title: A novel T-type overhangs improve the enzyme-free cloning of PCR products.
abstract: PCR product cloning is the foundational technology for almost all fields in the life sciences. Numerous innovative methods have been designed during the past few decades. Enzyme-free cloning is the only one that avoids post-amplification enzymatic treatments, making the technique reliable and cost effective. However, the complementary staggered overhangs used in enzyme-free cloning tend to result in self-ligation of the vector under some circumstances. Here, we describe a "T-type" enzyme-free cloning method: instead of designing the complementary staggered overhangs used in conventional enzyme-free cloning, we create "T-type" overhangs that reduce the possibility of self-ligation and are more convenient for multi-vector cloning. In this study, we systematically optimize "T-type" enzyme-free cloning, compare its cloning background with that in conventional enzyme-free cloning, and demonstrate a promising application of this technique in multi-vector cloning. Our method simplifies post-amplification procedures and greatly reduces cost, offering a competitive option for PCR product cloning.
authors.affiliation: Key Laboratory of Non-coding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
authors.forename: Ren-Bin
authors.initials: RB
authors.lastname: Yang
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Bi
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods* / Q000592:standards