DNA Analysis of PCR-Inhibitory Forensic Samples

Abstract: DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, crime scene samples often contain extraneous substances that may interfere with the PCR-based forensic analysis, resulting in partial or negative DNA profiles. Extensive DNA purification may remove inhibitors, but involves the risk of DNA loss. In this work, pre-PCR processing was applied to improve the success rate of forensic DNA analysis of “dirty” samples without interfering with the composition of the samples. An experimental model system was developed to screen for inhibitor-tolerant DNA polymerase-buffer systems. The best-performing polymerases, Bio-X-Act Short, ExTaq HS and PicoMaxx HF, were applied in STR DNA analysis of PCRinhibitory crime scenes samples, i.e. samples that failed to produce complete DNA profiles in routine casework despite containing acceptable levels of DNA. A ranking index, called the forensic DNA profile index (FI), was developed to quantitatively describe DNA profile quality. The application of analysis of variance (ANOVA) to FI values confirmed that the three alternative polymerases significantly improved DNA profile quality for 20 of 32 problematic samples, compared with the standard polymerase AmpliTaq Gold. ExTaq HS and PicoMaxx HF showed complementary inhibitor-relieving properties. A blend of the two polymerases exhibited tolerance to a broader range of extraneous compounds, improving DNA profile quality in 34 of 42 PCR-inhibitory forensic samples. When used separately, ExTaq HS and PicoMaxx HF improved the results of analysis for 26 and 23 samples, respectively. Apart from their complementarity, synergy between the polymerases was mathematically proven by calculating the geometric mean values of FI and applying ANOVA. In November, 2010, the customised DNA polymerase blend was introduced in routine casework at the Swedish National Laboratory of Forensic Science, increasing the proportion of complete DNA profiles generated from impure samples from 38% to 87% for saliva, and from 69% to 94% for blood. Many presumed saliva crime scene stains give negative DNA results if presumptive testing is not performed. In this work, amylase activity testing was evaluated as a tool for saliva screening. No direct correlation was found between amylase activity and the DNA content of saliva. However, the sensitivity of the developed swab screening procedure (positive results for 0.5 μL of dried saliva) makes it applicable in cases where the number of DNA analyses is limited due to cost.