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学科主题基础医学
Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR
Liu, Jia1; Song, Hongshuo2; Liu, Donglai2,3; Zuo, Tao2,3; Lu, Fengmin1; Zhuang, Hui1; Gao, Feng2,3
刊名PLOS ONE
2014-09-11
DOI10.1371/journal.pone.0106658
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
WOS记录号WOS:000341855900022
项目编号81101238 ; GM065057
资助机构National Natural Science Foundation of China ; National Institutes of Health
引用统计
被引频次:9[WOS]   [WOS记录]     [WOS相关记录]
文献类型期刊论文
版本出版稿
条目标识符http://ir.bjmu.edu.cn/handle/400002259/67404
专题北京大学基础医学院_病原生物学系
北京大学第一临床医学院_神经内科
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
推荐引用方式
GB/T 7714
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).
APA Liu, Jia.,Song, Hongshuo.,Liu, Donglai.,Zuo, Tao.,Lu, Fengmin.,...&Gao, Feng.(2014).Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR.PLOS ONE,9(9).
MLA Liu, Jia,et al."Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR".PLOS ONE 9.9(2014).
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