news
Sorting out mtDNA with MS DNA evidence can make or break a case, potentially placing a suspect at the scene of a crime, exonerating an innocent person, or identifying the remains of a victim. Sometimes, investigators must use mitochondrial DNA (mtDNA) because nuclear DNA is not available. Analysis of mtDNA can be a challenge because some samples contain multiple types of mtDNA, which can’t be resolved using standard sequencing methods. Now, a study in AC (2009, DOI 10.1021/ ac901222y) describes an automated method that uses MS to unravel these mtDNA mixtures. People perform mtDNA analysis when the sample is highly degraded or present in very small amounts, says Steven Hofstadler from Ibis Biosciences, Inc. Hofstadler coauthored the paper with colleagues at Ibis Biosciences, the Federal Bureau of Investigation (FBI), the Armed Forces DNA Identification Laboratory, and the University of North Texas Health Science Center. “The problem with that is that mitochondrial DNA can be highly variable, even within one cell.... When you sequence through that, you can get a real mess.” That variability comes in two flavors: length heteroplasmy and point heteroplasmy. Length heteroplasmy in mtDNA is generally due to variability in the number of cytosines (Cs) in a poly-C stretch, according to Hofstadler. Point heteroplasmy occurs at particular positions along the DNA strand, for example, in single nucleotide polymorphisms. Another potential hurdle is that forensic samples may be contaminated and thus contain a mixture of mtDNA from different people. Contamination can occur in the field or in the laboratory and complicates sequence analysis. “This technique allows the forensic scientist to analyze the components of mixed DNA samples, which is not possible with fluorescent dye terminator sequencing methods,” says Alice Isenberg, who is acting chief of the FBI Laboratory’s bio-
metrics analysis section and was not involved with the study. The key to the method is that, instead of sequencing the DNA, each am-
Mass spectrum of amplification products from a region of mtDNA, which reveals point heteroplasmy. One mtDNA species has a cytosine where the other has a tyrosine. The difference in peak amplitudes shows that the strand with cytosine is the dominant species.
plified region is measured by ESI MS. Because the masses for individual nucleotides are known, it is trivial to calculate the base-pair composition for a given amplicon mass, according to Hofstadler. In the study, the researchers used 24 primer pairs to amplify 24 overlapping segments from a section of the mtDNA that includes, but is slightly larger than, the two hypervariable regions (HV1 and HV2) that are typically sequenced in forensic analyses. Everything post-PCR, including amplicon purification and mass spectral analysis, was automated. The researchers used a 96-well plate format, adding mtDNA to each well. With each sample occupying 8 wells with 3 multiplexed primer pairs per well, each plate can analyze 12 samples at just under 1 minute per well. A mass spectrum of an amplicon from a sample containing a single mtDNA
10.1021/AC901883W 2009 AMERICAN CHEMICAL SOCIETY
Published on Web 09/02/2009
species contains two peaks of similar intensities: one peak for each strand of DNA. With heteroplasmy, the spectra become slightly more complex, containing a pair of peaks for each species. After determining base compositions, though, the nature of the heteroplasmy is apparent. MS data also provide an opportunity to determine the relative abundance of each mtDNA species in a sample because the peak intensities correspond to initial concentration, a feature that also helps with assignments. “When we have multiple sets of peaks and one set is of lower amplitude than the other, we can confirm that a mixture is present. And we can use the relative peak heights to pull the two profiles apart,” says Hofstadler. One well-documented problem with DNA analysis for forensics is that errors can be introduced during the often-laborious workflow. “The increased level of automation in this procedure may prevent certain errors in the lab, such as sample mix-ups,” says Isenberg. But can base composition give as much information as sequencing? According to the study, 94% of the information that allows individuals to be differentiated by sequencing was retained by the mass-based method. “This [6%] loss can be regained by analyzing larger areas of the mtDNA genome,” says Isenberg. Even so, an inherent limitation to this method is that, unlike sequencing, it may not be able to separate out two mtDNA species that contain multiple point differences that are reciprocal or complementary to each other. “That would give the same mass signature but differ in the actual sequence of DNA bases,” says Phil Danielson from the University of Denver. “It will be interesting to see how the scientific and legal communities weigh the potential advantages and limitations of this technology in the years to come.” —Erika Gebel
OCTOBER 1, 2009 / ANALYTICAL CHEMISTRY
7861
