Qiu X, Wu L, Huang H, McDonel P E, Palumbo A V, Tiedje J M, Zhou J
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 38831, USA.
Appl Environ Microbiol. 2001 Feb;67(2):880-7. doi: 10.1128/AEM.67.2.880-887.2001.
To evaluate PCR-generated artifacts (i.e., chimeras, mutations, and heteroduplexes) with the 16S ribosomal DNA (rDNA)-based cloning approach, a model community of four species was constructed from alpha, beta, and gamma subdivisions of the division Proteobacteria as well as gram-positive bacterium, all of which could be distinguished by HhaI restriction digestion patterns. The overall PCR artifacts were significantly different among the three Taq DNA polymerases examined: 20% for Z-Taq, with the highest processitivity; 15% for LA-Taq, with the highest fidelity and intermediate processitivity; and 7% for the conventionally used DNA polymerase, AmpliTaq. In contrast to the theoretical prediction, the frequency of chimeras for both Z-Taq (8.7%) and LA-Taq (6.2%) was higher than that for AmpliTaq (2.5%). The frequencies of chimeras and of heteroduplexes for Z-Taq were almost three times higher than those of AmpliTaq. The total PCR artifacts increased as PCR cycles and template concentrations increased and decreased as elongation time increased. Generally the frequency of chimeras was lower than that of mutations but higher than that of heteroduplexes. The total PCR artifacts as well as the frequency of heteroduplexes increased as the species diversity increased. PCR artifacts were significantly reduced by using AmpliTaq and fewer PCR cycles (fewer than 20 cycles), and the heteroduplexes could be effectively removed from PCR products prior to cloning by polyacrylamide gel purification or T7 endonuclease I digestion. Based upon these results, an optimal approach is proposed to minimize PCR artifacts in 16S rDNA-based microbial community studies.
为了用基于16S核糖体DNA（rDNA）的克隆方法评估聚合酶链式反应（PCR）产生的假象（即嵌合体、突变和异源双链体），构建了一个由变形菌门的α、β和γ亚群以及革兰氏阳性菌组成的四个物种的模型群落，所有这些物种都可以通过HhaI限制性酶切图谱区分。在所检测的三种Taq DNA聚合酶中，总体PCR假象存在显著差异：Z-Taq的假象率为20%，其合成能力最高；LA-Taq的假象率为15%，保真度最高且合成能力中等；传统使用的DNA聚合酶AmpliTaq的假象率为7%。与理论预测相反，Z-Taq（8.7%）和LA-Taq（6.2%）的嵌合体频率高于AmpliTaq（2.5%）。Z-Taq的嵌合体和异源双链体频率几乎是AmpliTaq的三倍。随着PCR循环数和模板浓度的增加，总PCR假象增加，而随着延伸时间的增加，总PCR假象减少。一般来说，嵌合体的频率低于突变的频率，但高于异源双链体的频率。随着物种多样性的增加，总PCR假象以及异源双链体的频率增加。使用AmpliTaq和较少的PCR循环（少于20个循环）可显著减少PCR假象，并且在克隆之前，通过聚丙烯酰胺凝胶纯化或T7核酸内切酶I消化可有效去除PCR产物中的异源双链体。基于这些结果，提出了一种优化方法，以尽量减少基于16S rDNA的微生物群落研究中的PCR假象。
