Sequencing from single epithelial cells. HV1 and HV2 regions of mitochondrial DNA

The smallest morphologically defined unit of biological trace evidence is a single cell. In most cases these are exfoliated epithelial cells or cells from secretions. With the aid of a micromanipulator, such as is used for intracytoplasmic sperm injections (ICSI) and self-made pipettes single cells were isolated, or lifted from smear preparations and both hypervariable regions (HV1 and HV2) of mitochondrial DNA (mtDNA) were subsequently amplified and sequenced. In total 175 single epithelial cells from 6 different subjects were examined. Single PCR for the HV1 region was performed for 57 of the single cells and PCR products could be demonstrated in 77% of cases. This procedure was also performed for the HV2 region from 22 single cells with PCR products being demonstrated in 90% of cases. For a further 96 single cells combined amplification of the HV1 and HV2 regions was performed in one PCR reaction and PCR products could be demonstrated in 80% of cases. During all experimental settings no contamination occurred meaning that none of the samples examined yielded any other sequence than the expected one and did not show a mixture of samples. The presented method can be applied to cases in which STR typing and conventional sequencing of mtDNA is limited or impossible. This particularly applies to mixed stains and examinations assessing heteroplasmy in mtDNA.