The application of reduced-size short tandem repeat primer sets in the analysis of human DNA from degraded and compromised samples

The purpose of this research was to demonstrate the applicability of reduced-size STR (Miniplex) primer sets to challenging samples and to provide the forensic community with new information regarding the analysis of degraded and inhibited DNA. The Miniplex primer sets were validated in accordance with guidelines set forth by the Scientific Working Group on DNA Analysis Methods (SWGDAM) in order to demonstrate the scientific validity of the kits. The Miniplex sets were also used in the analysis of DNA extracted from human skeletal remains and telogen hair. In addition, a method for evaluating the mechanism of PCR inhibition was developed using qPCR. The Miniplexes were demonstrated to be a robust and sensitive tool for the analysis of DNA with as low as 100 pg of template DNA. They also proved to be better than commercial kits in the analysis of DNA from human skeletal remains, with 64% of samples tested producing full profiles, compared to 16% for a commercial kit. The Miniplexes also produced amplification of nuclear DNA from human telogen hairs, with partial profiles obtained from as low as 60 pg of template DNA. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for forensic analysis of degraded DNA from human skeletal remains, telogen hairs, and other challenging samples. In the evaluation of inhibition by qPCR, the effect of amplicon length and primer melting temperature was evaluated in order to determine the binding mechanisms of different PCR inhibitors. Several mechanisms were indicated by the inhibitors tested, including binding of the polymerase, binding to the DNA, and effects on the processivity of the polymerase during primer extension. The data obtained from qPCR illustrated a method by which the type of inhibitor could be inferred in forensic samples, and some methods of reducing inhibition for specific inhibitors were demonstrated. An understanding of the mechanism of the inhibitors found in forensic samples will allow analysts to select the proper methods for inhibition removal or the type of analysis that can be performed, and will increase the information that can be obtained from inhibited samples.

Genetic individualization of Cannabis sativa by a short tandem repeat multiplex system

Cannabis sativa is the most frequently used of all illicit drugs in the United States. Cannabis has been used throughout history for its stems in the production of hemp fiber, for its seed for oil and food, and for its buds and leaves as a psychoactive drug. Short tandem repeats (STRs), were chosen as molecular markers because of their distinct advantages over other genetic methods. STRs are co-dominant, can be standardized such that reproducibility between laboratories can be easily achieved, have a high discrimination power and can be multiplexed. ^ In this study, six STR markers previously described for Cannabis were multiplexed into one reaction. The multiplex reaction was able to individualize 98 Cannabis samples (14 hemp and 84 marijuana, authenticated as originating from 33 of the 50 United States) and detect 29 alleles averaging 4.8 alleles per loci. The data did not relate the samples from the same state to each other. This is the first study to report a single reaction six-plex and apply it to the analysis of almost 100 Cannabis samples of known geographic collection site. ^

Substructuring in four populations of African descent

When a suspect's DNA profile is admitted into court as a match to evidence the probability of the perpetrator being another individual must be calculated from database allele frequencies. The two methods used for this calculation are phenotypic frequency and likelihood ratio. Neither of these calculations takes into account substructuring within populations. In these substructured populations the frequency of homozygotes increases and that of heterozygotes usually decreases. The departure from Hardy- Weinberg expectation in a sample population can be estimated using Sewall Wright's Fst statistic. Fst values were calculated in four populations of African descent by comparing allele frequencies at three short tandem repeat loci. This was done by amplifying the three loci in each sample using the Polymerase Chain Reaction and separating these fragments using polyacrylamide gel electrophoresis. The gels were then silver stained and autoradiograms taken, from which allele frequencies were estimated. Fst values averaged 0.007+- 0.005 within populations of African descent and 0.02+- 0.01 between white and black populations.