Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

doi:10.1007/s12033-017-0050-7

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	Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing
	Liu, Yilan 1; Chen, Jinjin 2,3; Thygesen, Anders 4
	2018-02-01
发表期刊	MOLECULAR BIOTECHNOLOGY
ISSN	1073-6085
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
DOI	10.1007/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
推荐引用方式 GB/T 7714	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.