DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.

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a b s t r a c tThe success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substances present in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene saliva stains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNA polymerase-buffer systems (Hedman et al., BioTechniques 47 (2009) 951-958). Here we show that blending inhibitor-resistant DNA polymerase-buffer systems further increases the success rate of PCR for various types of real crime scene samples showing inhibition. For 34 of 42 ''inhibited" crime scene stains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq Hot Start and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmed by analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerases used separately for all tested sample types. When used separately, the performance of the DNA polymerases varied depending on the nature of the sample. The superiority of the blend is discussed in terms of complementary effects and synergy between the DNA polymerase-buffer systems.

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BackgroundAssessment of DNA profile quality is vital in forensic DNA analysis, both in order to determine the evidentiary value of DNA results and to compare the performance of different DNA analysis protocols. Generally the quality assessment is performed through manual examination of the DNA profiles based on empirical knowledge, or by comparing the intensities (allelic peak heights) of the capillary electrophoresis electropherograms.ResultsWe recently developed a ranking index for unbiased and quantitative quality assessment of forensic DNA profiles, the forensic DNA profile index (FI) (Hedman et al. Improved forensic DNA analysis through the use of alternative DNA polymerases and statistical modeling of DNA profiles, Biotechniques 47 (2009) 951-958). FI uses electropherogram data to combine the intensities of the allelic peaks with the balances within and between loci, using Principal Components Analysis. Here we present the construction of FI. We explain the mathematical and statistical methodologies used and present details about the applied data reduction method. Thereby we show how to adapt the ranking index for any Short Tandem Repeat-based forensic DNA typing system through validation against a manual grading scale and calibration against a specific set of DNA profiles.ConclusionsThe developed tool provides unbiased quality assessment of forensic DNA profiles. It can be applied for any DNA profiling system based on Short Tandem Repeat markers. Apart from crime related DNA analysis, FI can therefore be used as a quality tool in paternal or familial testing as well as in disaster victim identification.

A reported likelihood ratio for the value of evidence is very often a point estimate based on various types of reference data. When presented in court, such frequentist likelihood ratio gets a higher scientific value if it is accompanied by an error bound. This becomes particularly important when the magnitude of the likelihood ratio is modest and thus is giving less support for the forwarded proposition. Here, we investigate methods for error bound estimation for the specific case of digital camera identification. The underlying probability distributions are continuous and previously proposed models for those are used, but the derived methodology is otherwise general. Both asymptotic and resampling distributions are applied in combination with different types of point estimators. The results show that resampling is preferable for assessment based on asymptotic distributions. Further, assessment of parametric estimators is superior to evaluation of kernel estimators when background data are limited.

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