DNA Identification in Mass Fatality Incidents
Short Tandem Repeat (STR) DNA Amplification and Analysis
In general, polymerase chain reaction (PCR) issues in a mass fatality identification effort are no different than in any other situation, except for the greater number of samples. Although different analytical approaches may eventually be required to make identifications, it is most expedient to use familiar and well-established technologies (i.e., short tandem repeat (STR) typing) as the method of first analysis. In fact, many disaster samples may be wholly typable by STR analysis.
It should be remembered when performing extractions, however, that additional testing may be needed; therefore, extraction techniques that will accommodate other testing methods—such as mitochondrial DNA (mtDNA) sequencing—should be considered.
After extraction, the template DNA is subjected to PCR, which is particularly useful for analyzing materials that may contain degraded DNA. A typical PCR requires three steps and is based on specific annealing and extension of oligonucleotide primers (two per marker) that flank a defined target DNA segment. The template DNA to be amplified by the PCR is first denatured, usually by heating the sample to 95 degrees Centigrade.
After denaturation, the two primers hybridize to the separated strands at a given locus. Primer annealing is accomplished by lowering the temperature to a defined point, typically between 45–65 degrees Centigrade. The next phase in the PCR process, primer extension, is generally carried out at 72 degrees Centigrade, the temperature at which Thermus aquaticus DNA polymerase can most effectively copy the original template DNA by extending the primers and making complementary copies of the original template DNA. These three steps (denaturation, primer annealing, and primer extension) represent a single PCR cycle.
Upon repeated cycles of the PCR, an exponential accumulation of a discrete DNA fragment containing the genetic marker of interest is achieved. Thus, PCR generates large amounts of specific DNA sequences from relatively small (picogram or nanogram) quantities of genomic DNA. Amplification of target sequences of DNA is primarily a technique to prepare the sample for typing.
Only a limited template may be available, and inhibitors to PCR may further reduce the yield of PCR product. Efforts should be made to optimize the components of the PCR to overcome the vagaries of environmental contamination. Some practices used by laboratories during routine analyses—using reduced reaction volumes, for example—may not be appropriate when samples are compromised. A larger reaction volume may dilute inhibitors to the point that the PCR can be successful. Additional enhancements to reduce the impact of inhibitors, such as Bovine Serum Albumen, may be considered part of the protocol for maximizing DNA yields from compromised samples.
