Leverhulme Research Centre for Forensic Science, University of Dundee, Dundee, Scotland.
Int J Legal Med. 2021 Jan;135(1):63-72. doi: 10.1007/s00414-020-02363-4. Epub 2020 Jul 3.
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.
金属离子对 PCR 的抑制作用可能会给法医 DNA 分析过程带来严重挑战。犯罪现场遇到的各种类型的金属离子污染的样本包括来自金属表面（如子弹、弹壳、武器（包括枪支和刀具）、金属丝和表面以及含有钙的骨样本）的拭子。金属离子对 DNA 回收、提取和随后扩增的影响的机制尚未完全了解。在这项研究中，我们评估了常见金属对 DNA 扩增的抑制作用。在测试的九种金属中，锌、锡、铁(II)和铜表现出最强的抑制特性，其 IC50 值明显低于 1mM。在研究的第二部分，我们测试了三种市售的 DNA 聚合酶对金属抑制的敏感性。与 Q5 和 Taq 聚合酶相比，我们发现 KOD 聚合酶对金属抑制的抵抗力最强。我们还展示了如何使用钙螯合剂乙二醇双(2-氨基乙基醚)-N,N,N',N'-四乙酸（EGTA）作为一种简单且非破坏性的方法来逆转钙诱导的 PCR 反应抑制。
