Abstract
Candidate long mtDNA targets ∼300 bp in length were identified on the revised Cambridge mtDNA reference sequence using Primer Express software (Applied Biosystems) with modified default analysis settings. The primer and hydrolysis probe sequences for the resultant three (3) targets were queried in the Mitomap database [1] to avoid common single nucleotide polymorphisms (SNPs) which, if present in a sample, could reduce binding to template and therefore result in inefficient amplification. Primers and probes identified by Primer Express, some synthesized degenerate to mitigate the presence of certain SNPs, were utilized in a Fast Advanced Master Mix (Applied Biosystems) reaction which was amplified on a 7500 Real Time PCR System using HID Real Time PCR Software v1.2 (Applied Biosystems) to collect and analyze the qPCR data. QPCR reaction conditions and software analysis settings were optimized and modified to yield efficient amplification and robust results. QPCR experiments were exported into Excel (Microsoft Corp.) for additional analyses and evaluation. The data was used to develop a triplex qPCR method, which includes amplification of one of the long targets, to quantify and assess degradation of human mtDNA, the results of which were previously published [2]. That triplex method also incorporated an internal positive control to test for the presence of amplification inhibitors in the sample [3]. The data presented herein may be used to develop alternative amplification methods for user-specific biomedical applications.