Sequence analysis of mitochondrial DNA hypervariable regions using infrared fluorescence detection

The non-coding region of the mitochondrial genome provides an attractive target for human forensic identification studies. Two hypervariable (HV) regions, each approximately 250-350 bp in length, contain the majority of mitochondrial DNA (mtDNA) sequence variability among different individuals. Various approaches to determine mtDNA sequence were evaluated utilizing highly sensitive infrared (IR) fluorescence detection. HV regions were amplified either together or separately and cycle-sequenced using a Thermo Sequenase protocol. An M13 universal primer sequence tail covalently attached to the 5' terminus of an amplification primer facilitated electrophoretic analysis and direct sequencing of the amplification products using IR detection.