Ishar, Seri Mirianti
(2018)
Development of mitochondrial DNA typing method for human identification.
Masters thesis, Universiti Sains Malaysia.
Abstract
Detection of mutations in human mtDNA is useful for the purpose of medical
reports, molecular genetic analysis, haplogroup variations, forensic search and many
other applications. In this project, Single Nucleotide Polymorphims (SNPs) from
coding and control regions of mtDNA were selected for development of mtDNA
typing kit. The main objective of this kit is to provide a simple, cost effective yet robust
identification of human remains. A total of 30 SNPs that are specific for Southeast
Asian haplogroups such as M, B, F, E and N were selected. The first round Polymerase
Chain Reaction (PCR) was performed using 25 sets of in house designed primers. The
purified amplified products were used in second round PCR known as allele specific
PCR (asPCR) and also sent for sequencing to confirm the sequence of the amplified
products. In asPCR, two types of allele specific primers (ASPs) for wild type ASP
(wtASP) and variant type ASP (vtASP) were designed to identify the polymorphisms
within the target fragments. A total of 20 ASPs were successfully designed and able
to amplify the targeted SNPs. For quality control purpose and also to avoid false
positive or false negative results in asPCR, an internal positive PCR control (IPC) was
developed and incorporated together in the same tube during asPCR. A validation
study for this mtDNA typing kit was carried out using human DNA samples (Malay,
Chinese and Indian), mammals, insects and several types of bacteria. Sensitivity study
was carried out using DNA extracted from ancient bones and serial dilution of freshhuman DNA sample. The ASPs are only specific for human DNA as no amplification
was observed when using DNA template from other species. Based on the sensitivity
validation study, the minimum amount of genomic DNA template required for
optimum amplification of first round PCR is 1ng/μl. Based on the successful
amplifications of both round PCRs using in house designed primers and the validation
studies results, a simple yet effective mtDNA typing kit for human identification has
been successfully developed in this study. This kit would be very much useful to
narrow down individual classification especially in cases of mass disaster, forensic
crime and plane crash.
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