A Two Stage Strategy for the Automated Analysis of Mitochondrial DNA

DNA amplification and sequencing of the mitochondrial (not) non-coding region provides a highly sensitive and discriminating test for individual identification which is ideal for the analysis of severely degraded human remains (Sullivan et al. 1994, Gill et al. 1994). A major drawback with DNA sequencing as a forensic tool is the labour-intensive nature and therefore high cost of the technique, which presently limits its application to the investigation of serious crime. Alternative strategies to DNA sequencing have been developed to characterise sequence polymorphisms in mtDNA products including hybridisation with allele-specific probes (Stoneking et al. 1991), detection of length polymorphisms (Bodenteich et al. 1992) and oligonucleotide ligation assay (Sullivan et al 1993). More recently, the novel technique of multiplex solid phase fluorescent minisequencing has been developed (Tully et al. 1995) which detects both sequence and length polymorphisms. The latter is being developed by our laboratory as a primary screen in a two stage strategy for the automated analysis of mtDNA. The minisequencing technique enables the majority of non-related individuals to be eliminated in a rapid and straightforward test so that the remaining samples can then be distinguished further by the application of full DNA sequencing, which is more discriminating than minisequencing but also much more labour-intensive to perform.