STR analysis of inhibited crime scene saliva samples
The statistical assessment tool described in the “Materials and methods” section was applied to the 32 inhibited crime scene samples. The forensic DNA profile index (FI) geometric means were used for pair-wise comparisons of the alternative polymerases to AmpliTaq Gold. Twenty out of 32 inhibited crime scene saliva samples showed statistically significant electropherogram/DNA profile improvements with at least one alternative polymerase. Table 2 provides examples, while the complete list of results can be found in Supplementary Table 1. Samples from cigarette butts showed improvements in 11 of 16 cases, and those from cotton swabs showed improvements in 9 of 16 cases. Figure 2 shows an example in which the profile was clearly improved from partial to complete when PicoMaxx High Fidelity was used instead of AmpliTaq Gold. The respective FI values for this sample were 0.97 for AmpliTaq Gold and 3.37 for PicoMaxx High Fidelity. The alternative polymerase–buffer systems Bio-X-Act Short, ExTaq Hot Start, and PicoMaxx High Fidelity provided improved forensic DNA profiles in 20, 11, and 14 samples, respectively. No statistically significant differences were found between the three alternative polymerases. When the DNA profiles were ranked manually by an experienced reporting officer, 28 samples were categorized as showing higher quality profiles with two or three of the alternative polymerases, compared with AmpliTaq Gold.
The success rate of routine DNA analysis at SKL shows the need for a PCR that is more robust to inhibitors. Using Chelex extraction, a widely used method in forensic DNA laboratories (14,21), 12% of crime scene saliva samples that contained detectable amounts of DNA (0.025–0.25 ng/µL) still produced blank electropherograms/DNA profiles. The reason for only studying samples in the concentration range of 0.025–0.25 ng/µL is that these samples are usually analyzed undiluted, and it is thus possible that PCR inhibitors will have considerable effects.
When analyzing real crime scene trace samples, their content of inhibitory components is generally unknown. However, knowledge about the origin of the sample can provide information about its possible inhibitor content. Aluminum cans may release Al3+ ions, which can inhibit PCR (22), while tobacco contains about 4000 chemical compounds, several of which may have inhibitory effects, such as formaldehyde (23) and phenols (24). Cigarette filter and paper may also have a negative effect since components of wood are known to inhibit PCR (25).
In forensic DNA analysis, commercial DNA typing kits including primers, DNA polymerase, and buffer are used almost exclusively throughout the world. AmpFlSTR SGM Plus and PowerPlex 16 are two of the most commonly used kits. Such DNA typing kits simplify the standardization and comparison of DNA profiles across borders, and enable relatively straightforward validation for each laboratory, compared with the use of in-house assays. However, the complete chemical content of the kits is not disclosed, making PCR chemistry a “black box.” Due to the wide use of analysis kits, modifying PCR chemistry to reduce the effects of inhibition has become rare in the field of diagnostic PCR (2). In forensics, only some minor DNA typing studies with alternative polymerases have been performed, and mainly on pure standard DNA (26).
We started this study by investigating the AE, the dynamic range of amplification, and the detection limit for nine different DNA polymerases using standardized mock crime scene saliva samples in a forensic singleplex real-time PCR assay. The ideal value of AE is 1.0, which corresponds to exponential amplification. Deviating values indicate inefficient amplification, due to either non-optimal PCR conditions or the presence of PCR inhibitors. The values above 1.0 obtained for AmpliTaq Gold, rTth, Taq, and Tth are the effect of postponed amplification (elevated Cp) due to inhibitory compounds in the saliva extracts; an effect that increases with increasing amount of saliva, and a flattening of the amplification curves for samples approaching the detection limit, giving lower Cp values than would be obtained from ideal sigmoid curves. Three of the polymerases producing AE values close to 1.0—Bio-X-Act Short, ExTaq Hot Start, and PicoMaxx High Fidelity—were virtually unaffected by inhibitors in the saliva extract, and their real-time PCR amplification curves retained the sigmoid shape and an amplitude well above the baseline, even for samples close to the detection limit.
Using AmpliTaq Gold, the three samples with the highest saliva/cell amounts were detected at higher Cp values than the fourth strongest sample (Figure 1B). For the other polymerases, the higher the saliva amount, the lower the Cp (Figure 1, A, C, and D). The AmpliTaq Gold results are counterintuitive, but since a higher amount of cells also means a higher amount of PCR inhibitors, it is in fact a sign that the polymerase does not perform ideally in the presence of inhibitors present in saliva. However, the detection limit with AmpliTaq Gold is low, and is only surpassed by Bio-X-Act Short (Table 1). When the amount of inhibitors is small, the enzyme readily amplifies even very small amounts of DNA. The sensitivity of AmpliTaq Gold to inhibitors is consistent with the results found in previous studies (10,24,27).
Differences in the AE between DNA polymerases (Table 1) can be explained in part by the presence of PCR facilitators in the different buffer systems (28). BSA is a well-documented PCR facilitator known to reduce the inhibition resulting from a range of substances, such as human bone (29), phenols (30), hemoglobin (31), and proteases (32). Here, BSA was added to the PCR master mixes with all nine DNA polymerases, to make the conditions for the DNA polymerases more similar.
In the second part of the study, the alternative DNA polymerases were compared with AmpliTaq Gold using real crime scene saliva samples. Bio-X-Act Short provided the highest number of improved DNA profiles, but also showed somewhat uneven amplification between replicates (Table 2, Supplementary Table 1). ExTaq Hot Start and PicoMaxx High Fidelity gave more reproducible results. The robustness to inhibitors from the sample matrices seemed to differ between the three polymerases, even though the differences could not be verified statistically. ExTaq Hot Start performed better for swabs from cans and bottles, and PicoMaxx High Fidelity performed better for cigarette ends.
Crime scene DNA samples are routinely quantified at SKL using the Quantifiler Human DNA quantification kit (Applied Biosystems), which includes an internal amplification control indicating inhibition by an elevated Cp value. However, the Quantifiler kit only detected inhibition in 4 of 32 clearly inhibited samples in this study; the others appeared pure (results not shown). This depends to some extent on the difference in the template-to-reaction volume ratio between the assays. For the Quantifiler kit, 2 µL of template is used in 25-µL reactions (Quantifiler Kit User's Manual), whereas for AmpFlSTR SGM Plus, ≤5× more template is added to make up the same final reaction volume (AmpFlSTR SGM Plus PCR Amplification Kit User's Manual), giving a five-fold higher PCR inhibitor concentration. The real-time PCR assay used here to evaluate the polymerases on mock crime scene samples was modified by using the same template-to-reaction volume as for AmpFlSTR SGM Plus, to provide similar inhibitory effects.
The proposed statistical model will improve the quality control of forensic DNA profiles, and aid in the evaluation of novel forensic DNA analysis procedures. In particular, it provides a tool for objective assessment that can both save time and ensure the overall quality of DNA profiling within the laboratory. The strength of the model is that it combines the heights of allelic peaks with the balance between peaks within a STR marker, as well as the balance between markers. Peak heights alone can sometimes give a decent picture of the performance of PCR. However, since preferential amplification of some alleles cannot be completely avoided—especially in samples with low levels of DNA—peak heights need to be complemented with balance. An increase in the FI can be interpreted as an improvement of the DNA profile; however, we emphasize that statistically significant increases in FI must be based on at least two electrophero-grams from the same sample.
The results from real crime scene saliva samples revealed that Bio-X-Act Short, ExTaq Hot Start, and PicoMaxx High Fidelity—as alternatives to AmpliTaqGold—all produced capillary electrophoresis electropherograms of improved quality with correctly labeled allelic peaks. Modifying PCR chemistry by employing alternative DNA polymerases and PCR facilitators thus proved to be a successful means of circumventing PCR inhibition in crime scene saliva samples. Moreover, a robust and sensitive amplification step will eliminate the need for complicated, time-consuming pretreatment of PCR samples, and reduce the risk of losing evidentiary DNA during sample preparation.
In conclusion, we show that the polymerase AmpliTaq Gold, commonly used in the forensic community, is not the optimal choice for crime scene samples showing PCR inhibition. Further studies on the mechanisms behind PCR inhibition in crime scene stains, and on the use of alternative DNA polymerase–buffer systems such as the ones described here, will benefit justice by rendering useful DNA profiles from a significantly higher percentage of forensic samples.