Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase
Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase
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DOI：
10.2144/000113219
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发表时间：
2009-10-01
期刊：
影响因子：
2.7
通讯作者：
Katz, Laura A.
中科院分区：
文献类型：
--
作者：
Lahr, Daniel J. G.;Katz, Laura A.
PCR 介导的重组可以极大地影响环境研究和基因家族进化分析中多样性的估计。在这里，我们通过分析从变形虫中分离出的八个部分肌动蛋白序列的混合物来测量嵌合体（PCR 介导的重组）的形成，这些肌动蛋白序列是在模拟标准实验室情况的各种条件下扩增的。我们进一步将新一代校对加工能力增强的聚合酶与标准校对酶和之前发表的结果进行比较。在必须进行进化推断的情况下，校对聚合酶优于其他聚合酶。我们的分析表明，降低初始模板浓度与减少循环次数对于减少嵌合体形成和提高准确性同样重要。此外，评估原始单倍型的恢复效率表明，需要多次 PCR 反应才能捕获样品的实际遗传多样性。最后，实验证实，持续合成能力增强的聚合酶可以通过降低起始模板浓度来大幅减少 PCR 介导的重组，而不会影响 PCR 反应的稳健性。
PCR-mediated recombination can greatly impact estimates of diversity, both in environmental studies and in analyses of gene family evolution. Here we measure chimera (PCR-mediated recombinant) formation by analyzing a mixture of eight partial actin sequences isolated from the amoeba Arcella hemisphaerica amplified under a variety of conditions that mimic standard laboratory situations. We further compare a new-generation proofreading processivity-enhanced polymerase to both a standard proofreading enzyme and previously published results. Proofreading polymerases are preferred over other polymerases in instances where evolutionary inferences must be made. Our analyses reveal that reducing the initial template concentration is as critical as reducing the number of cycles for decreasing chimera formation and improving accuracy. Furthermore, assessing the efficiency of recovery of original haplotypes demonstrates that multiple PCR reactions are required to capture the actual genetic diversity of a sample. Finally, the experiments confirm that processivity-enhanced polymerases enable a substantial decrease in PCR-mediated recombination through reducing starting template concentration, without compromising the robustness of PCR reactions.