King Christine, Debruyne Régis, Kuch Melanie, Schwarz Carsten, Poinar Hendrik
McMaster Ancient DNA Centre, Department of Anthropology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada.
Biotechniques. 2009 Nov;47(5):941-9. doi: 10.2144/000113244.
Inhibition is problematic in many applications of PCR, particularly those involving degraded or low amounts of template DNA, when simply diluting the extract is undesirable. Two basic approaches to monitoring inhibition in such samples using real-time or quantitative PCR (qPCR) have been proposed. The first method analyzes the quantification cycle (Cq) deviation of a spiked internal positive control. The second method considers variations in reaction efficiency based on the slopes of individual amplification plots. In combining these methods, we observed increased Cq values together with reduced amplification efficiencies in some samples, as expected; however, deviations from this pattern in other samples support the use of both measurements. Repeat inhibition testing enables optimization of PCR facilitator combinations and sample dilution such that DNA yields and/or quantitative accuracy can be maximized in subsequent PCR runs. Although some trends were apparent within sample types, differences in inhibition levels, optimal reactions conditions, and expected recovery of DNA under these conditions suggest that all samples be routinely tested with this approach.
在PCR的许多应用中，抑制作用是个问题，特别是在涉及降解的模板DNA或模板DNA量较少的情况下，此时简单稀释提取物并不可取。已经提出了两种使用实时或定量PCR（qPCR）监测此类样品中抑制作用的基本方法。第一种方法分析加标的内部阳性对照的定量循环（Cq）偏差。第二种方法考虑基于各个扩增曲线斜率的反应效率变化。在结合这些方法时，正如预期的那样，我们在一些样品中观察到Cq值增加以及扩增效率降低；然而，其他样品中偏离这种模式的情况支持同时使用这两种测量方法。重复抑制测试能够优化PCR促进剂组合和样品稀释，从而在后续的PCR运行中使DNA产量和/或定量准确性最大化。尽管在样品类型中一些趋势很明显，但抑制水平、最佳反应条件以及在这些条件下预期的DNA回收率的差异表明，所有样品都应常规使用这种方法进行测试。
