Hedman Johannes, Nordgaard Anders, Rasmusson Birgitta, Ansell Ricky, Rådström Peter
Department of Applied Microbiology, Lund University, Lund, Sweden.
Biotechniques. 2009 Nov;47(5):951-8. doi: 10.2144/000113246.
DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.
将犯罪者与犯罪现场联系起来的DNA证据是许多法律程序的核心。然而，犯罪现场的DNA样本通常含有PCR抑制物质，这可能会产生空白或不完整的DNA图谱。为了去除样本中的这些抑制剂，可能需要进行广泛的DNA纯化，尽管这些程序会增加DNA丢失的风险。大多数法医实验室使用以DNA聚合酶AmpliTaq Gold作为金标准的商业DNA扩增试剂盒（如AmpFlSTR SGM Plus）。在此，我们表明，替代的DNA聚合酶-缓冲液系统可以提高法医DNA分析的质量，并有效规避犯罪现场样本中的PCR抑制，而无需额外的样本制备。使用Bio-X-Act Short、ExTaq Hot Start或PicoMaxx High Fidelity而非AmpliTaq Gold，32个完全或部分受抑制的犯罪现场唾液样本中的20个样本的DNA图谱得到了显著改善。开发了一种用于法医DNA图谱无偏质量控制的统计模型来量化结果。我们的研究证明了调整PCR化学以增强法医DNA分析和诊断性PCR的重要性，为繁琐的样本制备方案提供了一种替代方法。
