Robert J. Steighner¹, Lois Tully², Valerie Prenger², Mitchell Holland¹, Phillip Belgrader^1
1 Armed Foreces DNA Identification Laboratory (AFDIL), Office of the Armed Forces Medical Examiner, The Armed Forces Institute of Pathology, Rockville, MD
² University of Maryland at Baltimore (U.M.A.B.), School of Medicine, Division of Human Genetics,
Baltimore, MD

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Mitochondrial DNA (mtDNA) typing is increasingly employed in the forensic identification of poorly preserved samples such as those obtained from skeletal remains. Current procedures require the amplification and sequencing of the entire mitochondrial hypervariable regions one (HV1) and two (HV2) from both test and reference samples before any comparisons may be made. DGGE of PCR products provides an accurate, inexpensive, and high throughput alternative to sequencing for testing the uniqueness of two mtDNA samples. Additional information derived from DGGE includes the identification of heteroplasmic variants (length or sequence-based) and contaminated samples. Typing of these samples is still possible since the individual species are resolved from one another in this procedure.

DGGE exploits the sensitivity of specified regions of DNA, defined by sequence, to denaturation under specific chemical denaturant concentrations. Differences in base-stacking energies induced by a single polymorphism between two otherwise identical fragments, result in a differential susceptibility to denaturation in the gradient. Sequence homology between reference and test samples is determined by mixing, heat denaturing, and allowing the sequences to reanneal (heteroduplexing). If a single matching band is observed by DGGE in both the heteroduplexed and independent sample lanes, the two input sequences are identical. If more than one sequence is present, multiple bands will be seen in the heteroduplexed lane and the uniqueness of the sequences established. Heteroplasmy or contamination will result in multiple bands in the independent sample lanes.

To test the sensitivity of the technique, twenty-five pairs of samples, each differing by a single unique polymorphism were amplified with primers designed for DGGE. The polymorphisms were distributed throughout the HV1 region. Twenty-three of the polymorphisms were transitions and two were transversions. The amplified products encompassed either the entire HV1 region, or alternatively, HV1 was divided into two smaller regions which were amplified and analyzed separately. All twenty-five pairs were resolved by DGGE, with the smaller fragments displaying superior resolution. Suspected heteroplasmic samples were also separated by DGGE and the heteroplasmic positions identified by sequence analysis following excision of the bands from the gel. These results strongly support the efficacy of the method. Sample pairs differing by multiple polymorphisms (as is seen with unrelated individuals) would be more unstable than those tested in this study and are expected to result in even easier resolution. We believe the technique represents a viable screening strategy for comparing two PCR products without sequencing.

The opinions and assertions expressed herein are solely those of the author and are not to be construed as official or as the views of the United States Department of Defense or the United States Department of the Army.