Rapid Mitochondrial DNA Typing Using Restriction Enzyme Digestion of PCR Amplicons and Capillary Electrophoresis

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Rapid Mitochondrial DNA Typing Using Restriction Enzyme Digestion of PCR Amplicons and Capillary Electrophoresis

John M. Butler*, Mark R. Wilson², and Dennis J. Reeder¹
¹Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899
²DNA Unit II, FBI Laboratory, Washington, D.C., 20535
*Current address: GeneTrace Systems, Inc., Menlo Park, CA 94025

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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 by the polymerase chain reaction (PCR). Typically, several hundred nucleotides are sequenced for sample comparison purposes. However, DNA sequencing remains labor intensive and expensive. More rapid screening assays would reduce both the effort and the expense of comparing two samples, especially if an exclusion could be made early on. A methodology has been developed that first uses restriction endonuclease digestion of PCR-amplified mtDNA with RsaI and MnlI. Capillary electrophoresis (CE) is then utilized to separate the generated DNA fragments and produce distinguishable patterns and thus differentiate samples. This rapid procedure offers an alternative method for screening polymorphisms in amplified mtDNA samples.

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