ArticleThe Indian journal of medical research2015
Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR.
Article in The Indian journal of medical research, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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5 citing papers in PubMed.
- A quantitative polymerase chain reaction approach for distinguishing the geographical origin ofFood science and biotechnology · 2025Article
- Identification ofPathogens (Basel, Switzerland) · 2021Article
- Design and characterization of a SYBR Green I-based melting curve method for investigation of HER2I655V polymorphism in breast cancer.Journal, genetic engineering & biotechnology · 2021Article
- Increased Expression of microRNA-141-3p Improves Necrotizing Enterocolitis of Neonates Through Targeting MNX1.Frontiers in pediatrics · 2020Article
- Identification of selected genetic polymorphisms in polycystic ovary syndrome in Sri Lankan women using low cost genotyping techniques.PloS one · 2018Article
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5 authors.
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Abstract
BACKGROUND &
objectivesGenotyping 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.
methodsTwo 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.
resultsThe 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. INTERPRETATION &
conclusionsIn 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.
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