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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
Source PublicationMOLECULAR BIOTECHNOLOGY
ISSN1073-6085
Volume60Issue:2Pages:92-99
AbstractGene 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.
KeywordFusion Pcr Dual-asymmetric Primers Two-step Annealing
SubtypeArticle
WOS HeadingsScience & Technology ; Life Sciences & Biomedicine
DOI10.1007/s12033-017-0050-7
Indexed BySCI
Language英语
WOS KeywordOVERLAP EXTENSION ; GENE ; DNA ; POLYMERASE
WOS Research AreaBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
WOS SubjectBiochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
Funding OrganizationNational Basic Research Program (973 Program)(2011CBA00800)
WOS IDWOS:000424251300002
Citation statistics
Document Type期刊论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/24097
Collection生化工程国家重点实验室
Affiliation1.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
Recommended Citation
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.
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