title: A restriction-free method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends.
abstract: Restriction-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.
  
  
  
  Double-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.
  
  
  
  Our 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.
authors.affiliation: College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China.
authors.forename: Fanli
authors.initials: F
authors.lastname: Zeng
authors.affiliation: College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Hao
authors.affiliation: College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China.
authors.forename: Pan
authors.initials: P
authors.lastname: Li
authors.affiliation: College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Meng
authors.affiliation: College of Life Sciences, Hebei Agricultural University, Baoding, 071001, China. shmdjg@hebau.edu.cn.
authors.forename: Jingao
authors.initials: J
authors.lastname: Dong
authors.affiliation: the University of Texas Houston McGovern Medical School, Houston, 77030, USA. Yibin.lin@uth.tmc.edu.
authors.forename: Yibin
authors.identifier: 0000-0003-0608-7875
authors.initials: Y
authors.lastname: Lin
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004247:DNA; D004262:DNA Restriction Enzymes
keywords: Gene reconstitution; High-throughput cloning; Restriction-free cloning; Single-primer PCR