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学科主题: 基础医学
题名:
Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR
作者: Liu, Jia1; Song, Hongshuo2; Liu, Donglai2,3; Zuo, Tao2,3; Lu, Fengmin1; Zhuang, Hui1; Gao, Feng2,3
刊名: PLOS ONE
发表日期: 2014-09-11
DOI: 10.1371/journal.pone.0106658
卷: 9, 期:9
收录类别: SCI
文章类型: Article
WOS标题词: Science & Technology
类目[WOS]: Multidisciplinary Sciences
研究领域[WOS]: Science & Technology - Other Topics
关键词[WOS]: IMMUNODEFICIENCY-VIRUS TYPE-1 ; DRUG-RESISTANCE MUTATIONS ; POLYMERASE-CHAIN-REACTION ; MEDIATED RECOMBINATION ; DNA-POLYMERASE ; VIRAL POPULATIONS ; AMPLIFICATION ; DIVERSITY ; EVOLUTION ; ANTIBODIES
英文摘要:

Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 10(13)-10(14) using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations.

语种: 英语
所属项目编号: 81101238 ; GM065057
项目资助者: National Natural Science Foundation of China ; National Institutes of Health
WOS记录号: WOS:000341855900022
Citation statistics:
内容类型: 期刊论文
版本: 出版稿
URI标识: http://ir.bjmu.edu.cn/handle/400002259/67404
Appears in Collections:基础医学院_病原生物学系_期刊论文

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作者单位: 1.Duke Univ, Med Ctr, Dept Med, Durham, NC 27710 USA
2.Peking Univ, Hlth Sci Ctr, Dept Microbiol, Beijing 100871, Peoples R China
3.Jilin Univ, Coll Life Sci, Natl Engn Lab AIDS Vaccine, Changchun 130023, Jilin, Peoples R China

Recommended Citation:
Liu, Jia,Song, Hongshuo,Liu, Donglai,et al. Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR[J]. PLOS ONE,2014,9(9).
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