The typing of single nucleotide polymorphisms (SNPs) located throughout the mitochondrial genome (mtGenome) allows for differentiation between individulas possessing an identical HVa/HV2 sequence. A set of 11 SNPs selected for separating the most common Caucasian HV1/HV2 mitotype were incorporated in an allele-specific primer extension assay. The 11 plex assay probed SNPs located at positions 477, 2010, 4530, 4793, 5004, 7028, 7202, 10211, 12858, 14470 and 16519 in the mtGenome. The assay was optimized for multiplex detection of these SNPs. Primers were designed to allow for the simultaneous PCR amplification of 11 unique regions in the mtGenome. Locus specific primers of varying lengths were employed in multiplex primer extension reactions. Extension primers binding 5' adjacent to the SNP sige of interest were enzymatically extended using fluorescently labeled dideoxynucleotides (ddNTPs). Resolution and detection of each extended fragment was achieved by analysis on a capillary-based electrophoresis (CE) platform. The electrophoretic mobility for the extension primers was compared in denaturing POP4 and POP6 CE running buffers. Empirical adjustment of extension primer concentrations resulted in even signal intensity for the 11 loci probed. The development of the mtSNP 11plex assay has resulted in an accurate method for typing sequence variant mtSnPs on a platform common to forensic laboratories.
Citation: International Journal of Legal Medicine
Pub Type: Journals
allele specific primer extension, capillary electrophoresis, mitochondrial DNA, multiplex PCR, single nucleotide polymorphism