Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Microarray-based DNA resequencing using 3' blocked primers.

ABSTRACT: To exceed the throughput and accuracy of conventional sequencing technologies, we tested a method (pyrophosphorolysis-activated polymerization [PAP]) of nucleic acid amplification that uses 3' blocked primers (P*s). As proof-of-principle, we resequenced a 20-bp region of the factor IX gene with a microarray of P*s. P*s discriminate 3' end mismatches with ultra-high specificity as well as mismatches along their lengths with high specificity. We correctly identified two wild-type samples as well as all mismatches, including three single-base substitutions, one microdeletion, one microinsertion, and one heterozygous mutation. Despite limitations in the primer purity, the signal/noise ratio between the matched and mismatched P*s sometimes exceeded 1000. Thus, PAP resequencing shows great potential for accurate and high-throughput microarray-based resequencing.

SUBMITTER: Sram J

PROVIDER: S-EPMC2292398 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Microarray-based DNA resequencing using 3' blocked primers.

Sram Jakub J Sommer Steve S SS Liu Qiang Q

Analytical biochemistry 20071104 1

To exceed the throughput and accuracy of conventional sequencing technologies, we tested a method (pyrophosphorolysis-activated polymerization [PAP]) of nucleic acid amplification that uses 3' blocked primers (P*s). As proof-of-principle, we resequenced a 20-bp region of the factor IX gene with a microarray of P*s. P*s discriminate 3' end mismatches with ultra-high specificity as well as mismatches along their lengths with high specificity. We correctly identified two wild-type samples as well a ...[more]

PMID: 18023400

Similar Datasets

Molecular characterization of Neisseria meningitidis isolates using a resequencing DNA microarray.

Project description:Neisseria meningitidis is a major cause of both meningitis and septicemia. Typically, isolates are characterized by using a combination of immunological phenotyping, using monoclonal and polyclonal antisera, and Sanger nucleotide sequencing of epitope-encoding variable regions, although these methods can be both time-consuming and limited by reagent availability. Herein, we describe and evaluate a novel microarray to define the porB and porA serotypes of N. meningitidis by the resequencing of variable regions in a single hybridization reaction. PCR products for each gene were amplified, pooled in equimolar concentrations, hybridized to the microarray, and analyzed using Affymetrix GeneChip DNA Analysis Software. Resequencing of the microarray data was then validated by comparison with sequencing data. Molecular profiles were generated for 50 isolates that were combinations of phenotypically typeable (ie, PorA and PorB) and non-typeable (PorB only) isolates. Microarray-generated profiles from isolates with a PorB phenotype were concordant with predicted profiles compared with a previously described typing scheme. In addition, 42% (8 of 19) of previously non-typeable samples were assigned a PorB type when tested using the microarray. The remaining isolates were novel types for which no typing antisera are currently available. The porA data were 97% concordant with Sanger nucleotide sequencing. These results suggest that that microarray resequencing may be a useful tool for the characterization of meningococci, particularly for those isolates that cannot be phenotyped, offering an alternative to conventional sequencing methods.

| S-EPMC2329792 | biostudies-literature

Microarray-based resequencing of multiple Bacillus anthracis isolates.

Project description:We used custom-designed resequencing arrays to generate 3.1 Mb of genomic sequence from a panel of 56 Bacillus anthracis strains. Sequence quality was shown to be very high by replication (discrepancy rate of 7.4 x 10(-7)) and by comparison to independently generated shotgun sequence (discrepancy rate < 2.5 x 10(-6)). Population genomics studies of microbial pathogens using rapid resequencing technologies such as resequencing arrays are critical for recognizing newly emerging or genetically engineered strains.

| S-EPMC549062 | biostudies-literature

Genotyping of Bacillus cereus strains by microarray-based resequencing.

Project description:The ability to distinguish microbial pathogens from closely related but nonpathogenic strains is key to understanding the population biology of these organisms. In this regard, Bacillus anthracis, the bacterium that causes inhalational anthrax, is of interest because it is closely related and often difficult to distinguish from other members of the B. cereus group that can cause diverse diseases. We employed custom-designed resequencing arrays (RAs) based on the genome sequence of Bacillus anthracis to generate 422 kb of genomic sequence from a panel of 41 Bacillus cereus sensu lato strains. Here we show that RAs represent a "one reaction" genotyping technology with the ability to discriminate between highly similar B. anthracis isolates and more divergent strains of the B. cereus s.l. Clade 1. Our data show that RAs can be an efficient genotyping technology for pre-screening the genetic diversity of large strain collections to selected the best candidates for whole genome sequencing.

| S-EPMC2438477 | biostudies-literature

RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers.

Project description:BACKGROUND: The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. RESULTS: Pyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. CONCLUSION: rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection.

| S-EPMC3224242 | biostudies-literature

Using a resequencing microarray as a multiple respiratory pathogen detection assay.

Project description:Simultaneous testing for detection of infectious pathogens that cause similar symptoms (e.g., acute respiratory infections) is invaluable for patient treatment, outbreak prevention, and efficient use of antibiotic and antiviral agents. In addition, such testing may provide information regarding possible coinfections or induced secondary infections, such as virally induced bacterial infections. Furthermore, in many cases, detection of a pathogen requires more than genus/species-level resolution, since harmful agents (e.g., avian influenza virus) are grouped with other, relatively benign common agents, and for every pathogen, finer resolution is useful to allow tracking of the location and nature of mutations leading to strain variations. In this study, a previously developed resequencing microarray that has been demonstrated to have these capabilities was further developed to provide individual detection sensitivity ranging from 10(1) to 10(3) genomic copies for more than 26 respiratory pathogens while still retaining the ability to detect and differentiate between close genetic neighbors. In addition, the study demonstrated that this system allows unambiguous and reproducible sequence-based strain identification of the mixed pathogens. Successful proof-of-concept experiments using clinical specimens show that this approach is potentially very useful for both diagnostics and epidemic surveillance.

| S-EPMC1829030 | biostudies-literature

Identification of Pork Adulteration in Processed Meat Products Using the Developed Mitochondrial DNA-Based Primers.

Project description:The identification of pork in commercially processed meats is one of the most crucial issues in the food industry because of religious food ethics, medical purposes, and intentional adulteration to decrease production cost. This study therefore aimed to develop a method for the detection of pork adulteration in meat products using primers specific for pig mitochondrial DNA. Mitochondrial DNA sequences for pig, cattle, chicken, and sheep were obtained from GenBank and aligned. The 294-bp mitochondrial DNA D-loop region was selected as the pig target DNA sequence and appropriate primers were designed using the MUSCLE program. To evaluate primer sensitivity, pork-beef-chicken mixtures were prepared as follows: i) 0% pork-50% beef-50% chicken, ii) 1% pork-49.5% beef-49.5% chicken, iii) 2% pork-49% beef-49% chicken, iv) 5% pork-47.5% beef-47.5% chicken, v) 10% pork-45% beef-45% chicken, and vi) 100% pork-0% beef-0% chicken. In addition, a total of 35 commercially packaged products, including patties, nuggets, meatballs, and sausages containing processed chicken, beef, or a mixture of various meats, were purchased from commercial markets. The primers developed in our study were able to detect as little as 1% pork in the heat treated pork-beef-chicken mixtures. Of the 35 processed products, three samples were pork positive despite being labeled as beef or chicken only or as a beef-chicken mix. These results indicate that the developed primers could be used to detect pork adulteration in various processed meat products for application in safeguarding religious food ethics, detecting allergens, and preventing food adulteration.

| S-EPMC5516074 | biostudies-literature

Targeted resequencing of HIV variants by microarray thermodynamics.

Project description:Within a single infected individual, a virus population can have a high genomic variability. In the case of HIV, several mutations can be present even in a small genomic window of 20-30 nucleotides. For diagnostics purposes, it is often needed to resequence genomic subsets where crucial mutations are known to occur. In this article, we address this issue using DNA microarrays and inputs from hybridization thermodynamics. Hybridization signals from multiple probes are analysed, including strong signals from perfectly matching (PM) probes and a large amount of weaker cross-hybridization signals from mismatching (MM) probes. The latter are crucial in the data analysis. Seven coded clinical samples (HIV-1) are analyzed, and the microarray results are in full concordance with Sanger sequencing data. Moreover, the thermodynamic analysis of microarray signals resolves inherent ambiguities in Sanger data of mixed samples and provides additional clinically relevant information. These results show the reliability and added value of DNA microarrays for point-of-care diagnostic purposes.

| S-EPMC3794611 | biostudies-literature

Application of high-density DNA resequencing microarray for detection and characterization of botulinum neurotoxin-producing clostridia.

Project description:BackgroundClostridium botulinum and related clostridia express extremely potent toxins known as botulinum neurotoxins (BoNTs) that cause severe, potentially lethal intoxications in humans. These BoNT-producing bacteria are categorized in seven major toxinotypes (A through G) and several subtypes. The high diversity in nucleotide sequence and genetic organization of the gene cluster encoding the BoNT components poses a great challenge for the screening and characterization of BoNT-producing strains.Methodology/principal findingsIn the present study, we designed and evaluated the performances of a resequencing microarray (RMA), the PathogenId v2.0, combined with an automated data approach for the simultaneous detection and characterization of BoNT-producing clostridia. The unique design of the PathogenID v2.0 array allows the simultaneous detection and characterization of 48 sequences targeting the BoNT gene cluster components. This approach allowed successful identification and typing of representative strains of the different toxinotypes and subtypes, as well as the neurotoxin-producing C. botulinum strain in a naturally contaminated food sample. Moreover, the method allowed fine characterization of the different neurotoxin gene cluster components of all studied strains, including genomic regions exhibiting up to 24.65% divergence with the sequences tiled on the arrays.Conclusions/significanceThe severity of the disease demands rapid and accurate means for performing risk assessments of BoNT-producing clostridia and for tracing potentials sources of contamination in outbreak situations. The RMA approach constitutes an essential higher echelon component in a diagnostics and surveillance pipeline. In addition, it is an important asset to characterise potential outbreak related strains, but also environment isolates, in order to obtain a better picture of the molecular epidemiology of BoNT-producing clostridia.

| S-EPMC3688605 | biostudies-literature

Reconstructed ancestral sequences improve pathogen identification using resequencing DNA microarrays.

Project description:We describe the benefit of using reconstructed ancestral sequences (RAS) on resequencing microarrays for rapid pathogen identification, with Enterobacteriaceae rpoB sequences as a model. Our results demonstrate a sharp improvement of call rate and accuracy when using RASs as compared to extant sequences. This improvement was attributed to the lower sequence divergence of RASs, which also expanded the sequence space covered by the microarray. Extension of this novel microarray design strategy to viruses, antimicrobial resistance elements or toxins is straightforward.

| S-EPMC3004854 | biostudies-literature

Broad-spectrum respiratory tract pathogen identification using resequencing DNA microarrays.

Project description:The exponential growth of pathogen nucleic acid sequences available in public domain databases has invited their direct use in pathogen detection, identification, and surveillance strategies. DNA microarray technology has offered the potential for the direct DNA sequence analysis of a broad spectrum of pathogens of interest. However, to achieve the practical attainment of this potential, numerous technical issues, especially nucleic acid amplification, probe specificity, and interpretation strategies of sequence detection, need to be addressed. In this report, we demonstrate an approach that combines the use of a custom-designed Affymetrix resequencing Respiratory Pathogen Microarray (RPM v.1) with methods for microbial nucleic acid enrichment, random nucleic acid amplification, and automated sequence similarity searching for broad-spectrum respiratory pathogen surveillance. Successful proof-of-concept experiments, utilizing clinical samples obtained from patients presenting adenovirus or influenza virus-induced febrile respiratory illness (FRI), demonstrate the ability of this approach for correct species- and strain-level identification with unambiguous statistical interpretation at clinically relevant sensitivity levels. Our results underscore the feasibility of using this approach to expedite the early surveillance of diseases, and provide new information on the incidence of multiple pathogens.

| S-EPMC1457032 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data