LA Tully, FP Schwarz, and BC Levin
Biotechnology Division, NIST

The National Institute of Standards and Technology (NIST) is developing a heteroplasmic human mitochondrial DNA (mtDNA) standard reference material (SRM) to provide quality control to medical, forensic, and toxicological scientists who wish to determine their detection limits when examining low frequency mutations or heteroplasmic sites in DNA. The detection of a mutation or polymorphism that is present in every mtDNA molecule is routine. However, in many cases, especially disease-prone individuals, the disease does not become apparent until a significant number of mtDNA molecules contain the mutation. The proportion of mutant mtDNA genomes appears to increase with age (i.e., the mutation becomes more predominant in older individuals, who then exhibit the disease symptoms). Heteroplasmy has also become problematic to the forensic community. It is very difficult to detect heteroplasmy present at low concentrations. Different hairs from the same individual can have different proportions of the base pairs contributing to the heteroplasmy. The presence of such heteroplasmic differences may result in an exclusion rather than a match. Toxicologists would like to detect mutations that arise in mtDNA from environmental exposures. With the present state-of-the-art techniques, however, low frequency mutations scattered throughout the DNA are almost impossible to detect.


Mitochondrial DNA mixtures containing a polymorphic/ wild-type site in different percentages (e.g., 1, 2.5, 5, 10, 20, 30, 40, and 50%) have been constructed from PCR products from two different cell culture lines which differed by one base pair in the amplified region. Various mutation detection techniques [e.g., automated sequencing, denaturant gradient gel electrophoresis (DGGE), peptide-nucleic acid (PNA)-directed PCR clamping] were used to determine the lowest detectable level of heteroplasmy in our mixtures.


With automated sequencing, we were able to unambiguously detect the polymorphism present at the 30% level. Although visible at the 10% and 20% concentrations, it was difficult to distinguish the polymorphism from the background. Using both DGGE and PNA-directed PCR clamping followed by sequencing, our resolution was increased to 5%.


We will conduct an Interlaboratory Evaluation (ILE) on these heteroplasmic mixtures to determine if other laboratories can detect the polymorphism. They will be free to use any technique that they may have developed to detect low frequency mutations or polymorphisms. The final mtDNA heteroplasmic SRM will include PCR products that contain different concentrations of a heteroplasmic site. Investigators will be able to use this SRM to determine the resolution of their mutation detection techniques and allow them to perfect even more sensitive methods. In-house research will assure that each step of the process is reliable, simple, and efficient and that the components of the SRM provide results which are trustworthy and cost-effective.