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A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the
Peter M Vallone1, Rebecca S Just, Michael D Coble
1Biotechnology Division, National Institute of Standards and Technology, 100 Bureau Drive, Mail Stop 8311, Gaithersburg, MD 20899, USA. peter.vallone@nist.gov
Abstract:
The typing of single nucleotide polymorphisms (SNPs) located throughout the mitochondrial genome (mtGenome) can help resolve individuals with an identical HV1/HV2 mitotype. A set of 11 SNPs selected for distinguishing individuals of the most common Caucasian HV1/HV2 mitotype were incorporated in an allele specific primer extension assay. The assay was optimized for multiplex detection of SNPs at positions 3010, 4793, 10211, 5004, 7028, 7202, 16519, 12858, 4580, 477 and 14470 in the mtGenome. Primers were designed to allow for simultaneous PCR amplification of 11 unique regions in the mtGenome and subsequent primer extension. By enzymatically incorporating fluorescently labeled dideoxynucleotides (ddNTPs) onto the 3' end of the extension primer, detection can be accomplished with a capillary-based electrophoresis (CE) platform common in most forensic laboratories. 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. We demonstrate that the assay performs well for heteroplasmy and mixture detection, and for typical mtDNA casework samples with highly degraded DNA.