Optimization of allele specific pcr for the development of human mitochondrial DNA typing method

Seri Mirianti Ishar, Rus Dina Rus Din

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish
Pages (from-to)151-157
Number of pages7
JournalBiotechnology
Volume17
Issue number3-4
DOIs
Publication statusPublished - 1 Jan 2018

Fingerprint

DNA Fingerprinting
Polymerase chain reaction
Human Development
Mitochondrial DNA
Single Nucleotide Polymorphism
Alleles
Polymerase Chain Reaction
Crime
Bearings (structural)
DNA
Population
Forensic Anthropology
Mutation
Polymorphism
Ultraviolet radiation
Disasters
Ultraviolet Rays
Molecular Biology
Costs and Cost Analysis
Costs

Keywords

  • Haplogroup variations
  • Human population
  • Mitochondrial DNA
  • Molecular genetic analysis
  • Mutation
  • Polymorphisms
  • Variant allele

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Optimization of allele specific pcr for the development of human mitochondrial DNA typing method. / Ishar, Seri Mirianti; Din, Rus Dina Rus.

In: Biotechnology, Vol. 17, No. 3-4, 01.01.2018, p. 151-157.

Research output: Contribution to journalArticle

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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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N2 - 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.

AB - 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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KW - Mitochondrial DNA

KW - Molecular genetic analysis

KW - Mutation

KW - Polymorphisms

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