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Biotechnology

Year: 2018 | Volume: 17 | Issue: 3 | Page No.: 151-157
DOI: 10.3923/biotech.2018.151.157
Optimization of Allele Specific PCR for the Development of Human Mitochondrial DNA Typing Method
Seri Mirianti Ishar and Rus Dina Rus Din

Abstract: Background and Objective: Human DNA is categorized into nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Mutation occurred in these two types of human DNA can be utilized for the purpose of medical reports, molecular genetic analysis, haplogroup variations, forensic crime search and many others. The polymorphisms were selected from mtDNA in both coding and control regions. Main objective of this project was to produce a simple, cost effective yet robust typing method for human identification using allele specific PCR (asPCR) specifically designed for southeast Asian population. Materials and Methods: A total of 60 subjects with ethical approval were randomly collected with 30, 20 and 10 of them represented Malay, Chinese and Indian population. Polymerase Chain Reaction (PCR) was carried out using 25 sets of primers, that amplify the fragments bearing the selected SNPs. In the second round PCR, two types of allele specific primers labelled as wild type allele specific primer (wtASP) and variant type ASP (vtASP) were used to amplify both Cambridge Reference Sequence (CRS) and variant sequence. The PCR was used for first round and for the second round, the as PCR was applied. Finally, the amplified products were directly viewed using UV light. Results: Overall, out of 30 selected SNPs, the designed ASP managed to amplify only 20 SNPs. The selected SNPs in this study were SNP 146, 195, 1709,1719, 1872, 3705, 3027, 3552, 4491, 7684, 9080, 8440, 13626, 16108, 16291, 16274, 16355, 16093, 16335 and 16148 that reported to belong to macrohaplo group M, B, F, E and N. All the amplified products of selected mtSNPs were observed in wild type lane except for SNP 195 that the amplified product was observed in variant lane. Conclusion: The variant allele managed to be amplified with simple technique yet robust to be brought on site. Generally, mutation found using this technique may narrow down the individual and also population hence it is beneficial in cases such as forensic crime and mass disaster.

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How to cite this article
Seri Mirianti Ishar and Rus Dina Rus Din, 2018. Optimization of Allele Specific PCR for the Development of Human Mitochondrial DNA Typing Method. Biotechnology, 17: 151-157.

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