PCR inhibition mechanisms in forensic DNA analysis

Abstract: In forensic DNA analysis, the polymerase chain reaction (PCR) enables DNAanalysis of minute biological crime scene traces. PCR is an enzymatic reaction for amplification of specific DNA fragments involving both biochemical andbiophysical processes. The main analytical challenge is posed by the nature of the samples of interest. Crime scene samples are heterogeneous and often contain background matrices, e.g. PCR inhibitors that impair the limit of detection. The objective of the work described in this Doctoral thesis was to obtain a greater understanding of how relevant PCR inhibitors impact the PCR. The focus was to study PCR inhibition mechanisms using the DNA analysis techniques qPCR, digital PCR (dPCR) and massively parallel sequencing (MPS). The importance of applying a DNA polymerase-buffer system that is compatible with the sample matrix was shown through screening of 16 DNA polymerase-buffer systems for qPCR analysis of environmental samples containing humic substances. In dPCR, the tolerance to humic acid was elevated 48 times when an alternative DNA polymerase-buffer system was used. Also, a statistical Bayesian model was developed to provide an objective dPCR data analysis method. Detailed PCR inhibition mechanisms were elucidated for the main inhibitory molecules in blood and soil matrices. IgG binds to genomic single-stranded DNA, thereby hindering primer annealing and causing delayed amplification. Haemoglobin and its derivative haematin negatively impact the DNA polymerase activity. When studying the PCR inhibition mechanisms of humic acid using a robust DNA polymerase-buffer system, a new bottleneck in the analysis was identified. Namely, that humic acid causes fluorescence quenching, thus hindering amplicon detection. Further studies revealed that not only humic acid, but also haemoglobin causes static quenching of DNA-binding dyes. MPS is finding its way into forensic casework, due to the many opportunities that the technique offers. I could showt hat humic acid and haematin have a considerable negative impact on the multiplex PCR used to prepare the DNA for sequencing. Further, the inhibitor-tolerance of the state-of-the art MPS method is poor, but could be improved through the addition of BSA in the initial PCR. In summary, I have elucidated the PCR inhibition mechanisms of the relevant PCR inhibitors in soil and blood matrices. I have identified and characterized inhibitors affecting DNA polymerase activity, nucleic acids and fluorescence detection. The knowledge gained can be used for developing accurate and reliable DNA analysis methods for forensic DNA analysis as well as for other fields where challenging samples from surfaces or the environment are analysed.