Mitochondrial DNA Testing on Evidentiary Specimens in the FBI Laboratory: Casework Examples and Future Directions
Mark R. Wilson, Joseph A. DiZinno, John E.B. Stewart, Eric Pokorak, Deborah Polanskey, and Bruce Budowle
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The F.B.I. Laboratory began mitochondrial DNA (mtDNA) testing on casework specimens in June, 1996. Since that time, approximately seventy (70) cases have been processed. This technique widens the applicability of DNA typing to forensic specimens such as bones and teeth, which have not previously been amenable to DNA typing.
Two major areas of concern in mtDNA testing are the potential for external contamination and heteroplasmy. Potential contamination is monitored through the use of reagent blank and negative control amplification reactions. Heteroplasmy is a biological aspect of mtDNA genetics, and is becoming better understood by both the general scientific and forensic communities. In heteroplasmic individuals, point or length mutations are observed in differing ratios from tissue to tissue, and, in some cases, from hair to hair within an individual. The presence of heteroplasmy can complicate sequence comparisons, and hence match criteria. However, careful assessment of known samples, combined with an understanding of the segregational mechanics of heteroplasmy and mtDNA population genetics, can enhance the power of a sequence match in some instances where heteroplasmy is observed.
Recent advances in fluorescent sequencing chemistry have greatly enhanced the quality of mtDNA sequence data. This enhanced quality results in less interpretation difficulty arising from comparisons which include individuals from heteroplasmic lineages. It is anticipated that future technological developments will continue this trend. Figure 1 shows the presence of heteroplasmy at position 16355 in hypervariable region I of the human mtDNA control region. The light strand sequence is shown from two separate sequencing reactions from amplified blood sample extracts. The Taq FS dye terminator chemistry from Perkin-Elmer/Applied Biosystems Division (PE/ABD) was employed in this case.
Figure 1. Two separate extractions from blood samples of a heteroplasmic individual at position 16355 of hypervariable region I of the mtDNA control region. The chemistry used in this set of experiments was Taq FS dye terminator chemistry from PE/ABD.
Figure 2 shows the sequencing results from three separate hair samples from the same individual. Again, position 16355 shows heteroplasmy, but the pattern is markedly different from that of blood samples. In some hairs, thymine predominates, cytosine in others, and a mixture is present in some hairs. It is obvious from observing the chromatograms in Figure 2 that clean, robust fluorescent sequencing is imperative in these types of analyses, especially when subtle mixtures due to heteroplasmy are encountered.
PE/ABD has since released a new dye terminator chemistry, utilizing dichloro-Rhodamine terminators. In our hands, these dyes offer a substantial improvement in the quality of sequence chromatograms, greatly enhancing the ease of mtDNA sequence analysis. Figure 3 shows a direct comparison of Taq FS dye-terminator chemistry to the new di-chloroRhodamine chemistry. Not only is the signal enhanced with the new dyes, but less background noise is also observed. Enhancements of this kind greatly facilitate interpretation, as well as simplify sequence comparisons when heteroplasmy is present.
Figure 3. The top panel shows a segment of hypervariable region I sequenced with Taq FS terminator chemistry. The same template, sequenced with dichloro Rhodamine terminators, is shown in the lower panel.
The forensic mtDNA typing community is actively tracking instances where heteroplasmy is encountered, both in controlled studies and casework. Specific patterns are beginning to emerge. For instance, position16093 in hypervariable region I has been identified as a "hot spot", subject to a substantially higher rate of heteroplasmy than other positions. Future discoveries in this vein should not only assist to elucidate the mechanism of mutation in the human mtDNA control region, but at the same time enhance the comparison process in forensic
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