Competitive enzymatic reaction to control allele-specific extensions.
ABSTRACT: Here, we present a novel method for SNP genotyping based on protease-mediated allele-specific primer extension (PrASE), where the two allele-specific extension primers only differ in their 3'-positions. As reported previously [Ahmadian,A., Gharizadeh,B., O'Meara,D., Odeberg,J. and Lundeberg,J. (2001), Nucleic Acids Res., 29, e121], the kinetics of perfectly matched primer extension is faster than mismatched primer extension. In this study, we have utilized this difference in kinetics by adding protease, a protein-degrading enzyme, to discriminate between the extension reactions. The competition between the polymerase activity and the enzymatic degradation yields extension of the perfectly matched primer, while the slower extension of mismatched primer is eliminated. To allow multiplex and simultaneous detection of the investigated single nucleotide polymorphisms (SNPs), each extension primer was given a unique signature tag sequence on its 5' end, complementary to a tag on a generic array. A multiplex nested PCR with 13 SNPs was performed in a total of 36 individuals and their alleles were scored. To demonstrate the improvements in scoring SNPs by PrASE, we also genotyped the individuals without inclusion of protease in the extension. We conclude that the developed assay is highly allele-specific, with excellent multiplex SNP capabilities.
Project description:BACKGROUND: There is an imperative need for SNP genotyping technologies that are cost-effective per sample with retained high accuracy, throughput and flexibility. We have developed a microarray-based technique and compared it to Pyrosequencing. In the protease-mediated allele-specific extension (PrASE), the protease constrains the elongation reaction and thus prevents incorrect nucleotide incorporation to mismatched 3'-termini primers. RESULTS: The assay is automated for 48 genotyping reactions in parallel followed by a tag-microarray detection system. A script automatically visualizes the results in cluster diagrams and assigns the genotypes. Ten polymorphic positions suggested as prothrombotic genetic variations were analyzed with Pyrosequencing and PrASE technologies in 442 samples and 99.8 % concordance was achieved. In addition to accuracy, the robustness and reproducibility of the technique has been investigated. CONCLUSION: The results of this study strongly indicate that the PrASE technology can offer significant improvements in terms of accuracy and robustness and thereof increased number of typeable SNPs.
Project description:Single nucleotide polymorphisms (SNPs) of genes that affect cytokine production and function are known to influence the susceptibility and progression of immune-related conditions such as infection, autoimmune diseases, transplantation, and cancer. We established a multiplex genotyping method to analyze the SNPs of cytokine genes by combining the multiplex PCR and bead array platform. Thirteen cytokine gene regions, including 20 SNPs, were amplified, and allele-specific primer extension was performed in a single tube. High-quality allele-specific primers were selected for signals greater than 1000 median fluorescence intensity (MFI) for positive alleles, and less than 500 MFI for negative alleles. To select and improve the extension primers, modifications for the reverse direction, length or refractory were performed. 24 primers in the forward or reverse direction step and 12 primers in length or refractory modifications were selected and showed high concordance with results by nucleotide sequencing. Among the 13 candidate cytokine genes, the SNPs of 12 cytokine genes, including IL-1?, IL-1R, IL-1RA, IL-1?, IL-2, IL-4, IL-4R?, IL-6, IL-10, IL-12, TGF-?1, and TNF-?, were successfully defined with the selected allele-specific primers in healthy Korean subjects. Our genotyping system provides a fast and accurate detection for SNPs of multiple cytokine genes to investigate their association with immune-related diseases and transplantation outcomes.
Project description:An increasing number of biological and epidemiological evidence suggests that c.919-2A?>?G and c.2168A?>?G variants of solute carrier family 26, member 4 (SLC26A4) gene play a critical role in the development of large vestibular aqueduct syndrome (LVAS). In this study, we developed a rapid genotyping method for discriminating LVAS-associated high-frequency variants in SLC26A4 gene. The genotyping technique consists of 3' terminal exonuclease-resistant phosphorothioate-modified allele specific primer extension mediated by exo+ polymerase. In PCR amplification by Pfu polymerase, allelic specific primers perfectly matching wild type allele were extended while no specific products were yielded from primers targeting variant allele. Similarly, allelic specific primers perfectly matching variant allele were extended and no specific products were observed from primers targeting wild type allele. The clinical application of 3' terminal phosphorothioate-modified allele specific primer extension mediated by Pfu polymerase identified both homozygous for SLC26A4 gene c.919-2A?>?G variant in two patients clinically diagnosed as LVAS by temporal bone CT scan. The genetic results from this method are consistent with that of DNA sequencing. The data suggest that exo+ polymerase-mediated 3' terminal phosphorothioate-modified primer extension is reliable in the identification of SLC26A4 gene high-frequency variant prior to high-resolution CT scan. The method is extremely suitable for quickly molecular etiologic screening and early diagnosis and aggressive prevention therapy of LVAS.
Project description:Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems.
Project description:A novel primer extension-based, multiplex minisequencing assay targeting six highly informative single nucleotide polymorphisms (SNPs) in four virulence genes correctly identified and differentiated all four epidemic clones (ECs) of Listeria monocytogenes and 9 other strains initially misclassified as non-ECs. This assay allows rapid, accurate, and high-throughput screening for all known ECs of L. monocytogenes.
Project description:We have developed a locus-specific DNA target preparation method for highly multiplexed single nucleotide polymorphism (SNP) genotyping called MARA (Multiplexed Anchored Runoff Amplification). The approach uses a single primer per SNP in conjunction with restriction enzyme digested, adapter-ligated human genomic DNA. Each primer is composed of common sequence at the 5' end followed by locus-specific sequence at the 3' end. Following a primary reaction in which locus-specific products are generated, a secondary universal amplification is carried out using a generic primer pair corresponding to the oligonucleotide and genomic DNA adapter sequences. Allele discrimination is achieved by hybridization to high-density DNA oligonucleotide arrays. Initial multiplex reactions containing either 250 primers or 750 primers across nine DNA samples demonstrated an average sample call rate of approximately 95% for 250- and 750-plex MARA. We have also evaluated >1000- and 4000-primer plex MARA to genotype SNPs from human chromosome 21. We have identified a subset of SNPs corresponding to a primer conversion rate of approximately 75%, which show an average call rate over 95% and concordance >99% across seven DNA samples. Thus, MARA may potentially improve the throughput of SNP genotyping when coupled with allele discrimination on high-density arrays by allowing levels of multiplexing during target generation that far exceed the capacity of traditional multiplex PCR.
Project description:The ability to detect and monitor single nucleotide polymorphisms (SNPs) in biological samples is an enabling research and clinical tool. We have developed a surprising, inexpensive primer design method that provides exquisite discrimination between SNPs. The field of DNA computation is largely reliant on using so-called toeholds to initiate strand displacement reactions, leading to the execution of kinetically trapped circuits. We have now similarly found that the short toehold sequence to a target of interest can initiate both strand displacement within the hairpin and extension of the primer by a polymerase, both of which will further stabilize the primer:template complex. However, if the short toehold does not bind, neither of these events can readily occur and thus amplification should not occur. Toehold hairpin primers were used to detect drug resistance alleles in two genes, rpoB and katG, in the Mycobacterium tuberculosis genome, and ten alleles in the Escherichia coli genome. During real-time PCR, the primers discriminate between mismatched templates with Cq delays that are frequently so large that the presence or absence of mismatches is essentially a 'yes/no' answer.
Project description:Efforts to correlate genetic variations with phenotypic differences are intensifying due to the availability of high-density maps of single nucleotide polymorphisms (SNPs) and the development of high throughput scoring methods. These recent advances have led to an increased interest for improved multiplex preparations of genetic material to facilitate such whole genome analyses. Here we propose a strategy for the parallel amplification of polymorphic loci based on a reduced set of nucleotides. The technique denoted Tri-nucleotide Threading (TnT), allows SNPs to be amplified via controlled linear amplification followed by complete removal of the target material and subsequent amplification with a pair of universal primers. A dedicated software tool was developed for this purpose and variable positions in genes associated with different forms of cancer were analyzed using sub-nanogram amounts of starting material. The amplified fragments were then successfully scored using a microarray-based PrASE technique. The results of this study, in which 75 SNPs were analyzed, show that the TnT technique circumvents potential problems associated with multiplex amplification of SNPs from minute amounts of material. The technique is specific, sensitive and can be readily adapted to equipment and genotyping techniques used in other research laboratories without requiring changes to the preferred typing method.
Project description:Multiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step.Herein, we report multiplex one-step RT-PCR experiments in which the PCR primers contain thermolabile phosphotriester modification groups. The presence of these groups minimizes PCR primer extension during the RT step and allows for control of PCR primer extension until the more stringent, elevated temperatures of PCR are reached. Results reveal that the use of primers whose extension can be controlled in a temperature-mediated way provides improved one-step RT-PCR specificity in both singleplex and multiplex reaction formats.The need for an accurate and sensitive technique to quantify mRNA expression levels makes the described modified primer technology a promising tool for use in multiplex one-step RT-PCR. A more accurate representation of the abundances in initial template sample is feasible with modified primers, as artifacts of biased PCR are reduced because of greater improvements in reaction specificity.
Project description:BACKGROUND: Innate immune system is the first line of research when studying immune response to diverse infections and autoimmune/inflammatory diseases. This immune response has been reported to be genetically diverse, due to polymorphisms coded by different genes. For this reason, our purpose was to develop a multiplex assay that allows the genotyping of candidate single nucleotide polymorphisms (SNPs) in innate immune genes. FINDINGS: We developed three multiplex PCR panels coupled with the minisequencing (SNaPshot) technique (multiplex PCR, multiplex primer extension, and capillary electrophoresis). The panels were tested in a sample set composed of 100 anonymous DNAs from healthy blood donors living in São Miguel Island (Azores, Portugal). Sixteen relevant SNPs among nine genes of the innate immune system--IL1?, IL1?, IL6, IL10, IL12RB1, TLR2, TLR4, TLR9 and CD14--were genotyped and validated by direct sequencing, with the exception of one that was undetected by minisequencing. We suggest that these panels can be used in future studies for detection of risk gene variants in several populations and/or diseases. CONCLUSIONS: In summary, we propose a multiplex assay that is able to identify the most frequent candidate SNPs in innate immune genes, using a medium scale genotyping platform. The assays can be used to evaluate the risk gene variants in populations of various geographic origins.