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Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing
Liu, Yilan1; Chen, Jinjin2,3; Thygesen, Anders4
2018-02-01
发表期刊MOLECULAR BIOTECHNOLOGY
ISSN1073-6085
卷号60期号:2页码:92-99
摘要Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized. In addition, the fusion products produced with this method were successfully applied in seamless genome editing. The fusion of two fragments by this method takes less than 0.5 day. The method is expected to facilitate various kinds of complex genetic engineering projects with enhanced efficiency.
关键词Fusion Pcr Dual-asymmetric Primers Two-step Annealing
文章类型Article
WOS标题词Science & Technology ; Life Sciences & Biomedicine
DOI10.1007/s12033-017-0050-7
收录类别SCI
语种英语
关键词[WOS]OVERLAP EXTENSION ; GENE ; DNA ; POLYMERASE
WOS研究方向Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS类目Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
项目资助者National Basic Research Program (973 Program)(2011CBA00800)
WOS记录号WOS:000424251300002
引用统计
文献类型期刊论文
条目标识符http://ir.ipe.ac.cn/handle/122111/24097
专题生化工程国家重点实验室
作者单位1.Univ Nebraska Lincoln, Dept Chem & Biomol Engn, Lincoln, NE 68588 USA
2.Chinese Acad Sci, Inst Proc Engn, State Key Lab Biochem Engn, Beijing 100190, Peoples R China
3.Univ Nebraska Lincoln, Dept Food Sci & Technol, Lincoln, NE 68588 USA
4.Tech Univ Denmark, Dept Chem & Biochem Engn, Ctr Bioproc Engn, DK-2800 Lyngby, Denmark
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Liu, Yilan,Chen, Jinjin,Thygesen, Anders. Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing[J]. MOLECULAR BIOTECHNOLOGY,2018,60(2):92-99.
APA Liu, Yilan,Chen, Jinjin,&Thygesen, Anders.(2018).Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing.MOLECULAR BIOTECHNOLOGY,60(2),92-99.
MLA Liu, Yilan,et al."Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing".MOLECULAR BIOTECHNOLOGY 60.2(2018):92-99.
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