A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends

BMC Biotechnology

Methodology Article

Fanli Zeng¹  Zhimin Hao¹  Jingao Dong¹  Yanan Meng¹  Pan Li¹  Yibin Lin² 

[1] College of Life Sciences, Hebei Agricultural University, 071001, Baoding, China;the University of Texas Houston McGovern Medical School, 77030, Houston, USA;

关键词: Restriction-free cloning;    Gene reconstitution;    Single-primer PCR;    High-throughput cloning;   

DOI  :  10.1186/s12896-017-0346-5

 received in 2016-12-08, accepted in 2017-03-01,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundRestriction-free (RF) cloning, a PCR-based method for the creation of custom DNA plasmids, allows for the insertion of any sequence into any plasmid vector at any desired position, independent of restriction sites and/or ligation. Here, we describe a simple and fast method for performing gene reconstitution by modified RF cloning.ResultsDouble-stranded inserts and acceptors were first amplified by regular PCR. The amplified fragments were then used as the templates in two separate linear amplification reactions containing either forward or reverse primer to generate two single-strand reverse-complement counterparts, which could anneal to each other. The annealed inserts and acceptors with 5’ and 3’ cohesive ends were sealed by ligation reaction. Using this method, we made 46 constructs containing insertions of up to 20 kb. The average cloning efficiency was higher than 85%, as confirmed by colony PCR and sequencing of the inserts.ConclusionsOur method provides an alternative cloning method capable of inserting any DNA fragment of up to at least 20 kb into a plasmid, with high efficiency. This new method does not require restriction sites or alterations of the plasmid or the gene of interest, or additional treatments. The simplicity of both primer design and the procedure itself makes the method suitable for high-throughput cloning and structural genomics.

【 授权许可】

CC BY   
© The Author(s). 2017