasses the Bayesian method of Rannala and Mountain (single nucleotide polymorphisms = 0.56, short tandem repeats = 0.83) for accurate assignment of individuals to the correct breed. Additionally, a post-assignment verification step with the five phenotypic single nucleotide polymorphisms accurately identified between 0.31 and 0.58 of the mis-assigned individuals raising the sensitivity of assignment with the frequentist method to 0.89 and 0.92 single nucleotide polymorphisms and short tandem repeats respectively. This study provides a novel multi-step assignment strategy and suggests that, despite their short breed history and breed family groupings, a majority of cats can be assigned to their proper breed or population of origin, i.e. race. PMID:23171373

An Integrated Quantum Dot Barcode Smartphone Optical Device for Wireless Multiplexed Diagnosis of Infected Patients

NASA Astrophysics Data System (ADS)

Ming, Kevin

Integrating mobile-cellular devices with multiplex molecular diagnostics can potentially provide the most powerful platform for tracking, managing and preventing the transmission of infectious diseases. With over 6.9 billion subscriptions globally, handheld mobile-cellular devices can be programmed to spatially map, temporally track, and transmit information on infections over wide geographical space and boundaries. Current cell phone diagnostic technologies have poor limit of detection, dynamic range, and cannot detect multiple pathogen targets simultaneously, limiting their utility to single infections with high load. Here we combined recent advances in quantum dot barcode technology for molecular detection with smartphones to engineer a simple and low-cost chip-based wireless multiplex diagnostic device. We validated our device using a variety of synthetic genomic targets for the respiratory virus and blood-borne pathogens, and demonstrated that it could detect clinical samples after simple amplification. More importantly, we confirmed that the device is capable of detecting patients infected with a single or multiple infectious pathogens (e.g., HIV and hepatitis B) in a single test. This device advances the capacity for global surveillance of infectious diseases and has the potential to accelerate knowledge exchange-transfer of emerging or exigent disease threats with healthcare and military organizations in real-time.

Detection of BRAF mutations from solid tumors using Tumorplex™ technology

PubMed Central

Yo, Jacob; Hay, Katie S.L.; Vinayagamoorthy, Dilanthi; Maryanski, Danielle; Carter, Mark; Wiegel, Joseph; Vinayagamoorthy, Thuraiayah

2015-01-01

Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. • Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal. • Tumorplex™ provides a high level of sensitivity. • Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA. PMID:26258049

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree

PubMed Central

2013-01-01

Background With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. Results The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. Conclusions Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology. PMID:23497206

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.

Next generation sequencing of SNPs for non-invasive prenatal diagnosis: challenges and feasibility as illustrated by an application to β-thalassaemia

PubMed Central

Papasavva, Thessalia; van IJcken, Wilfred F J; Kockx, Christel E M; van den Hout, Mirjam C G N; Kountouris, Petros; Kythreotis, Loukas; Kalogirou, Eleni; Grosveld, Frank G; Kleanthous, Marina

2013-01-01

β-Thalassaemia is one of the most common autosomal recessive single-gene disorder worldwide, with a carrier frequency of 12% in Cyprus. Prenatal tests for at risk pregnancies use invasive methods and development of a non-invasive prenatal diagnostic (NIPD) method is of paramount importance to prevent unnecessary risks inherent to invasive methods. Here, we describe such a method by assessing a modified version of next generation sequencing (NGS) using the Illumina platform, called ‘targeted sequencing', based on the detection of paternally inherited fetal alleles in maternal plasma. We selected four single-nucleotide polymorphisms (SNPs) located in the β-globin locus with a high degree of heterozygosity in the Cypriot population. Spiked genomic samples were used to determine the specificity of the platform. We could detect the minor alleles in the expected ratio, showing the specificity of the platform. We then developed a multiplexed format for the selected SNPs and analysed ten maternal plasma samples from pregnancies at risk. The presence or absence of the paternal mutant allele was correctly determined in 27 out of 34 samples analysed. With haplotype analysis, NIPD was possible on eight out of ten families. This is the first study carried out for the NIPD of β-thalassaemia using targeted NGS and haplotype analysis. Preliminary results show that NGS is effective in detecting paternally inherited alleles in the maternal plasma. PMID:23572027

Imputation of single nucleotide polymorhpism genotypes of Hereford cattle: reference panel size, family relationship and population structure

USDA-ARS?s Scientific Manuscript database

The objective of this study is to investigate single nucleotide polymorphism (SNP) genotypes imputation of Hereford cattle. Purebred Herefords were from two sources, Line 1 Hereford (N=240) and representatives of Industry Herefords (N=311). Using different reference panels of 62 and 494 males with 1...

A resource of single-nucleotide polymorphisms for rainbow trout generated by restriction-site associated DNA sequencing of doubled haploids

USDA-ARS?s Scientific Manuscript database

Salmonid genomes are considered to be in a pseudo-tetraploid state as a result of an evolutionarily recent genome duplication event. This situation complicates single nucleotide polymorphism (SNP) discovery in rainbow trout as many putative SNPs are actually paralogous sequence variants (PSVs) and ...

Single nucleotide polymorphisms in candidate genes associated with fertilizing ability of sperm and subsequent embryonic development in cattle

USDA-ARS?s Scientific Manuscript database

Fertilization and development of the preimplantation embryo is under genetic control. The goal of the current study was to test 434 single nucleotide polymorphisms (SNPs) for association with genetic variation in fertilization and early embryonic development. The approach was to produce embryos from...

Prospects for inferring pairwise relationships with single nucleotide polymorphisms

Treesearch

Jeffery C. Glaubitz; O. Eugene, Jr. Rhodes; J. Andrew DeWoody

2003-01-01

An extraordinarily large number of single nucleotide polymorphisms (SNPs) are now available in humans as well as in other model organisms. Technological advancements may soon make it feasible to assay hundreds of SNPs in virtually any organism of interest. One potential application of SNPs is the determination of pairwise genetic relationships in populations without...

Short communication: Relationship of call rate and accuracy of single nucleotide polymorphism genotypes in dairy cattle

USDA-ARS?s Scientific Manuscript database

Call rate has been used as a measure of quality on both a single nucleotide polymorphism (SNP) and animal basis since SNP genotypes were first used in genomic evaluation of dairy cattle. The genotyping laboratories perform initial quality control screening and genotypes that fail are usually exclude...

Single nucleotide polymorphisms generated by genotyping by sequencing to characterize genome-wide diversity, linkage disequilibrium, and selective sweeps in cultivated watermelon

USDA-ARS?s Scientific Manuscript database

Large datasets containing single nucleotide polymorphisms (SNPs) are used to analyze genome-wide diversity in a robust collection of cultivars from representative accessions, across the world. The extent of linkage disequilibrium (LD) within a population determines the number of markers required fo...

Lack of Association Between Polymorphisms in Dopa Decarboxylase and Dopamine Receptor-1 Genes With Childhood Autism in Chinese Han Population.

PubMed

Yu, Hong; Liu, Jun; Yang, Aiping; Yang, Guohui; Yang, Wenjun; Lei, Heyue; Quan, Jianjun; Zhang, Zengyu

2016-04-01

Genetic factors play an important role in childhood autism. This study is to determine the association of single-nucleotide polymorphisms in dopa decarboxylase (DDC) and dopamine receptor-1 (DRD1) genes with childhood autism, in a Chinese Han population. A total of 211 autistic children and 250 age- and gender-matched healthy controls were recruited. The severity of disease was determined by Children Autism Rating Scale scores. TaqMan Probe by real-time polymerase chain reaction was used to determine genotypes and allele frequencies of single-nucleotide polymorphism rs6592961 in DDC and rs251937 in DRD1. Case-control and case-only studies were respectively performed, to determine the contribution of both single-nucleotide polymorphisms to the predisposition of disease and its severity. Our results showed that there was no significant association of the genotypes and allele frequencies of both single-nucleotide polymorphisms concerning childhood autism and its severity. More studies with larger samples are needed to corroborate their predicting roles. © The Author(s) 2015.

Single-molecule comparison of DNA Pol I activity with native and analog nucleotides

NASA Astrophysics Data System (ADS)

Gul, Osman; Olsen, Tivoli; Choi, Yongki; Corso, Brad; Weiss, Gregory; Collins, Philip

2014-03-01

DNA polymerases are critical enzymes for DNA replication, and because of their complex catalytic cycle they are excellent targets for investigation by single-molecule experimental techniques. Recently, we studied the Klenow fragment (KF) of DNA polymerase I using a label-free, electronic technique involving single KF molecules attached to carbon nanotube transistors. The electronic technique allowed long-duration monitoring of a single KF molecule while processing thousands of template strands. Processivity of up to 42 nucleotide bases was directly observed, and statistical analysis of the recordings determined key kinetic parameters for the enzyme's open and closed conformations. Subsequently, we have used the same technique to compare the incorporation of canonical nucleotides like dATP to analogs like 1-thio-2'-dATP. The analog had almost no affect on duration of the closed conformation, during which the nucleotide is incorporated. On the other hand, the analog increased the rate-limiting duration of the open conformation by almost 40%. We propose that the thiolated analog interferes with KF's recognition and binding, two key steps that determine its ensemble turnover rate.

Multiplexed aberration measurement for deep tissue imaging in vivo

PubMed Central

Wang, Chen; Liu, Rui; Milkie, Daniel E.; Sun, Wenzhi; Tan, Zhongchao; Kerlin, Aaron; Chen, Tsai-Wen; Kim, Douglas S.; Ji, Na

2014-01-01

We describe a multiplexed aberration measurement method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine their phase gradients. Applicable to fluorescent-protein-labeled structures of arbitrary complexity, it allows us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improves structural and functional imaging of fine neuronal processes over a large imaging volume. PMID:25128976

AFTI/F16 Automated Maneuvering Attack System Test Reports/Special Technologies and Outlook.

DTIC Science & Technology

1986-07-11

Multiplex Data Bus A-A Air-To-Air A-S Air-to-Surface AFTI Advanced Fighter Technology Integration SYSTEM DESIGN AGL Above-Ground-Level AMAS Automated...Maneuvering Attack System Design requirements for the AFTI/F-16 are driven AMUX Avionics Multiplex Data Bus by realistic air combat scenarios and are...the avionics subsystem IFIM and avionics systems are single-thread, much of the sensed various flight control sensors. Additionally, along with data

Single Multiplex PCR Assay To Identify Simultaneously the Six Categories of Diarrheagenic Escherichia coli Associated with Enteric Infections

PubMed Central

Vidal, Maricel; Kruger, Eileen; Durán, Claudia; Lagos, Rosanna; Levine, Myron; Prado, Valeria; Toro, Cecilia; Vidal, Roberto

2005-01-01

We designed a multiplex PCR for the detection of all categories of diarrheagenic Escherichia coli. This method proved to be specific and rapid in detecting virulence genes from Shiga toxin-producing (stx1, stx2, and eae), enteropathogenic (eae and bfp), enterotoxigenic (stII and lt), enteroinvasive (virF and ipaH), enteroaggregative (aafII), and diffuse adherent (daaE) Escherichia coli in stool samples. PMID:16208019

Uplink transmission of a 60-km-reach WDM/OCDM-PON using a spectrum-sliced pulse source

NASA Astrophysics Data System (ADS)

Choi, Yong-Kyu; Hanawa, Masanori; Park, Chang-Soo

2014-02-01

We propose and experimentally demonstrate the uplink transmission of a 60-km-reach wavelength division multiplexing/optical code division multiplexing (WDM/OCDM) passive optical network (PON) using a spectrum-sliced pulse source. As a single light source, a broadband pulse source with a bandwidth of 6.5 nm and a repetition rate of 1.25 GHz is generated at a central office and supplied to a remote node (RN) through a 50-km fiber link. At the RN, narrow-band pulses (as a source for uplink transmission) are obtained by spectrum slicing the broadband pulse source with a cyclic arrayed waveguide grating and are then supplied to all optical network units (ONUs) via 1×4 power splitters and 10-km drop fibers. Eight wavelengths are obtained with a 6.5-nm bandwidth of the broadband pulse source, and the qualities of the pulses with a repetition rate of 1.25 GHz and a pulse width of 45 ps for the eight wavelengths are sufficient for four-chip OCDM encoding at the ONUs. In our experiments, four signals are multiplexed by OCDM at one wavelength, and another encoded signal is also multiplexed by WDM. The bit error rates (BERs) of the signals exhibit error-free transmission (BER<10-9) over a 60-km single-mode fiber at 1.25 Gb/s.

Characterization of LDPC-coded orbital angular momentum modes transmission and multiplexing over a 50-km fiber.

PubMed

Wang, Andong; Zhu, Long; Chen, Shi; Du, Cheng; Mo, Qi; Wang, Jian

2016-05-30

Mode-division multiplexing over fibers has attracted increasing attention over the last few years as a potential solution to further increase fiber transmission capacity. In this paper, we demonstrate the viability of orbital angular momentum (OAM) modes transmission over a 50-km few-mode fiber (FMF). By analyzing mode properties of eigen modes in an FMF, we study the inner mode group differential modal delay (DMD) in FMF, which may influence the transmission capacity in long-distance OAM modes transmission and multiplexing. To mitigate the impact of large inner mode group DMD in long-distance fiber-based OAM modes transmission, we use low-density parity-check (LDPC) codes to increase the system reliability. By evaluating the performance of LDPC-coded single OAM mode transmission over 50-km fiber, significant coding gains of >4 dB, 8 dB and 14 dB are demonstrated for 1-Gbaud, 2-Gbaud and 5-Gbaud quadrature phase-shift keying (QPSK) signals, respectively. Furthermore, in order to verify and compare the influence of DMD in long-distance fiber transmission, single OAM mode transmission over 10-km FMF is also demonstrated in the experiment. Finally, we experimentally demonstrate OAM multiplexing and transmission over a 50-km FMF using LDPC-coded 1-Gbaud QPSK signals to compensate the influence of mode crosstalk and DMD in the 50 km FMF.

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue

PubMed Central

Gerdes, Michael J.; Sevinsky, Christopher J.; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O.; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J.; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Lowes, Christina; McCulloch, Colin C.; McDonough, Elizabeth; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D.; Seel, Maximilian L.; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-01-01

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics. PMID:23818604

Highly multiplexed single-cell analysis of formalin-fixed, paraffin-embedded cancer tissue.

PubMed

Gerdes, Michael J; Sevinsky, Christopher J; Sood, Anup; Adak, Sudeshna; Bello, Musodiq O; Bordwell, Alexander; Can, Ali; Corwin, Alex; Dinn, Sean; Filkins, Robert J; Hollman, Denise; Kamath, Vidya; Kaanumalle, Sireesha; Kenny, Kevin; Larsen, Melinda; Lazare, Michael; Li, Qing; Lowes, Christina; McCulloch, Colin C; McDonough, Elizabeth; Montalto, Michael C; Pang, Zhengyu; Rittscher, Jens; Santamaria-Pang, Alberto; Sarachan, Brion D; Seel, Maximilian L; Seppo, Antti; Shaikh, Kashan; Sui, Yunxia; Zhang, Jingyu; Ginty, Fiona

2013-07-16

Limitations on the number of unique protein and DNA molecules that can be characterized microscopically in a single tissue specimen impede advances in understanding the biological basis of health and disease. Here we present a multiplexed fluorescence microscopy method (MxIF) for quantitative, single-cell, and subcellular characterization of multiple analytes in formalin-fixed paraffin-embedded tissue. Chemical inactivation of fluorescent dyes after each image acquisition round allows reuse of common dyes in iterative staining and imaging cycles. The mild inactivation chemistry is compatible with total and phosphoprotein detection, as well as DNA FISH. Accurate computational registration of sequential images is achieved by aligning nuclear counterstain-derived fiducial points. Individual cells, plasma membrane, cytoplasm, nucleus, tumor, and stromal regions are segmented to achieve cellular and subcellular quantification of multiplexed targets. In a comparison of pathologist scoring of diaminobenzidine staining of serial sections and automated MxIF scoring of a single section, human epidermal growth factor receptor 2, estrogen receptor, p53, and androgen receptor staining by diaminobenzidine and MxIF methods yielded similar results. Single-cell staining patterns of 61 protein antigens by MxIF in 747 colorectal cancer subjects reveals extensive tumor heterogeneity, and cluster analysis of divergent signaling through ERK1/2, S6 kinase 1, and 4E binding protein 1 provides insights into the spatial organization of mechanistic target of rapamycin and MAPK signal transduction. Our results suggest MxIF should be broadly applicable to problems in the fields of basic biological research, drug discovery and development, and clinical diagnostics.

Transcript-specific, single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.).

PubMed

Allen, Alexandra M; Barker, Gary L A; Berry, Simon T; Coghill, Jane A; Gwilliam, Rhian; Kirby, Susan; Robinson, Phil; Brenchley, Rachel C; D'Amore, Rosalinda; McKenzie, Neil; Waite, Darren; Hall, Anthony; Bevan, Michael; Hall, Neil; Edwards, Keith J

2011-12-01

Food security is a global concern and substantial yield increases in cereal crops are required to feed the growing world population. Wheat is one of the three most important crops for human and livestock feed. However, the complexity of the genome coupled with a decline in genetic diversity within modern elite cultivars has hindered the application of marker-assisted selection (MAS) in breeding programmes. A crucial step in the successful application of MAS in breeding programmes is the development of cheap and easy to use molecular markers, such as single-nucleotide polymorphisms. To mine selected elite wheat germplasm for intervarietal single-nucleotide polymorphisms, we have used expressed sequence tags derived from public sequencing programmes and next-generation sequencing of normalized wheat complementary DNA libraries, in combination with a novel sequence alignment and assembly approach. Here, we describe the development and validation of a panel of 1114 single-nucleotide polymorphisms in hexaploid bread wheat using competitive allele-specific polymerase chain reaction genotyping technology. We report the genotyping results of these markers on 23 wheat varieties, selected to represent a broad cross-section of wheat germplasm including a number of elite UK varieties. Finally, we show that, using relatively simple technology, it is possible to rapidly generate a linkage map containing several hundred single-nucleotide polymorphism markers in the doubled haploid mapping population of Avalon × Cadenza. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

TPH-2 Polymorphisms Interact with Early Life Stress to Influence Response to Treatment with Antidepressant Drugs.

PubMed

Xu, Zhi; Reynolds, Gavin P; Yuan, Yonggui; Shi, Yanyan; Pu, Mengjia; Zhang, Zhijun

2016-11-01

Variation in genes implicated in monoamine neurotransmission may interact with environmental factors to influence antidepressant response. We aimed to determine how a range of single nucleotide polymorphisms in monoaminergic genes influence this response to treatment and how they interact with childhood trauma and recent life stress in a Chinese sample. An initial study of monoaminergic coding region single nucleotide polymorphisms identified significant associations of TPH2 and HTR1B single nucleotide polymorphisms with treatment response that showed interactions with childhood and recent life stress, respectively (Xu et al., 2012). A total of 47 further single nucleotide polymorphisms in 17 candidate monoaminergic genes were genotyped in 281 Chinese Han patients with major depressive disorder. Response to 6 weeks' antidepressant treatment was determined by change in the 17-item Hamilton Depression Rating Scale score, and previous stressful events were evaluated by the Life Events Scale and Childhood Trauma Questionnaire-Short Form. Three TPH2 single nucleotide polymorphisms (rs11178998, rs7963717, and rs2171363) were significantly associated with antidepressant response in this Chinese sample, as was a haplotype in TPH2 (rs2171363 and rs1487278). One of these, rs2171363, showed a significant interaction with childhood adversity in its association with antidepressant response. These findings provide further evidence that variation in TPH2 is associated with antidepressant response and may also interact with childhood trauma to influence outcome of antidepressant treatment. © The Author 2016. Published by Oxford University Press on behalf of CINP.

TPH-2 Polymorphisms Interact with Early Life Stress to Influence Response to Treatment with Antidepressant Drugs

PubMed Central

Reynolds, Gavin P.; Yuan, Yonggui; Shi, Yanyan; Pu, Mengjia; Zhang, Zhijun

2016-01-01

Background: Variation in genes implicated in monoamine neurotransmission may interact with environmental factors to influence antidepressant response. We aimed to determine how a range of single nucleotide polymorphisms in monoaminergic genes influence this response to treatment and how they interact with childhood trauma and recent life stress in a Chinese sample. An initial study of monoaminergic coding region single nucleotide polymorphisms identified significant associations of TPH2 and HTR1B single nucleotide polymorphisms with treatment response that showed interactions with childhood and recent life stress, respectively (Xu et al., 2012). Methods: A total of 47 further single nucleotide polymorphisms in 17 candidate monoaminergic genes were genotyped in 281 Chinese Han patients with major depressive disorder. Response to 6 weeks’ antidepressant treatment was determined by change in the 17-item Hamilton Depression Rating Scale score, and previous stressful events were evaluated by the Life Events Scale and Childhood Trauma Questionnaire-Short Form. Results: Three TPH2 single nucleotide polymorphisms (rs11178998, rs7963717, and rs2171363) were significantly associated with antidepressant response in this Chinese sample, as was a haplotype in TPH2 (rs2171363 and rs1487278). One of these, rs2171363, showed a significant interaction with childhood adversity in its association with antidepressant response. Conclusions: These findings provide further evidence that variation in TPH2 is associated with antidepressant response and may also interact with childhood trauma to influence outcome of antidepressant treatment. PMID:27521242

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

PubMed

Niu, Chenqi; Xu, Yuancong; Zhang, Chao; Zhu, Pengyu; Huang, Kunlun; Luo, Yunbo; Xu, Wentao

2018-05-01

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

Integration of Multiplexed Microfluidic Electrokinetic Concentrators with a Morpholino Microarray via Reversible Surface Bonding for Enhanced DNA Hybridization.

PubMed

Martins, Diogo; Wei, Xi; Levicky, Rastislav; Song, Yong-Ak

2016-04-05

We describe a microfluidic concentration device to accelerate the surface hybridization reaction between DNA and morpholinos (MOs) for enhanced detection. The microfluidic concentrator comprises a single polydimethylsiloxane (PDMS) microchannel onto which an ion-selective layer of conductive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) ( PSS) was directly printed and then reversibly surface bonded onto a morpholino microarray for hybridization. Using this electrokinetic trapping concentrator, we could achieve a maximum concentration factor of ∼800 for DNA and a limit of detection of 10 nM within 15 min. In terms of the detection speed, it enabled faster hybridization by around 10-fold when compared to conventional diffusion-based hybridization. A significant advantage of our approach is that the fabrication of the microfluidic concentrator is completely decoupled from the microarray; by eliminating the need to deposit an ion-selective layer on the microarray surface prior to device integration, interfacing between both modules, the PDMS chip for electrokinetic concentration and the substrate for DNA sensing are easier and applicable to any microarray platform. Furthermore, this fabrication strategy facilitates a multiplexing of concentrators. We have demonstrated the proof-of-concept for multiplexing by building a device with 5 parallel concentrators connected to a single inlet/outlet and applying it to parallel concentration and hybridization. Such device yielded similar concentration and hybridization efficiency compared to that of a single-channel device without adding any complexity to the fabrication and setup. These results demonstrate that our concentrator concept can be applied to the development of a highly multiplexed concentrator-enhanced microarray detection system for either genetic analysis or other diagnostic assays.

Homeostatic plasticity for single node delay-coupled reservoir computing.

PubMed

Toutounji, Hazem; Schumacher, Johannes; Pipa, Gordon

2015-06-01

Supplementing a differential equation with delays results in an infinite-dimensional dynamical system. This property provides the basis for a reservoir computing architecture, where the recurrent neural network is replaced by a single nonlinear node, delay-coupled to itself. Instead of the spatial topology of a network, subunits in the delay-coupled reservoir are multiplexed in time along one delay span of the system. The computational power of the reservoir is contingent on this temporal multiplexing. Here, we learn optimal temporal multiplexing by means of a biologically inspired homeostatic plasticity mechanism. Plasticity acts locally and changes the distances between the subunits along the delay, depending on how responsive these subunits are to the input. After analytically deriving the learning mechanism, we illustrate its role in improving the reservoir's computational power. To this end, we investigate, first, the increase of the reservoir's memory capacity. Second, we predict a NARMA-10 time series, showing that plasticity reduces the normalized root-mean-square error by more than 20%. Third, we discuss plasticity's influence on the reservoir's input-information capacity, the coupling strength between subunits, and the distribution of the readout coefficients.

Method and Apparatus of Multiplexing and Acquiring Data from Multiple Optical Fibers Using a Single Data Channel of an Optical Frequency-Domain Reflectometry (OFDR) System

NASA Technical Reports Server (NTRS)

Parker, Jr., Allen R (Inventor); Chan, Hon Man (Inventor); Piazza, Anthony (Nino) (Inventor); Richards, William Lance (Inventor)

2014-01-01

A method and system for multiplexing a network of parallel fiber Bragg grating (FBG) sensor-fibers to a single acquisition channel of a closed Michelson interferometer system via a fiber splitter by distinguishing each branch of fiber sensors in the spatial domain. On each branch of the splitter, the fibers have a specific pre-determined length, effectively separating each branch of fiber sensors spatially. In the spatial domain the fiber branches are seen as part of one acquisition channel on the interrogation system. However, the FBG-reference arm beat frequency information for each fiber is retained. Since the beat frequency is generated between the reference arm, the effective fiber length of each successive branch includes the entire length of the preceding branch. The multiple branches are seen as one fiber having three segments where the segments can be resolved. This greatly simplifies optical, electronic and computational complexity, and is especially suited for use in multiplexed or branched OFS networks for SHM of large and/or distributed structures which need a lot of measurement points.

Development and validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples.

PubMed

Wang, Dennis Y; Chang, Chien-Wei; Lagacé, Robert E; Oldroyd, Nicola J; Hennessy, Lori K

2011-07-01

The AmpFℓSTR(®) Identifiler(®) Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single-source blood and buccal samples on FTA(®) card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay's sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA(®) cards, and the assay's specificity was verified by establishing minimal cross-reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA(®) substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler(®) Direct Kit for forensic standards and database samples genotyping. © 2011 American Academy of Forensic Sciences.

Simple and fast multiplex PCR method for detection of species origin in meat products.

PubMed

Izadpanah, Mehrnaz; Mohebali, Nazanin; Elyasi Gorji, Zahra; Farzaneh, Parvaneh; Vakhshiteh, Faezeh; Shahzadeh Fazeli, Seyed Abolhassan

2018-02-01

Identification of animal species is one of the major concerns in food regulatory control and quality assurance system. Different approaches have been used for species identification in animal origin of feedstuff. This study aimed to develop a multiplex PCR approach to detect the origin of meat and meat products. Specific primers were designed based on the conserved region of mitochondrial Cytochrome C Oxidase subunit I ( COX1 ) gene. This method could successfully distinguish the origin of the pig, camel, sheep, donkey, goat, cow, and chicken in one single reaction. Since PCR products derived from each species represent unique molecular weight, the amplified products could be identified by electrophoresis and analyzed based on their size. Due to the synchronized amplification of segments within a single PCR reaction, multiplex PCR is considered to be a simple, fast, and inexpensive technique that can be applied for identification of meat products in food industries. Nowadays, this technique has been considered as a practical method to identify the species origin, which could further applied for animal feedstuffs identification.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo

PubMed Central

Chu, Jun; Oh, Young-Hee; Sens, Alex; Ataie, Niloufar; Dana, Hod; Macklin, John J.; Laviv, Tal; Welf, Erik S.; Dean, Kevin M.; Zhang, Feijie; Kim, Benjamin B.; Tang, Clement Tran; Hu, Michelle; Baird, Michelle A.; Davidson, Michael W.; Kay, Mark A.; Fiolka, Reto; Yasuda, Ryohei; Kim, Douglas S.; Ng, Ho-Leung; Lin, Michael Z.

2016-01-01

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins. PMID:27240196

Multiplex engineering of industrial yeast genomes using CRISPRm.

PubMed

Ryan, Owen W; Cate, Jamie H D

2014-01-01

Global demand has driven the use of industrial strains of the yeast Saccharomyces cerevisiae for large-scale production of biofuels and renewable chemicals. However, the genetic basis of desired domestication traits is poorly understood because robust genetic tools do not exist for industrial hosts. We present an efficient, marker-free, high-throughput, and multiplexed genome editing platform for industrial strains of S. cerevisiae that uses plasmid-based expression of the CRISPR/Cas9 endonuclease and multiple ribozyme-protected single guide RNAs. With this multiplex CRISPR (CRISPRm) system, it is possible to integrate DNA libraries into the chromosome for evolution experiments, and to engineer multiple loci simultaneously. The CRISPRm tools should therefore find use in many higher-order synthetic biology applications to accelerate improvements in industrial microorganisms.

Experimental study on the statistic characteristics of a 3x3 RF MIMO channel over a single conventional multimode fiber.

PubMed

Lei, Yi; Li, Jianqiang; Wu, Rui; Fan, Yuting; Fu, Songnian; Yin, Feifei; Dai, Yitang; Xu, Kun

2017-06-01

Based on the observed random fluctuation phenomenon of speckle pattern across multimode fiber (MMF) facet and received optical power distribution across three output ports, we experimentally investigate the statistic characteristics of a 3×3 radio frequency multiple-input multiple-output (MIMO) channel enabled by mode division multiplexing in a conventional 50 µm MMF using non-mode-selective three-dimensional waveguide photonic lanterns as mode multiplexer and demultiplexer. The impacts of mode coupling on the MIMO channel coefficients, channel matrix, and channel capacity have been analyzed over different fiber lengths. The results indicate that spatial multiplexing benefits from the greater fiber length with stronger mode coupling, despite a higher optical loss.

A single-step polymerase chain reaction for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae.

PubMed

Kunthalert, Duangkamol; Henghiranyawong, Kritsada; Sistayanarain, Anchalee; Khoothiam, Krissana

2013-02-01

The critically high prevalence of bacterial otitis media worldwide has prompted a proper disease management. While vaccine development for otitis media is promising, the reliable and effective methods for diagnosis of such etiologic agents are of importance. We developed a multiplex polymerase chain reaction assay for simultaneous detection and differentiation of nontypeable and serotypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae. Five primer pairs targeting genes fumarate reductase (H. influenzae), outer membrane protein B (M. catarrhalis), major autolysin (S. pneumoniae), capsulation-associated BexA protein (all encapsulated H. influenzae) and 16S rRNA were incorporated in this single-step PCR. Validation of the multiplex PCR was also performed on clinical isolates. The developed multiplex PCR was highly specific, enabling the detection of the target pathogens in a specific manner, either individually or as a mixture of all target organisms. The assay was also found to be sensitive with the lowest detection limit of 1 ng of bacterial DNA. When applied to clinical isolates from diverse specimen sources, the multiplex PCR developed in this study correctly identified each microorganism individually or in a combination of two or more target organisms. All results matched with conventional culture identification. In addition, the ability of such assay to differentiate H. influenzae encapsulation from the study clinical isolates was 100%. Our multiplex PCR provides a rapid and accurate diagnostic tool for detection of the 4 target organisms. Such assay would serve as a useful tool for clinicians and epidemiologists in their efforts to the proper treatment and disease management caused by these organisms. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Bidirectional optical subassembly-shaped 20-Gbit/s compact single-mode four-channel wavelength-division multiplexing optical modules for optical multimedia interfaces

NASA Astrophysics Data System (ADS)

Lim, Kwon-Seob; Yu, Hong-Yeon; Park, Hyoung-Jun; Kang, Hyun Seo; Jang, Jae-Hyung

2016-06-01

Low-cost single-mode four-channel optical transmitter and receiver modules using the wavelength-division multiplexing (WDM) method have been developed for long-reach fiber optic applications. The single-mode four-channel WDM optical transmitter and receiver modules consist of two dual-wavelength optical transmitter and receiver submodules, respectively. The integration of two channels in a glass-sealed transistor outline-can package is an effective way to reduce cost and size and to extend the number of channels. The clear eye diagrams with more than about 6 dB of the extinction ratio and the minimum receiver sensitivity of lower than -16 dBm at a bit error rate of 10-12 have been obtained for the transmitter and receiver modules, respectively, at 5 Gbps/channel. The 4K ultrahigh definition contents have been transmitted over a 1-km-long single-mode fiber using a pair of proposed four-channel transmitter optical subassembly and receiver optical subassembly.

Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells.

PubMed

Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana; Niklasson, Mia; Segerman, Anna; Flamourakis, Georgios; Fredriksson, Simon; Assarsson, Erika; Lundberg, Martin; Nelander, Sven; Westermark, Bengt; Landegren, Ulf

2016-01-12

Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A multiplexed magnetic tweezer with precision particle tracking and bi-directional force control.

PubMed

Johnson, Keith C; Clemmens, Emilie; Mahmoud, Hani; Kirkpatrick, Robin; Vizcarra, Juan C; Thomas, Wendy E

2017-01-01

In the past two decades, methods have been developed to measure the mechanical properties of single biomolecules. One of these methods, Magnetic tweezers, is amenable to aquisition of data on many single molecules simultaneously, but to take full advantage of this "multiplexing" ability, it is necessary to simultaneously incorprorate many capabilities that ahve been only demonstrated separately. Our custom built magnetic tweezer combines high multiplexing, precision bead tracking, and bi-directional force control into a flexible and stable platform for examining single molecule behavior. This was accomplished using electromagnets, which provide high temporal control of force while achieving force levels similar to permanent magnets via large paramagnetic beads. Here we describe the instrument and its ability to apply 2-260 pN of force on up to 120 beads simultaneously, with a maximum spatial precision of 12 nm using a variety of bead sizes and experimental techniques. We also demonstrate a novel method for increasing the precision of force estimations on heterogeneous paramagnetic beads using a combination of density separation and bi-directional force correlation which reduces the coefficient of variation of force from 27% to 6%. We then use the instrument to examine the force dependence of uncoiling and recoiling velocity of type 1 fimbriae from Eschericia coli ( E. coli ) bacteria, and see similar results to previous studies. This platform provides a simple, effective, and flexible method for efficiently gathering single molecule force spectroscopy measurements.

Single-shot ultrafast tomographic imaging by spectral multiplexing

NASA Astrophysics Data System (ADS)

Matlis, N. H.; Axley, A.; Leemans, W. P.

2012-10-01

Computed tomography has profoundly impacted science, medicine and technology by using projection measurements scanned over multiple angles to permit cross-sectional imaging of an object. The application of computed tomography to moving or dynamically varying objects, however, has been limited by the temporal resolution of the technique, which is set by the time required to complete the scan. For objects that vary on ultrafast timescales, traditional scanning methods are not an option. Here we present a non-scanning method capable of resolving structure on femtosecond timescales by using spectral multiplexing of a single laser beam to perform tomographic imaging over a continuous range of angles simultaneously. We use this technique to demonstrate the first single-shot ultrafast computed tomography reconstructions and obtain previously inaccessible structure and position information for laser-induced plasma filaments. This development enables real-time tomographic imaging for ultrafast science, and offers a potential solution to the challenging problem of imaging through scattering surfaces.

Multiplexed Simultaneous High Sensitivity Sensors with High-Order Mode Based on the Integration of Photonic Crystal 1 × 3 Beam Splitter and Three Different Single-Slot PCNCs.

PubMed

Zhou, Jian; Huang, Lijun; Fu, Zhongyuan; Sun, Fujun; Tian, Huiping

2016-07-07

We simulated an efficient method for the sensor array of high-sensitivity single-slot photonic crystal nanobeam cavities (PCNCs) on a silicon platform. With the combination of a well-designed photonic crystal waveguide (PhCW) filter and an elaborate single-slot PCNC, a specific high-order resonant mode was filtered for sensing. A 1 × 3 beam splitter carefully established was implemented to split channels and integrate three sensors to realize microarrays. By applying the three-dimensional finite-difference-time-domain (3D-FDTD) method, the sensitivities calculated were S₁ = 492 nm/RIU, S₂ = 244 nm/RIU, and S₃ = 552 nm/RIU, respectively. To the best of our knowledge, this is the first multiplexing design in which each sensor cite features such a high sensitivity simultaneously.

Multiplexed Simultaneous High Sensitivity Sensors with High-Order Mode Based on the Integration of Photonic Crystal 1 × 3 Beam Splitter and Three Different Single-Slot PCNCs

PubMed Central

Zhou, Jian; Huang, Lijun; Fu, Zhongyuan; Sun, Fujun; Tian, Huiping

2016-01-01

We simulated an efficient method for the sensor array of high-sensitivity single-slot photonic crystal nanobeam cavities (PCNCs) on a silicon platform. With the combination of a well-designed photonic crystal waveguide (PhCW) filter and an elaborate single-slot PCNC, a specific high-order resonant mode was filtered for sensing. A 1 × 3 beam splitter carefully established was implemented to split channels and integrate three sensors to realize microarrays. By applying the three-dimensional finite-difference-time-domain (3D-FDTD) method, the sensitivities calculated were S1 = 492 nm/RIU, S2 = 244 nm/RIU, and S3 = 552 nm/RIU, respectively. To the best of our knowledge, this is the first multiplexing design in which each sensor cite features such a high sensitivity simultaneously. PMID:27399712

Progress Towards High-Sensitivity Arrays of Detectors of Sub-mm Radiation Using Superconducting Tunnel Junctions with Integrated Radio Frequency Single-Electron Transistors

NASA Technical Reports Server (NTRS)

Stevenson, T. R.; Hsieh, W.-T.; Li, M. J.; Prober, D. E.; Rhee, K. W.; Schoelkopf, R. J.; Stahle, C. M.; Teufel, J.; Wollack, E. J.

2004-01-01

For high resolution imaging and spectroscopy in the FIR and submillimeter, space observatories will demand sensitive, fast, compact, low-power detector arrays with 104 pixels and sensitivity less than 10(exp -20) W/Hz(sup 0.5). Antenna-coupled superconducting tunnel junctions with integrated rf single-electron transistor readout amplifiers have the potential for achieving this high level of sensitivity, and can take advantage of an rf multiplexing technique. The device consists of an antenna to couple radiation into a small superconducting volume and cause quasiparticle excitations, and a single-electron transistor to measure current through junctions contacting the absorber. We describe optimization of device parameters, and results on fabrication techniques for producing devices with high yield for detector arrays. We also present modeling of expected saturation power levels, antenna coupling, and rf multiplexing schemes.

Single Nucleotide Polymorphisms Predict Symptom Severity of Autism Spectrum Disorder

ERIC Educational Resources Information Center

Jiao, Yun; Chen, Rong; Ke, Xiaoyan; Cheng, Lu; Chu, Kangkang; Lu, Zuhong; Herskovits, Edward H.

2012-01-01

Autism is widely believed to be a heterogeneous disorder; diagnosis is currently based solely on clinical criteria, although genetic, as well as environmental, influences are thought to be prominent factors in the etiology of most forms of autism. Our goal is to determine whether a predictive model based on single-nucleotide polymorphisms (SNPs)…

Single nucleotide polymorphisms in uracil-processing genes, intake of one-carbon nutrients and breast cancer risk

USDA-ARS?s Scientific Manuscript database

Background/Objectives: The misincorporation of uracil into DNA leads to genomic instability. In a previous study, some of us identified four common single nucleotide polymorphisms (SNPs) in uracil-processing genes (rs2029166 and rs7296239 in SMUG1, rs34259 in UNG and rs4775748 in DUT) that were asso...

Single nucleotide polymorphisms in specific candidate genes are associated with phenotypic differences in days open for first lactation in Holstein cows

USDA-ARS?s Scientific Manuscript database

Previously, a candidate gene approach identified 51 single nucleotide polymorphisms (SNP) associated with genetic merit for reproductive traits and 26 associated with genetic merit for production in dairy bulls. We evaluated association of the 77 SNPs with days open (DO) for first lactation in a pop...

An integrated genetic linkage map of watermelon and genetic diversity based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers

USDA-ARS?s Scientific Manuscript database

Watermelon (Citrullus lanatus var. lanatus) is an important vegetable fruit throughout the world. A high number of single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers should provide large coverage of the watermelon genome and high phylogenetic resolution of germplasm acces...

Identification and characterization of single nucleotide polymorphisms (SNPs) in Culex theileri (Diptera: Culicidae).

PubMed

Demirci, Berna; Lee, Yoosook; Lanzaro, Gregory C; Alten, Bulent

2012-05-01

Culex theileri Theobald (Diptera: Culicidae) is one of the most common mosquito species in northeastern Turkey and serves as a vector for various zoonotic diseases including West Nile virus. Although there have been some studies on the ecology of Cx. theileri, very little genetic data has been made available. We successfully sequenced 11 gene fragments from Cx. theileri specimens collected from the northeastern part of Turkey. On average, we found a Single nucleotide polymorphism every 45 bp. Transitions outnumbered transversions, at a ratio of 2:1. This is the first report of genetic polymorphisms in Cx. theileri and Single nucleotide polymorphism discovered from this study can be used to investigate population structure and gene-environmental interactions.

Multiplex coherent anti-Stokes Raman scattering microspectroscopy of brain tissue with higher ranking data classification for biomedical imaging

NASA Astrophysics Data System (ADS)

Pohling, Christoph; Bocklitz, Thomas; Duarte, Alex S.; Emmanuello, Cinzia; Ishikawa, Mariana S.; Dietzeck, Benjamin; Buckup, Tiago; Uckermann, Ortrud; Schackert, Gabriele; Kirsch, Matthias; Schmitt, Michael; Popp, Jürgen; Motzkus, Marcus

2017-06-01

Multiplex coherent anti-Stokes Raman scattering (MCARS) microscopy was carried out to map a solid tumor in mouse brain tissue. The border between normal and tumor tissue was visualized using support vector machines (SVM) as a higher ranking type of data classification. Training data were collected separately in both tissue types, and the image contrast is based on class affiliation of the single spectra. Color coding in the image generated by SVM is then related to pathological information instead of single spectral intensities or spectral differences within the data set. The results show good agreement with the H&E stained reference and spontaneous Raman microscopy, proving the validity of the MCARS approach in combination with SVM.

Multiplex Detection of Aspergillus Species.

PubMed

Martínez-Culebras, Pedro; Selma, María Victoria; Aznar, Rosa

2017-01-01

Multiplex real-time polymerase chain reaction (PCR) provides a fast and accurate DNA-based tool for the simultaneous amplification of more than one target sequence in a single reaction. Here a duplex real-time PCR assay is described for the simultaneous detection of Aspergillus carbonarius and members of the Aspergillus niger aggregate, which are the main responsible species for ochratoxin A (OTA) contamination in grapes. This single tube reaction targets the beta-ketosynthase and the acyl transferase domains of the polyketide synthase of A. carbonarius and the A. niger aggregate, respectively.Besides, a rapid and efficient fungi DNA extraction procedure is described suitable to be applied in wine grapes. It includes a pulsifier equipment to remove conidia from grapes which prevents releasing of PCR inhibitors.

Real-time multiplexed digital cavity-enhanced spectroscopy

DOE PAGES

Boyson, Toby K.; Dagdigian, Paul J.; Pavey, Karl D.; ...

2015-10-01

Cavity-enhanced spectroscopy is a sensitive optical absorption technique but one where the practical applications have been limited to studying small wavelength ranges. In addition, this Letter shows that wideband operation can be achieved by combining techniques usually reserved for the communications community with that of cavity-enhanced spectroscopy, producing a multiplexed real-time cavity-enhanced spectrometer. We use multiple collinear laser sources operating asynchronously and simultaneously while being detected on a single photodetector. This is synonymous with radio frequency (RF) cellular systems in which signals are detected on a single antenna but decoded uniquely. Here, we demonstrate results with spectra of methyl salicylatemore » and show parts-per-billion per root hertz sensitivity measured in real-time.« less

Three-dimensional particle tracking via tunable color-encoded multiplexing.

PubMed

Duocastella, Martí; Theriault, Christian; Arnold, Craig B

2016-03-01

We present a novel 3D tracking approach capable of locating single particles with nanometric precision over wide axial ranges. Our method uses a fast acousto-optic liquid lens implemented in a bright field microscope to multiplex light based on color into different and selectable focal planes. By separating the red, green, and blue channels from an image captured with a color camera, information from up to three focal planes can be retrieved. Multiplane information from the particle diffraction rings enables precisely locating and tracking individual objects up to an axial range about 5 times larger than conventional single-plane approaches. We apply our method to the 3D visualization of the well-known coffee-stain phenomenon in evaporating water droplets.

Large-memory real-time multichannel multiplexed pattern recognition

NASA Technical Reports Server (NTRS)

Gregory, D. A.; Liu, H. K.

1984-01-01

The principle and experimental design of a real-time multichannel multiplexed optical pattern recognition system via use of a 25-focus dichromated gelatin holographic lens (hololens) are described. Each of the 25 foci of the hololens may have a storage and matched filtering capability approaching that of a single-lens correlator. If the space-bandwidth product of an input image is limited, as is true in most practical cases, the 25-focus hololens system has 25 times the capability of a single lens. Experimental results have shown that the interfilter noise is not serious. The system has already demonstrated the storage and recognition of over 70 matched filters - which is a larger capacity than any optical pattern recognition system reported to date.

All optical OFDM transmission for passive optical networks

NASA Astrophysics Data System (ADS)

Kachare, Nitin; Ashik T., J.; Bai, K. Kalyani; Kumar, D. Sriram

2017-06-01

This paper demonstrates the idea of data transmission at a very higher rate (Tbits/s) through optical fibers in a passive optical network using the most efficient data transmission technique widely used in wireless communication that is orthogonal frequency division multiplexing. With an increase in internet users, data traffic has also increased significantly and the current dense wavelength division multiplexing (DWDM) systems may not support the next generation passive optical networks (PONs) requirements. The approach discussed in this paper allows to increase the downstream data rate per user and extend the standard single-mode fiber reach for future long-haul applications. All-optical OFDM is a promising solution for terabit per second capable single wavelength transmission, with high spectral efficiency and high tolerance to chromatic dispersion.

Single-shot thermal ghost imaging using wavelength-division multiplexing

NASA Astrophysics Data System (ADS)

Deng, Chao; Suo, Jinli; Wang, Yuwang; Zhang, Zhili; Dai, Qionghai

2018-01-01

Ghost imaging (GI) is an emerging technique that reconstructs the target scene from its correlated measurements with a sequence of patterns. Restricted by the multi-shot principle, GI usually requires long acquisition time and is limited in observation of dynamic scenes. To handle this problem, this paper proposes a single-shot thermal ghost imaging scheme via a wavelength-division multiplexing technique. Specifically, we generate thousands of correlated patterns simultaneously by modulating a broadband light source with a wavelength dependent diffuser. These patterns carry the scene's spatial information and then the correlated photons are coupled into a spectrometer for the final reconstruction. This technique increases the speed of ghost imaging and promotes the applications in dynamic ghost imaging with high scalability and compatibility.

Distinctive features of single nucleotide alterations in induced pluripotent stem cells with different types of DNA repair deficiency disorders

PubMed Central

Okamura, Kohji; Sakaguchi, Hironari; Sakamoto-Abutani, Rie; Nakanishi, Mahito; Nishimura, Ken; Yamazaki-Inoue, Mayu; Ohtaka, Manami; Periasamy, Vaiyapuri Subbarayan; Alshatwi, Ali Abdullah; Higuchi, Akon; Hanaoka, Kazunori; Nakabayashi, Kazuhiko; Takada, Shuji; Hata, Kenichiro; Toyoda, Masashi; Umezawa, Akihiro

2016-01-01

Disease-specific induced pluripotent stem cells (iPSCs) have been used as a model to analyze pathogenesis of disease. In this study, we generated iPSCs derived from a fibroblastic cell line of xeroderma pigmentosum (XP) group A (XPA-iPSCs), a rare autosomal recessive hereditary disease in which patients develop skin cancer in the areas of skin exposed to sunlight. XPA-iPSCs exhibited hypersensitivity to ultraviolet exposure and accumulation of single-nucleotide substitutions when compared with ataxia telangiectasia-derived iPSCs that were established in a previous study. However, XPA-iPSCs did not show any chromosomal instability in vitro, i.e. intact chromosomes were maintained. The results were mutually compensating for examining two major sources of mutations, nucleotide excision repair deficiency and double-strand break repair deficiency. Like XP patients, XPA-iPSCs accumulated single-nucleotide substitutions that are associated with malignant melanoma, a manifestation of XP. These results indicate that XPA-iPSCs may serve a monitoring tool (analogous to the Ames test but using mammalian cells) to measure single-nucleotide alterations, and may be a good model to clarify pathogenesis of XP. In addition, XPA-iPSCs may allow us to facilitate development of drugs that delay genetic alteration and decrease hypersensitivity to ultraviolet for therapeutic applications. PMID:27197874

Forensic validation of the SNPforID 52-plex assay.

PubMed

Musgrave-Brown, Esther; Ballard, David; Balogh, Kinga; Bender, Klaus; Berger, Burkhard; Bogus, Magdalena; Børsting, Claus; Brion, María; Fondevila, Manuel; Harrison, Cheryl; Oguzturun, Ceylan; Parson, Walther; Phillips, Chris; Proff, Carsten; Ramos-Luis, Eva; Sanchez, Juan J; Sánchez Diz, Paula; Sobrino Rey, Bea; Stradmann-Bellinghausen, Beate; Thacker, Catherine; Carracedo, Angel; Morling, Niels; Scheithauer, Richard; Schneider, Peter M; Syndercombe Court, Denise

2007-06-01

The advantages of single nucleotide polymorphism (SNP) typing in forensic genetics are well known and include a wider choice of high-throughput typing platforms, lower mutation rates, and improved analysis of degraded samples. However, if SNPs are to become a realistic supplement to current short tandem repeat (STR) typing methods, they must be shown to successfully and reliably analyse the challenging samples commonly encountered in casework situations. The European SNPforID consortium, supported by the EU GROWTH programme, has developed a multiplex of 52 SNPs for forensic analysis, with the amplification of all 52 loci in a single reaction followed by two single base extension (SBE) reactions which are detected with capillary electrophoresis. In order to validate this assay, a variety of DNA extracts were chosen to represent problems such as low copy number and degradation that are commonly seen in forensic casework. A total of 40 extracts were used in the study, each of which was sent to two of the five participating laboratories for typing in duplicate or triplicate. Laboratories were instructed to carry out their analyses as if they were dealing with normal casework samples. Results were reported back to the coordinating laboratory and compared with those obtained from traditional STR typing of the same extracts using Powerplex 16 (Promega). These results indicate that, although the ability to successfully type good quality, low copy number extracts is lower, the 52-plex SNP assay performed better than STR typing on degraded samples, and also on samples that were both degraded and of limited quantity, suggesting that SNP analysis can provide advantages over STR analysis in forensically relevant circumstances. However, there were also additional problems arising from contamination and primer quality issues and these are discussed.

Structure-function clustering in multiplex brain networks

NASA Astrophysics Data System (ADS)

Crofts, J. J.; Forrester, M.; O'Dea, R. D.

2016-10-01

A key question in neuroscience is to understand how a rich functional repertoire of brain activity arises within relatively static networks of structurally connected neural populations: elucidating the subtle interactions between evoked “functional connectivity” and the underlying “structural connectivity” has the potential to address this. These structural-functional networks (and neural networks more generally) are more naturally described using a multilayer or multiplex network approach, in favour of standard single-layer network analyses that are more typically applied to such systems. In this letter, we address such issues by exploring important structure-function relations in the Macaque cortical network by modelling it as a duplex network that comprises an anatomical layer, describing the known (macro-scale) network topology of the Macaque monkey, and a functional layer derived from simulated neural activity. We investigate and characterize correlations between structural and functional layers, as system parameters controlling simulated neural activity are varied, by employing recently described multiplex network measures. Moreover, we propose a novel measure of multiplex structure-function clustering which allows us to investigate the emergence of functional connections that are distinct from the underlying cortical structure, and to highlight the dependence of multiplex structure on the neural dynamical regime.

A method of multiplex PCR for detection of field released Beauveria bassiana, a fungal entomopathogen applied for pest management in jute (Corchorus olitorius).

PubMed

Biswas, Chinmay; Dey, Piyali; Gotyal, B S; Satpathy, Subrata

2015-04-01

The fungal entomopathogen Beauveria bassiana is a promising biocontrol agent for many pests. Some B. bassiana strains have been found effective against jute pests. To monitor the survival of field released B. bassiana a rapid and efficient detection technique is essential. Conventional methods such as plating method or direct culture method which are based on cultivation on selective media followed by microscopy are time consuming and not so sensitive. PCR based methods are rapid, sensitive and reliable. A single primer PCR may fail to amplify some of the strains. However, multiplex PCR increases the possibility of detection as it uses multiple primers. Therefore, in the present investigation a multiplex PCR protocol was developed by multiplexing three primers SCA 14, SCA 15 and SCB 9 to detect field released B. bassiana strains from soil as well as foliage of jute field. Using our multiplex PCR protocol all the five B. bassiana strains could be detected from soil and three strains viz., ITCC 6063, ITCC 4563 and ITCC 4796 could be detected even from the crop foliage after 45 days of spray.

Development of a multiplexing fingerprint and high wavenumber Raman spectroscopy technique for real-time in vivo tissue Raman measurements at endoscopy.

PubMed

Bergholt, Mads Sylvest; Zheng, Wei; Huang, Zhiwei

2013-03-01

We report on the development of a novel multiplexing Raman spectroscopy technique using a single laser light together with a volume phase holographic (VPH) grating that simultaneously acquires both fingerprint (FP) and high wavenumber (HW) tissue Raman spectra at endoscopy. We utilize a customized VPH dual-transmission grating, which disperses the incident Raman scattered light vertically onto two separate segments (i.e., -150 to 1950  cm⁻¹; 1750 to 3600  cm⁻¹) of a charge-coupled device camera. We demonstrate that the multiplexing Raman technique can acquire high quality in vivo tissue Raman spectra ranging from 800 to 3600  cm⁻¹ within 1.0 s with a spectral resolution of 3 to 6  cm⁻¹ during clinical endoscopy. The rapid multiplexing Raman spectroscopy technique covering both FP and HW ranges developed in this work has potential for improving in vivo tissue diagnosis and characterization at endoscopy.

How multiple social networks affect user awareness: The information diffusion process in multiplex networks

NASA Astrophysics Data System (ADS)

Li, Weihua; Tang, Shaoting; Fang, Wenyi; Guo, Quantong; Zhang, Xiao; Zheng, Zhiming

2015-10-01

The information diffusion process in single complex networks has been extensively studied, especially for modeling the spreading activities in online social networks. However, individuals usually use multiple social networks at the same time, and can share the information they have learned from one social network to another. This phenomenon gives rise to a new diffusion process on multiplex networks with more than one network layer. In this paper we account for this multiplex network spreading by proposing a model of information diffusion in two-layer multiplex networks. We develop a theoretical framework using bond percolation and cascading failure to describe the intralayer and interlayer diffusion. This allows us to obtain analytical solutions for the fraction of informed individuals as a function of transmissibility T and the interlayer transmission rate θ . Simulation results show that interaction between layers can greatly enhance the information diffusion process. And explosive diffusion can occur even if the transmissibility of the focal layer is under the critical threshold, due to interlayer transmission.

Multiplex network analysis of employee performance and employee social relationships

NASA Astrophysics Data System (ADS)

Cai, Meng; Wang, Wei; Cui, Ying; Stanley, H. Eugene

2018-01-01

In human resource management, employee performance is strongly affected by both formal and informal employee networks. Most previous research on employee performance has focused on monolayer networks that can represent only single categories of employee social relationships. We study employee performance by taking into account the entire multiplex structure of underlying employee social networks. We collect three datasets consisting of five different employee relationship categories in three firms, and predict employee performance using degree centrality and eigenvector centrality in a superimposed multiplex network (SMN) and an unfolded multiplex network (UMN). We use a quadratic assignment procedure (QAP) analysis and a regression analysis to demonstrate that the different categories of relationship are mutually embedded and that the strength of their impact on employee performance differs. We also use weighted/unweighted SMN/UMN to measure the predictive accuracy of this approach and find that employees with high centrality in a weighted UMN are more likely to perform well. Our results shed new light on how social structures affect employee performance.

Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR.

PubMed

Lee, Shin-Young; Kim, Mi-Ju; Kim, Hyun-Joong; Jeong, KwangCheol Casey; Kim, Hae-Yeong

2018-02-28

A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

Field Demonstration of a Multiplexed Point-of-Care Diagnostic Platform for Plant Pathogens.

PubMed

Lau, Han Yih; Wang, Yuling; Wee, Eugene J H; Botella, Jose R; Trau, Matt

2016-08-16

Effective disease management strategies to prevent catastrophic crop losses require rapid, sensitive, and multiplexed detection methods for timely decision making. To address this need, a rapid, highly specific and sensitive point-of-care method for multiplex detection of plant pathogens was developed by taking advantage of surface-enhanced Raman scattering (SERS) labeled nanotags and recombinase polymerase amplification (RPA), which is a rapid isothermal amplification method with high specificity. In this study, three agriculturally important plant pathogens (Botrytis cinerea, Pseudomonas syringae, and Fusarium oxysporum) were used to demonstrate potential translation into the field. The RPA-SERS method was faster, more sensitive than polymerase chain reaction, and could detect as little as 2 copies of B. cinerea DNA. Furthermore, multiplex detection of the three pathogens was demonstrated for complex systems such as the Arabidopsis thaliana plant and commercial tomato crops. To demonstrate the potential for on-site field applications, a rapid single-tube RPA/SERS assay was further developed and successfully performed for a specific target outside of a laboratory setting.

Multiplex Immunoassay Profiling of Hormones Involved in Metabolic Regulation.

PubMed

Stephen, Laurie; Guest, Paul C

2018-01-01

Multiplex immunoassays are used for rapid profiling of biomarker proteins and small molecules in biological fluids. The advantages over single immunoassays include lower sample consumption, cost, and labor. This chapter details a protocol to develop a 5-plex assay for glucagon-like peptide 1, growth hormone, insulin, leptin, and thyroid-stimulating hormone on the Luminex ® platform. The results of the analysis of insulin in normal control subjects are given due to the important role of this hormone in nutritional programming diseases.

Simultaneous off-axis multiplexed holography and regular fluorescence microscopy of biological cells.

PubMed

Nygate, Yoav N; Singh, Gyanendra; Barnea, Itay; Shaked, Natan T

2018-06-01

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

Wavelength-multiplexed fiber optic position encoder for aircraft control systems

NASA Astrophysics Data System (ADS)

Beheim, Glenn; Krasowski, Michael J.; Sotomayor, Jorge L.; Fritsch, Klaus; Flatico, Joseph M.; Bathurst, Richard L.; Eustace, John G.; Anthan, Donald J.

1991-02-01

NASA Lewis together with John Carroll University has worked for the last several years to develop wavelength-multiplexed digital position transducers for use in aircraft control systems. A prototype rotary encoder is being built for a demonstration program involving the control of a commercial transport''s turbofan engine. This encoder has eight bits of resolution a 90 degree range and is powered by a single LED. A compact electro-optics module is being developed to withstand the extremely hostile gas turbine environment.

Multichannel temperature controller for hot air solar house

NASA Technical Reports Server (NTRS)

Currie, J. R.

1979-01-01

This paper describes an electronic controller that is optimized to operate a hot air solar system. Thermal information is obtained from copper constantan thermocouples and a wall-type thermostat. The signals from the thermocouples are processed through a single amplifier using a multiplexing scheme. The multiplexing reduces the component count and automatically calibrates the thermocouple amplifier. The processed signals connect to some simple logic that selects one of the four operating modes. This simple, inexpensive, and reliable scheme is well suited to control hot air solar systems.

Microlaser-based compact optical neuro-processors (Invited Paper)

NASA Astrophysics Data System (ADS)

Paek, Eung Gi; Chan, Winston K.; Zah, Chung-En; Cheung, Kwok-wai; Curtis, L.; Chang-Hasnain, Constance J.

1992-10-01

This paper reviews the recent progress in the development of holographic neural networks using surface-emitting laser diode arrays (SELDAs). Since the previous work on ultrafast holographic memory readout system and a robust incoherent correlator, progress has been made in several areas: the use of an array of monolithic `neurons' to reconstruct holographic memories; two-dimensional (2-D) wavelength-division multiplexing (WDM) for image transmission through a single-mode fiber; and finally, an associative memory using time- division multiplexing (TDM). Experimental demonstrations on these are presented.

High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling

PubMed Central

Till, Bradley J.; Zerr, Troy; Bowers, Elisabeth; Greene, Elizabeth A.; Comai, Luca; Henikoff, Steven

2006-01-01

Human individuals differ from one another at only ∼0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease. PMID:16893952

Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F1-ATPase.

PubMed

Volkán-Kacsó, Sándor; Marcus, Rudolph A

2016-10-25

A recently proposed chemomechanical group transfer theory of rotary biomolecular motors is applied to treat single-molecule controlled rotation experiments. In these experiments, single-molecule fluorescence is used to measure the binding and release rate constants of nucleotides by monitoring the occupancy of binding sites. It is shown how missed events of nucleotide binding and release in these experiments can be corrected using theory, with F 1 -ATP synthase as an example. The missed events are significant when the reverse rate is very fast. Using the theory the actual rate constants in the controlled rotation experiments and the corrections are predicted from independent data, including other single-molecule rotation and ensemble biochemical experiments. The effective torsional elastic constant is found to depend on the binding/releasing nucleotide, and it is smaller for ADP than for ATP. There is a good agreement, with no adjustable parameters, between the theoretical and experimental results of controlled rotation experiments and stalling experiments, for the range of angles where the data overlap. This agreement is perhaps all the more surprising because it occurs even though the binding and release of fluorescent nucleotides is monitored at single-site occupancy concentrations, whereas the stalling and free rotation experiments have multiple-site occupancy.