Rapid mitochondrial DNA typing using restriction enzyme digestion of polymerase chain reaction amplications followed by capillary electrophoresis separation with laser‐induced fluorescence detection

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Abstract
The polymorphic control region of mitochondrial DNA (mtDNA) is becoming more commonly used in forensic applications to differentiate among individuals in a population. Two hypervariable regions (HV1 and HV2) are often sequenced following amplification of the mtDNA via the polymerase chain reaction (PCR). More rapid screening assays would reduce both the effort and the expenses of comparing two samples. A methodology has been developed that first uses restriction endonuclease digestion of the PCR-amplified mtDNA using RsaI and Mn/I and then capillary electrophoresis (CE) to separate and size the PCR-RFLP fragments. This rapid procedure offers an alternative method for screening of polymorphisms in amplified mtDNA samples. In addition, the presence of a T→C transition at position 16189, which gives rise to the so-called “C-stretch” in HV1, may be predicted from the presence of nonspecific PCR products in the CE results.