DNA Identification in Mass Fatality Incidents
Alternative Testing Methods
In the WTC identification effort, the OCME relied on the recommendations of the Kinship and Data Analysis Panel (KADAP) regarding new identification methods for analyzing compromised samples. In considering additional typing technologies and strategies, the KADAP considered the sufficiency of extracted material to support all attempted technologies, as well as any quality control issues that might arise. The KADAP also considered how to handle the statistical approach using other technologies, including linkage and haplotype/genotype comparisons.
Anne Walsh
The KADAP served as a model for scientific collaboration and peer review of method validation under the challenging and stressful conditions of responding to a mass fatality disaster.
Mitochondrial DNA Analysis
STRs reside in the human cell nucleus; outside the nucleus, in the cytoplasm, are mitochondria. Mitochondria are subcellular organelles that contain an extra chromosomal genome separate and distinct from the nuclear genome. Human mitochondrial DNA differs from nuclear DNA in that it is a closed, circular (rather than linear) molecule; it is smaller, consisting of approximately 16,569 base pairs; it is maternally inherited; it does not undergo recombination; and it is present in high copy number in a cell.
The maternal inheritance and lack of recombination characteristics are particularly helpful in identifying human remains. Associations can be made or refuted where known maternal relatives are the reference sample sources, even if they are several generations removed from the victim.
The primary advantage of using mtDNA (as opposed to nuclear DNA analysis) on compromised samples is the high copy number of mtDNA molecules in a cell. When the amount of extracted DNA is very small or degraded (as can be the case in mass disaster tissue samples of bone, teeth, and hair), an identification is more likely using mtDNA analysis than using the polymorphic markers found in nuclear DNA.
In the WTC identification effort, a number of samples could not be typed sufficiently with STR loci to identify the source with a high degree of confidence. In these cases, mtDNA sequencing was attempted to increase the discrimination power. Although the extraction process for mtDNA typically requires a relatively clean environment, this was not possible in the WTC identification effort, due to the number of samples. However, reasonable precautions were taken, including a reduction in the number of amplification cycles (28 or 29 instead of the typical 36). This reduced contamination issues, although at the expense of the sensitivity of detection.
Although not as informative as a battery of autosomal STR loci, a unique mitotype may be sufficient to make an identification, if the victims are from a closed population. The mitotype can be used to group individuals into smaller categories, narrowing the candidate pool. It may then be possible for a less informative partial STR profile to become a unique identifier within the mtDNA subcategory. Screening by mtDNA sequencing would be possible because of the availability of high-throughput analysis, coupled with software that automatically interprets mitotypes.
Repositioning Primers
In the WTC identification effort, recovered DNA was often too degraded and fragmented to produce STR results with standard commercial STR kits. However, by repositioning the primers so that they resided closer to the repeat region, the amplified product (or amplicon) was made smaller than some of the fragmented DNA template molecules, thus making genetic characterization of the sample possible for more STRs than when using traditional typing. These STR miniplexes were invaluable for analyzing the more degraded samples, and, in fact, results were obtained for some samples at loci that were not typable using commercially available kits.
The general assay procedure for the miniplex test used in the identification of WTC victims was similar to that used for forensically validated STRs. After evaluating the methods, reagents, and validation data, the KADAP determined that no additional equipment and training was necessary.
Single Nucleotide Polymorphism Analysis
The PCR amplicon size can be further reduced by amplifying regions that contain a class of genetic markers known as single nucleotide polymorphisms (SNPs). Although an abundant supply of SNPs exists for identity testing, most SNPs are biallelic and, therefore, not as informative for identity testing as STR loci. However, because the amplicon size can be reduced 60–80 base pairs in length, DNA that is degraded beyond the limits of STR typing may be typable.
In the WTC identification effort, an SNP typing method was validated for the more difficult-to-type samples. In fact, identifications that otherwise would not have been possible were made using this technology. Combining the features of a chip array, the primer extension assay, and universal tags, the multiplex assay method was carried out in a flat-bottom microplate, in which each well contained a total of 16 individual antitag sequences for 12 SNPs and 4 controls. (Basically, each PCR primer, about 45 bases long, is comprised of a 25-base-long segment that is complementary to the area immediately adjacent to the SNP extension site and a 20-base-long sequence—that is, the tag sequence—that is complementary to an antitag sequence attached to the bottom of a well.)
Using that process, the SNP extension product was transferred after PCR and allowed to hybridize in the array of antitags. A fluorescent detection system allowed typing of the two possible alleles at the SNP site by comparing signals from fluorescent dyes used to label the two different allelic products in the PCR extension reaction. With this technology, identifications were made on some very compromised samples that otherwise would not have been possible to identify.
