International Journal of Legal Medicine

, Volume 118, Issue 3, pp 147–157 | Cite as

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

  • Peter M. Vallone
  • Rebecca S. Just
  • Michael D. Coble
  • John M. Butler
  • Thomas J. Parsons
Original Article

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.

Keywords

Mitochondrial DNA Capillary electrophoresis Allele-specific primer extension Single nucleotide polymorphism Multiplex PCR 

Notes

Acknowledgements

We thank Suzanne M. Barritt of AFDIL for discussion and comments on the manuscript, as well as for providing non-probative casework samples. Jill Appleby, Tracey Johnson, Danielle Goldstein, and Jennifer Kappeller of AFDIL also assisted in providing casework samples. Jennifer O’Callaghan, Jessica Saunier, Allison McClure, and Devon Pierce, AFDIL, provided technical laboratory or data analysis assistance. We thank Brion C. Smith, James C. Canik, and Kevin S. Carrol, AFDIL, for administrative, logistical, and moral support, and James P. Ross for computer support.

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Peter M. Vallone
    • 1
  • Rebecca S. Just
    • 2
  • Michael D. Coble
    • 2
  • John M. Butler
    • 1
  • Thomas J. Parsons
    • 2
  1. 1.Biotechnology DivisionNational Institute of Standards and TechnologyGaithersburgUSA
  2. 2.Armed Forces DNA Identification Laboratory, RockvilleUSA

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