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The Use of a Comprehensive Approach for Neutralization of PCR Inhibitors Found in Forensic Samples and Its Use In a Homicide/Sexual Assault CaseJanice M. Williamson1, John S. Waye2, Pamela Newall3,
David H. Bing1 and Edward Blake4 Inhibition of the Taq DNA polymerase by exogenous materials such as hemin, organic compounds and soluble extracts of microorganisms that co-purify with DNA isolated from forensic samples is a frequently encountered problem that prevents successful PCR amplification of the template DNA. We report in this communication a systematic approach that was devised and successfully used to overcome this kind of inhibition of PCR amplification of DNA. The case involved a 9 year old female whose body was found in a wooded area about 3 months after her murder in 1984. Investigation at that time was consistent with her being stabbed and perhaps sexually assaulted. The son of neighbors of the child's parents was charged with the crime and convicted. One item of evidence collected at the crime scene consisted of a semen stain on the victim's underwear that was removed from her decomposing body. Microscopic examination of the stain showed it contained spermatozoa. In 1989 and 1991 a sample of that stain was differentially extracted into epithelial cell and male cell fractions, purified by organic extraction, washed on a Centricon 100 filter and analyzed with the HLA DQ Alpha Amplitype Kit independently by two of our laboratories. Both laboratories reported inconclusive results on the male cell fraction. Analysis of the DQ Alpha amplified product from that test indicated that there was inhibition of amplification. Because it appeared there was sufficient genomic DNA isolated from the underwear stain to undertake further types of PCR analysis, at the request of the prosecution and defense, a strategy was devised to address whether this inhibition could be overcome and successful amplification and typing could be achieved. The strategy was based on optimizing three different approaches that have previously been suggested for overcoming the effect of inhibitors of the Taq DNA Polymerase: (1) adding additional Taq to the PCR mixture prior to amplification, (2) adding varying concentrations of Bovine Serum Albumin to the amplification mixtures prior to amplification and (3) treating the DNA extracts with the affinity resin Thiopropyl Sepharose 6B. All DNA was quantitated with the D17Z1 Human DNA probe. The optimization of the best combination of conditions was then undertaken with the DNA isolated from the epithelial cell fraction of the stain, as it contained all the inhibitors present in the DNA isolated in the male fraction. The ability to overcome inhibition was based on analysis of a product gel from the Amplitype PM test results. Once the optimum conditions had been determined, the male fraction DNA was amplified and typed with the Amplitype HLA DQ Alpha and PM PCR Amplification and typing Kits. The conditions for over coming inhibition were determined to be to first absorb the extracts with Thiopropyl Sepharose 6B (400 uL of packed H20 washed resin for 1.25 to 1 ng of DNA) followed by concentrating the DNA to 10 uL with a Centricon 100 filter. Next, 16 ug of BSA and 10 U of Taq were added to the DNA to give a final volume of 20 uL which was then amplified. With these conditions, the DNA extracted from the male fraction from the semen stain was typed as follows: DQA1, 1.2/3; LDLR, BB; GYPA, BB; HBGG, AB; D7S8, BB, GC, AC. The DNA extracted from the blood of the suspect was typed as follows: DQA1, 1.2/1.3; LDLR, AA; GYPA, BB; HBGG, AA; D7S8, AB; GC, AC. Based on these results, the suspect was exonerated and the original conviction was reversed. Go to proceedings home page
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