Impact of metal ions on PCR inhibition and RT-PCR efficiency

Aga Kuffel, Alexander Gray (Lead / Corresponding author), Niamh Nic Daeid

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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.
Original languageEnglish
Number of pages10
JournalInternational Journal of Legal Medicine
Early online date3 Jul 2020
DOIs
Publication statusE-pub ahead of print - 3 Jul 2020

Keywords

  • DNA polymerase
  • EGTA
  • Metal ions
  • PCR inhibition

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