Abstract
Inhibition of PCR by metal ions can pose a serious challenge in the process of forensic DNA analysis. Samples contaminated with various types of metal ions encountered at crime scenes include swabs from metal surfaces such as bullets, cartridge casings, weapons (including guns and knives), metal wires and surfaces as well as bone samples which contain calcium. The mechanism behind the impact of metal ions on DNA recovery, extraction and subsequent amplification is not fully understood. In this study, we assessed the inhibitory effects of commonly encountered metals on DNA amplification. Of the nine tested metals, zinc, tin, iron(II) and copper were shown to have the strongest inhibitory properties having IC50 values significantly below 1 mM. In the second part of the study, three commercially available DNA polymerases were tested for their susceptibility to metal inhibition. We found that KOD polymerase was the most resistant to metal inhibition when compared with Q5 and Taq polymerase. We also demonstrate how the calcium chelator ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) can be used as an easy and non-destructive method of reversing calcium-induced inhibition of PCR reactions.
Highlights
The effect of metal ions on DNA and PCR amplification is not fully understood, despite there being a significant number of studies that focus on the inhibitory properties of metals
The degree of PCR inhibition by metal ions was determined by assessing the qPCR cycle threshold (Ct) values in the presence of added metal ions over a 4-log concentration range (0.002 to 5 mM) as detailed in “Materials and methods”
Despite many studies describing undesirable effects of metal ions on DNA and amplification process, there has been little research into ways of enhancing recovery or processing of DNA samples that have come into contact with a metal source
Summary
The effect of metal ions on DNA and PCR amplification is not fully understood, despite there being a significant number of studies that focus on the inhibitory properties of metals. The published research shows that metal ions can interfere with DNA analysis at various points; from extraction to PCR amplification [1,2,3,4,5,6] and as a result hinder subsequent DNA profiling. It is assumed that metal ions have an affinity for DNA mediated by the negative charge on the phosphate back bone of the DNA interacting with the positively charged metal ions. Some metals such as K+, Na+ and Mg2+ are essential for DNA stability [7].
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