Keith McKenney, Jingxiang Tian, Prasad Reddy and Dennis Reeder
DNA Technologies Group, Biotechnology Division, Chemical Science and Technology Laboratory,
National Institute of Standards and Technology, Gaithersburg, MD

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We have developed a human mitochondrial DNA reference material in response to requests from the forensics community. A survey of the forensics community defined the human mitochondrial DNA sequences between nucleotides 15,806 and 626 as essential reference material for DNA amplification and sequence determination. This sequence encodes a number of tRNA genes, the origin for H strand DNA replication, a number of control sites and is called the HV1+ HV2 region of the mitochondrial genome. Two synthetic oligonucleotide primers were designed to prime the amplification of a high quality 1390 base pair double stranded DNA product. The DNA product is easily purified with a Quiagen spin column. Both DNA strands are sequenced using a four primer set in conjunction with DNA polymerase and dideoxy dye terminators, providing high quality DNA sequence data for human identification. The four primers make three primer pairs that can be used to amplify and sequence the complete HV1 + 2 region as well as the individual HV1 and HV2 regions.