, Volume 118, Issue 3, pp 147-157
Date: 04 Feb 2004

A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome

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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.

Official disclaimers. Certain commercial equipment, instruments and materials are identified in order to specify experimental procedures as completely as possible. In no case does such identification imply a recommendation or endorsement by the National Institute of Standards and Technology nor does it imply that any of the materials, instruments or equipment identified are necessarily the best available for the purpose. The opinions and assertions contained herein are solely those of the authors and are not to be construed as official or as views of the U.S. Department of Defense, the U.S. Department of the Army, or the U.S. Department of Justice.
Sources of support. The National Institute of Justice (NIJ) funded the work described here through an interagency agreement with the NIST Office of Law Enforcement Standards. This work was also supported by the National Institute of Justice grant 2000-1J-CX-K010 to T.J.P.