Evaluation of PCR-generated chimeras: Mutations, and heteroduplexes with 16S rRNA gene-based cloning
Evaluation of PCR-generated chimeras: Mutations, and heteroduplexes with 16S rRNA gene-based cloning
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DOI：
10.1128/aem.67.2.880-887.2001
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发表时间：
2001-02-01
影响因子：
4.4
通讯作者：
Zhou, JZ
中科院分区：
文献类型：
--
作者：
Qiu, XY;Wu, LY;Zhou, JZ
为了使用基于 16S 核糖体 DNA (rDNA) 的克隆方法评估 PCR 产生的假象（即嵌合体、突变和异源双链体），我们从变形菌门的 α、β 和 γ 细分以及革兰氏阳性细菌构建了四个物种的模型群落，所有这些细菌都可以通过 HhaI 限制性消化模式进行区分。所检测的三种标签 DNA 聚合酶的总体 PCR 伪影存在显着差异：Z-Taq 为 20%，最高：持续合成能力； LA-Taq 为 15%，具有最高的保真度和中等的持续处理能力；传统使用的 DNA 聚合酶 AmpliTaq 为 7%。与理论预测相反，Z-Taq (8.7%) 和 LA-Taq (6.2%) 的嵌合体频率均高于 AmpliTaq (2.5%)。 Z-Taq 的嵌合体和异源双链体的频率几乎是 AmpliTaq 的三倍。总 PCR 伪影随着 PCR 循环而增加，模板浓度随着延伸时间的增加而增加和减少。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 伪影以及异源双链体的频率也增加。通过使用 AmpliTaq 和更少的 PCR 循环（少于 20 个循环），可以显着减少 PCR 伪影，并且在通过聚丙烯酰胺凝胶纯化或 T7 核酸内切酶 I 消化进行克隆之前，可以有效地从 PCR 产物中去除异源双链体。基于这些结果，提出了一种最佳方法，以最大限度地减少基于 16S rDNA 的微生物群落研究中的 PCR 伪影。
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 Tag 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.