Mitochondrial DNA SNP Detection: Design Issues and the Use of the Mass Spectrometer as an Analysis Platform

Introduction DNA typing has become widely accepted for the characterization of forensic biological evidence. The current genetic markers used, i.e., predominately short tandem repeat (STR) loci and to a lesser extent mitochondrial DNA (mtDNA), offer high levels of discrimination. In addition, the polymerase chain reaction (PCR) (1), the cornerstone of forensic DNA typing, provides a sensitivity of detection such that exceedingly small samples can be analyzed. Even with the successes encountered using the PCR and forensically validated genetic markers, there are samples that do not contain sufficient DNA or are too degraded to undergo DNA analysis. Strategies are being sought to type samples containing very minute amounts of DNA. The approach most widely used which typifies the strategy for attempting to analyze very limited amounts of DNA template is known as low copy number (LCN) typing (2-9). LCN typing, usually carried out with STR loci, is the analysis of any results below the stochastic threshold for normal interpretation. Typing can be achieved by increasing the number of PCR cycles, for example from 28 to 34; by reducing the PCR volume; by reducing salt concentration in the sample before capillary electrophoresis; by use of a formamide with a lower conductivity; by adding more amplified product to the denaturant formamide; and/or by increasing injection time (10). However, at these low levels of template DNA (usually less than 100 pg), stochastic effects can and do occur, resulting in either a substantial imbalance of two alleles at a given heterozygous locus or allelic dropout. Even though the assay may not always be reproducible and suffers from allele drop out, as well as allele drop in, LCN has been used for analysis of forensic evidence and for providing investigative leads (2-4, 7-9). A more robust approach than LCN typing is the interrogation of smaller DNA target regions. To improve success with STR typing of limited and/or degraded DNA, the PCR primers for the STR loci can be repositioned so they reside closer to the repeat region (11-14). Thus, the PCR amplicon(s) is reduced in length. If the amplicon to be generated is smaller than some of the fragmented DNA template molecules, 2 STR analysis of the degraded sample may then be possible. This approach has been successful for typing telogen hairs (12) and for typing some of the remains from the World Trade Center disaster (15). To reduce the amplicon size further another class of …