Barbara C. Levin,¹ Haiyan Cheng² and Dennis J. Reeder^1
1 Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD
² GEO-CENTERS, Inc., Newton Centre, MA

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Every human cell has from a few dozen to several thousand mitochondria, each of which contains mitochondrial DNA (mtDNA). The sequence of the entire human mtDNA (16,569 base pairs) was determined and published by Anderson et al. In 1981. MtDNA is being used by the forensic community for human identification and by the medical community for diagnoses of a number of human diseases now known to be associated with specific mutations and deletions of mtDNA. A third area of investigation, namely, examination of the mutagenic effects of environmental toxins on mtDNA is essentially unexplored. A mtDNA standard reference material (SRM) is being prepared by the National Institute of Standards and Technology to provide quality control for the scientific community when they sequence human mtDNA. The mtDNA standard reference material set will include all the components necessary to successfully go through the PCR amplification process (following the extraction of DNA from blood or tissue), cycle sequencing steps, gel separation and data analysis to the final DNA sequence. The extracted DNA from an Epstein-Barr virus transformed tissue culture cell line from blood and fifty-eight sets of unique primers will be supplied with the SRM to allow any area or all of the mtDNA to be amplified and cycle sequenced. Compared to the Anderson sequence, the mtDNA from this tissue culture cell line has six differences in the HV1 and seven in the HV2 region (the two areas of the non-coding region) and 33 differences in the coding regions. None of these differences correspond to any of the published mutations in mtDNA that have been correlated with specific disease states. The differences between the Anderson sequence and the SRM mtDNA make this cell line a good choice for use in the mtDNA SRM, since it provides multiple areas of comparison. Before release to the scientific community, an interlaboratory evaluation of the entire 16,569 base pair mtDNA SRM will be conducted by multiple laboratories to determine any difficulties other laboratories might encounter in amplification, sequencing and analysis of data. Investigators will be able to purchase this SRM from NIST and use it as a control when they amplify and sequence their experimental mtDNA. The correct results with the SRM will provide the quality assurance to the investigators that they are sequencing their experimental mtDNA correctly, providing the right identifications and determining the correct diagnoses.