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RESEARCH PROFILES New mitochondrial DNA profiling technique for forensics
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dundant samples quickly, before mtDNA sequencing is done. SNP-typing methods based on DNA microarrays and mini-sequencing lack the sensitivity and reproducibility needed for forensic work, though, and MALDI TOF requires excessively short PCR amplicons for analysis. So Parson’s team turned to ESI MS.
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Searching for new and efficient ways to analyze crime-scene specimens, Walther Parson and colleagues at the Institute of Legal Medicine at Innsbruck Medical University (Austria) have turned to mitochondrial DNA (mtDNA) and multiplex-PCR typing of single nucleotide polymorphisms (SNPs). Using ion-pair reversed-phase HPLC and ESI TOFMS (ICEMS), they analyzed PCR amplicons covering 23 known SNPs from a sample population. The work is described in the November 15 issue of Analytical Chemistry (pp 7816–7827). The researchers found that the 23-plex-PCR/ICEMS assay was effective at discriminating among mtDNA samples from 90 West Eurasian male individuals with a high power of discrimination (POD) and sensitivity. The technique also proved effective for analyzing samples from eight typical crime-scene DNA sources, including hair, chewing gum, and swabs from a mobile phone and other surfaces. “Forensic mtDNA typing is usually used to investigate samples that may not contain enough nuclear DNA, maybe due to degradation or sample size,” Parson says. But because one crime scene can generate ≥100 specimens to be typed, a rapid screening technique was needed to speed the process and clear sample backlogs. “Our method would help to sort out the samples that need special attention, such as sequencing, and exclude the majority of samples that play no role in the particular case,” says Parson. Standard forensic typing of mtDNA relies on sequence analysis of hypervariable segments (HVS-I and HVS-II) of the noncoding control region. HVSI/HVS-II sequencing is currently the accepted standard for crime-scene identification in international courts, Parson notes, but that analysis is “laborious, time-consuming, and expensive.” Because SNP patterns can be used to determine the population genetic background of a sample, SNP typing represents a promising rapid-screening method to weed out irrelevant or re-
The power of HPLC/MS combined with the selectivity of multiplex-PCR typing of SNPs yields a new mtDNA profiling technique.
Their approach has several advantages as an mtDNA screening tool. The 23 target SNPs were selected because of their known usefulness in forensic discrimination in West Eurasian populations, resulting in a POD of 94.8%. Although this falls short of the 98.2% POD achieved with direct sequencing of HVS-I/ HVS-II, Parson says their approach is clearly superior to other mtDNA screening methods. And because the 23 target SNPs lie outside HVS-I/HVS-II, the typing represents additional information that can be combined with direct sequencing data to further improve POD (to 98.6% in this study). With amplicon lengths of 55–131 bp, 627 total nucleotide positions were covered for each sample; 60 known polymorphic sites were screened. Most SNP-typing methods target single positions and report only positive or negative answers, but Parson’s approach capitalizes on all known SNPs in the screened regions as well as any unique mutations that may exist. A total of 14 different, nontarget sequence variations were detected in the current study. And although the current assay was opti-
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mized for discrimination in West Eurasian populations, Parson says his group is exploring marker multiplexes that would be suited to other populations. Additional benefits include very high sensitivity (unequivocal genotyping of 22 markers was achieved in one sample containing just 6 mt genomes/µL after 45 PCR cycles—approximately on par with direct sequencing of HVS-I/HVSII), relatively high throughput (12 min/sample after amplification), and low costs. The ICEMS mtDNA screening strategy does have some drawbacks, however. Detected mutations cannot be localized in the mt genome; thus, a single apparent phenotype can be caused by mutations at different positions. And a single amplicon with synonymous mutations cannot be discriminated from one without mutations. In the first case, says Parson, “One needs to bear in mind that categories can be ‘artificial’, and confirmation by sequencing may be required—though we observe that this is not necessary for most of the data we have analyzed so far.” In the second case, he says, careful amplicon design can almost entirely exclude the already rare situation of synonymous mutation. To test their assay’s applicability to real-world scenarios, Parson’s group successfully analyzed mixtures of DNA from 2 individuals, representing a range of mixing ratios down to 10% minority component. And they successfully typed mtDNA recovered from eight typical crime-scene specimens, including several that produced only incomplete profiles under routine typing by analysis of short tandem repeats. “We tested real forensic samples and found that the method works very well,” says Parson. That efficacy, he says, combined with the assay’s speed, ease of use, and low cost, should make multiplex-PCR/ICEMS mtDNA typing a useful prescreening complement to standard HVS-I/HVSII sequencing of forensic samples. a —Thomas Hayden
