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
刊名	MOLECULAR BIOTECHNOLOGY
	2018-02-01
卷号	60 期号:2 页码:92-99
关键词	Fusion Pcr Dual-asymmetric Primers Two-step Annealing
ISSN号	1073-6085
DOI	10.1007/s12033-017-0050-7
文献子类	Article
英文摘要	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.
WOS关键词	OVERLAP EXTENSION ; GENE ; DNA ; POLYMERASE
WOS研究方向	Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
语种	英语
WOS记录号	WOS:000424251300002
资助机构	National Basic Research Program (973 Program)(2011CBA00800)
内容类型	期刊论文
源URL	[http://ir.ipe.ac.cn/handle/122111/24097]
专题	过程工程研究所_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.