Dataset Information

Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR.

ABSTRACT: Genotyping has now become one of the major diagnostic means for almost all diseases. Among the advanced techniques that are used to study single nucleotide polymorphisms (SNPs), only a few are applicable for routine disease diagnosis. Their applicability mainly depends on three factors: cost, time, and accuracy. The primary objective of this study was to propose allele-specific real-time PCR as a rapid, low cost and simple genotyping method for routine diagnostics.Two SNPs, rs3014866 and rs2149356 were analysed using allele-specific real-time PCR. The polymerase chain reaction was carried out using RealQ PCR master mix containing SYBR Green DNA I dye followed by melt curve analysis. The results were validated by agarose gel electrophoresis and DNA sequencing.The allelic discrimination and zygosity of the two SNPs were assessed by combined cycle threshold (Ct) and melting temperature (T m ) values. Variations in Ct and T m values among the two alleles were observed in both rs3014866 (Ct: C allele - 24 ± 1, T allele - 27 ± 1; T m: C allele - 82.5 ± 0.3, T allele - 86.3 ± 0.2) and rs2149356 (Ct: C allele - 24 ± 1, A allele - 26 ± 1; T m: C allele - 79.4 ± 0.2, A allele - 80.4 ± 0.3). Based on the variations, homozygous and heterozygous alleles were detect ed. Agarose gel electrophoresis and DNA sequencing also confirmed the allelic variation and zygosity observed in real-time PCR.In diagnostic settings where a large number of samples are analysed daily, allele-specific real-time PCR assay may serve as a simple, low cost and efficient method of genotyping.

SUBMITTER: Dhas DB

PROVIDER: S-EPMC4743342 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR.

Dhas D Benet Bosco DB Ashmi A Hiasindh AH Bhat B Vishnu BV Parija Subash Chandra SC Banupriya N N

The Indian journal of medical research 20151101 5

<h4>Background & objectives</h4>Genotyping has now become one of the major diagnostic means for almost all diseases. Among the advanced techniques that are used to study single nucleotide polymorphisms (SNPs), only a few are applicable for routine disease diagnosis. Their applicability mainly depends on three factors: cost, time, and accuracy. The primary objective of this study was to propose allele-specific real-time PCR as a rapid, low cost and simple genotyping method for routine diagnostics ...[more]

PMID: 26658590

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Project description:BACKGROUND: Together single nucleotide substitutions and small insertion/deletion variants are the most common form of sequence variation in the human gene pool.High-resolution SNP profile and/or haplotype analyses enable the identification of modest-risk susceptibility genes to common diseases, genes that may modulate responses to pharmaceutical agents, and SNPs that can affect either their expression or function. In addition, sensitive techniques for germline or somatic mutation detection are important tools for characterizing sequence variations in genes responsible for tumor predisposition. Cost-effective methods are highly desirable. Many of the recently developed high-throughput technologies are geared toward industrial scale genetic studies and arguably do not provide useful solutions for small laboratory investigator-initiated projects. Recently, the use of new fluorescent dyes allowed the high-resolution analysis of DNA melting curves (HRM). RESULTS: Here, we compared the capacity of HRM, applicable to both genotyping and mutation scanning, to detect genetic variations in the tumor suppressor gene TP53 with that of mutation screening by full resequencing. We also assessed the performance of a variety of available HRM-based genotyping assays by genotyping 30 TP53 SNPs. We describe a series of solutions to handle the difficulties that may arise in large-scale application of HRM to mutation screening and genotyping at the TP53 locus. In particular, we developed specific HRM assays that render possible genotyping of 2 or more, sometimes closely spaced, polymorphisms within the same amplicon. We also show that simultaneous genotyping of 2 SNPs from 2 different amplicons using a multiplex PCR reaction is feasible; the data can be analyzed in a single HRM run, potentially improving the efficiency of HRM genotyping workflows. CONCLUSION: The HRM technique showed high sensitivity and specificity (1.0, and 0.8, respectively, for amplicons of <400 bp) for mutation screening and provided useful genotyping assays as assessed by comparing the results with those obtained with Sanger sequencing. Thus, HRM is particularly suitable for either performing mutation scanning of a large number of samples, even in the situation where the amplicon(s) of interest harbor a common variant that may disturb the analysis, or in a context where gathering common SNP genotypes is of interest.

Project description:BackgroundDNA extracted from historical samples is an important resource for understanding genetic consequences of anthropogenic influences and long-term environmental change. However, such samples generally yield DNA of a lower amount and quality, and the extent to which DNA degradation affects SNP genotyping success and allele frequency estimation is not well understood. We conducted high density SNP genotyping and allele frequency estimation in both individual DNA samples and pooled DNA samples extracted from dried Atlantic salmon (Salmo salar) scales stored at room temperature for up to 35 years, and assessed genotyping success, repeatability and accuracy of allele frequency estimation using a high density SNP genotyping array.ResultsIn individual DNA samples, genotyping success and repeatability was very high (> 0.973 and > 0.998, respectively) in samples stored for up to 35 years; both increased with the proportion of DNA of fragment size > 1000 bp. In pooled DNA samples, allele frequency estimation was highly repeatable (Repeatability = 0.986) and highly correlated with empirical allele frequency measures (Mean Adjusted R2 = 0.991); allele frequency could be accurately estimated in > 95% of pooled DNA samples with a reference group of at least 30 individuals. SNPs located in polyploid regions of the genome were more sensitive to DNA degradation: older samples had lower genotyping success at these loci, and a larger reference panel of individuals was required to accurately estimate allele frequencies.ConclusionsSNP genotyping was highly successful in degraded DNA samples, paving the way for the use of degraded samples in SNP genotyping projects. DNA pooling provides the potential for large scale population genetic studies with fewer assays, provided enough reference individuals are also genotyped and DNA quality is properly assessed beforehand. We provide recommendations for future studies intending to conduct high-throughput SNP genotyping and allele frequency estimation in historical samples.

Dataset Information

Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR.

Publications

Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR.

Similar Datasets

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