Dataset Information

A Mutagenic Primer Assay for Genotyping of the CRHR1 Gene Rare Variant rs1876828 (A/G) in Asians: A Cost-Effective SNP Typing.

ABSTRACT: BACKGROUND:Today, the genetic and genomic research entered in a new era of high-throughput genotyping technology. However, mutagenic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is still a choice of genotyping method in molecular epidemiological research. It has been extensively used for the detection of risk alleles, if the target SNP has no natural discriminating restriction site. We undertook this study to develop a mutagenic primer assay for a CRHR1 rare gene variant: rs1876828 (A/G) and to determine their allele frequency in north Indian children. METHODS:The mutagenic primers were designed and assay conditions were optimized to perform mutagenic PCR-RFLP in 550 subjects. The efficiency of assay and results were validated by sequencing. RESULTS:This study demonstrated that the mutagenic primer assay is feasible and applicable to discriminate CRHR1 gene rare variant rs1876828 (A/G) and the "frequency of allele "G" was 100% in north Indian asthmatics as well as normal subjects. CONCLUSION:This method can be used for both large- and small-scale study of complex genetic, where CRHR1 gene plays the pivotal roles.

SUBMITTER: Sharma N

PROVIDER: S-EPMC6807002 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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A Mutagenic Primer Assay for Genotyping of the CRHR1 Gene Rare Variant rs1876828 (A/G) in Asians: A Cost-Effective SNP Typing.

Sharma Neeraj N Sharma Neeraj N Awasthi Shally S Phadke Shubha R SR

Journal of clinical laboratory analysis 20141226 2

<h4>Background</h4>Today, the genetic and genomic research entered in a new era of high-throughput genotyping technology. However, mutagenic polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is still a choice of genotyping method in molecular epidemiological research. It has been extensively used for the detection of risk alleles, if the target SNP has no natural discriminating restriction site. We undertook this study to develop a mutagenic primer assay for a CRHR1 r ...[more]

PMID: 25546060

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Project description:Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which should prove useful to the wider scientific community.

Dataset Information

A Mutagenic Primer Assay for Genotyping of the CRHR1 Gene Rare Variant rs1876828 (A/G) in Asians: A Cost-Effective SNP Typing.

Publications

A Mutagenic Primer Assay for Genotyping of the CRHR1 Gene Rare Variant rs1876828 (A/G) in Asians: A Cost-Effective SNP Typing.

Similar Datasets

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