PCR inhibition mechanisms in forensic DNA analysis

In forensic DNA analysis, the polymerase chain reaction (PCR) enables DNA
analysis of minute biological crime scene traces. PCR is an enzymatic reaction for amplification of specific DNA fragments involving both biochemical and
biophysical 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 show that 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.

DNA is one of the key pieces of evidence collected at a crime scene. The results
from a forensic DNA analysis guide the court in determining guilt or innocence of a person. Therefore, it is imperative that the methods are efficient and accurate. At the Swedish National Forensic Centre (NFC) around 60,000 crime scene DNA samples are analysed each year. The main challenge is that the samples often contain low levels of DNA in combination with background material that may disturb the analysis. To be able to analyse challenging samples, an increased understanding of molecular inhibition mechanisms as well as rational analysis solutions to overcome inhibition is needed.

The technology that is used for forensic DNA analysis is called PCR (polymerase
chain reaction) and relies on a biochemical process to detect and multiply specific DNA fragments in crime scene samples. In this thesis, a combination of modern DNA analysis technology and other molecular techniques were used to investigate how molecules, called PCR inhibitors, from blood and soil mechanistically disturb the DNA analysis.

The main findings have been to show, for the first time in PCR-based analysis,
how certain molecules from blood and soil interfere with the light signal that is
used to detect the DNA. These molecules are more specifically humic acid in soil
and haemoglobin in blood. I have also concluded that these molecules disturb the copying of DNA through disrupting the DNA polymerase, the enzyme responsible for multiplying the DNA fragments. Immunoglobulin G, a molecule in blood, has another inhibition mechanism and disturbs the reaction by binding to single-stranded genomic DNA.

Moreover, I have in my research shown that the choice of DNA polymerase is
crucial for the generation of reliable results from challenging samples. An
alternative DNA polymerase was shown to handle 48 times more of the PCR
inhibitors than the regularly used enzyme.

A new technique called massively parallel sequencing (MPS) enables a broadened
analysis of DNA in criminal investigations. Thanks to MPS, more genetic information can be obtained which can be used to predict e.g. hair and eye colour and to reach further in separating DNA from different persons. I have in my thesis studied how PCR inhibitors impact MPS and I could show that the currently used method for MPS analysis of crime scene traces is very sensitive to inhibitors. Moreover, I could show that the MPS inhibition can be overcome with the same strategies as for PCR analysis, for example by adding a specific protein. More work is needed to design MPS methods for analysis of challenging samples.

The findings reported in this Doctoral thesis have already strengthened the forensic
routine analysis at NFC. For example, the acquired knowledge has led to the
identification of several analytical bottlenecks and has made trouble-shooting
more efficient. The findings have also improved diagnostic DNA analysis in the
fields of medicine, food and bioterrorism, for example by optimizing the DNA
analysis of disease-associated bacteria in environmental samples. Through
continued efforts on strategic research I believe that we in the future will be able
to analyse many samples that are currently too challenging due to low levels of
DNA as well as presence of PCR inhibitors. The overall aim is to better support
police investigations through informative and rational DNA analysis of
challenging crime scene samples.