Examining Sources of Error in PCR by Single-Molecule Sequencing.
Examining Sources of Error in PCR by Single-Molecule Sequencing.
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DOI：
10.1371/journal.pone.0169774
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Ong JL
中科院分区：
文献类型：
--
作者：
Potapov V;Ong JL
下一代测序技术已经能够检测罕见的遗传或体细胞突变，并有助于我们了解疾病的进展和演变。然而，许多下一代测序技术首先依赖于DNA扩增，通过聚合酶链式反应（PCR），作为样品制备工作流程的一部分。PCR过程中的错误会出现在测序数据中，并导致最终混淆遗传分析的假突变。在这份报告中，一个单分子测序分析被用来全面编目不同类型的错误在PCR过程中引入，包括聚合酶错误掺入，结构诱导的模板转换，PCR介导的重组和DNA损伤。除了充分表征的聚合酶碱基置换错误外，发现其他错误来源同样普遍。在单分子水平上观察到Taq聚合酶的PCR介导的重组，并且令人惊讶地发现其发生频率与聚合酶碱基置换错误一样高，表明其可能是多重扩增反应的错误来源。lacZ中的反向重复结构元件在复制期间引起顶部和底部链之间的聚合酶模板切换，并且测量不同聚合酶的这些事件的频率。对于非常精确的聚合酶，温度循环过程中引入的DNA损伤，而不是聚合酶碱基置换错误，似乎是扩增产物中发生突变的主要原因。总的来说，我们在单分子水平上分析了PCR产物，并在这里展示了DNA扩增过程中发生的错误类型的更完整的图片。
Next-generation sequencing technology has enabled the detection of rare genetic or somatic mutations and contributed to our understanding of disease progression and evolution. However, many next-generation sequencing technologies first rely on DNA amplification, via the Polymerase Chain Reaction (PCR), as part of sample preparation workflows. Mistakes made during PCR appear in sequencing data and contribute to false mutations that can ultimately confound genetic analysis. In this report, a single-molecule sequencing assay was used to comprehensively catalog the different types of errors introduced during PCR, including polymerase misincorporation, structure-induced template-switching, PCR-mediated recombination and DNA damage. In addition to well-characterized polymerase base substitution errors, other sources of error were found to be equally prevalent. PCR-mediated recombination by Taq polymerase was observed at the single-molecule level, and surprisingly found to occur as frequently as polymerase base substitution errors, suggesting it may be an underappreciated source of error for multiplex amplification reactions. Inverted repeat structural elements in lacZ caused polymerase template-switching between the top and bottom strands during replication and the frequency of these events were measured for different polymerases. For very accurate polymerases, DNA damage introduced during temperature cycling, and not polymerase base substitution errors, appeared to be the major contributor toward mutations occurring in amplification products. In total, we analyzed PCR products at the single-molecule level and present here a more complete picture of the types of mistakes that occur during DNA amplification.
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DOI：
10.1073/pnas.1210309109
发表时间：
2012-10-09
影响因子：
11.1
作者：
Lee, Heewook;Popodi, Ellen;Foster, Patricia L.
通讯作者：
Foster, Patricia L.
DOI：
10.1073/pnas.1208715109
发表时间：
2012-09-04
影响因子：
11.1
作者：
Schmitt, Michael W.;Kennedy, Scott R.;Loeb, Lawrence A.
通讯作者：
Loeb, Lawrence A.
影响因子：
14.9
作者：
Mytelka, DS;Chamberlin, MJ
通讯作者：
Chamberlin, MJ
影响因子：
14.9
作者：
ODELBERG, SJ;WEISS, RB;WHITE, R
通讯作者：
WHITE, R
影响因子：
2.7
作者：
Lahr, Daniel J. G.;Katz, Laura A.
通讯作者：
Katz, Laura A.