Participants: Peter M. Vallone, Michael D. Coble, Margaret C. Kline, and John M. Butler (AFDIL participants: Rebecca Just and Thomas Parsons)
Project Timeframe: 2001 to present
Purpose: The development of multiplex primer extension assays to probe coding region mitochondrial SNPs to help resolve common mitotypes.
Progress: The typing of single nucleotide polymorphisms (SNPs) located throughout the mitochondrial genome (mtGenome) allows for differentiation between individuals possessing an identical HV1/HV2 sequence. 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 11-plex assay probed SNPs located at positions 477, 3010, 4580, 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 polymerase chain reaction (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 site of interest were enzymatically extended using fluorescently labeled dideoxynucleotides (ddNTPs). Resolution and detection of each extended fragment were 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 11-plex assay has resulted in an accurate method for typing sequence variant mtSNPs on a platform common to forensic laboratories.
We are currently developing additional multiplex SNP panels to resolve other common mitotypes (Caucasian, Hispanic, and African American).
Publications Resulting From This Project:
Kline, M.C., Vallone, P.M., Redman, J.W., Duewer, D.L., Calloway, C.D., and Butler, J.M. (2005) Mitochondrial DNA typing screens with control region and coding region SNPs. J. Forensic Sci. 50(2): 377-385.
Just, R.S., Irwin, J.A., O'Callaghan, J.E., Saunier, J.L., Coble, M.D., Vallone, P.M., Butler, J.M., Barritt, S.M., and Parsons, T.J. (2004) Toward increased utility of mtDNA in forensic identifications. Forensic Sci. Int. 146S: S147-S149.
Vallone, P.M., Just, R.S., Coble, M.D., Butler, J.M., and Parsons, T.J. (2004) A multiplex allele-specific primer extension assay for forensically informative SNPs distributed throughout the mitochondrial genome. Int. J. Legal Med. 118: 147-157.
Coble, M.D., Just, R.S., O'Callaghan, J.E., Letmanyi, I.H., Peterson, C.T., Irwin, J.A., Parsons, T.J. (2004) Single nucleotide polymorphisms over the entire mtDNA genome that increase the power of forensic testing in Caucasians. Int. J. Legal Med. 118: 137-146.
This is a National Institute of Justice funded project conducted by the National Institute of Standards and Technology Human Identity Team. This project is supported by Grant Numbers 1999-IJ-R-A094 and 2003-IJ-R-029, which is an interagency agreement between NIJ and the NIST Office of Law Enforcement Standards, awarded by NIJ, Office of Justice Programs, U.S. Department of Justice. Points of view in this document are those of the authors and do not necessarily represent the official position or policies of the U.S. Department of Justice. 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 NIST nor does it imply that any of the materials, instruments, or equipment identified are necessarily the best available for the purpose.
