Homologous alignment cloning: a rapid, flexible and highly efficient general molecular cloning method

PeerJ

article

Lendl Tan¹  Emily J. Strong¹  Kyra Woods¹  Nicholas P. West¹ 

[1] School of Chemistry and Molecular Biosciences, Australian Infectious Diseases Research Centre, University of Queensland

关键词: Molecular cloning;    Low cost;    High efficiency;    Simple;    Ligation-independant;    High fidelity;    Rapid;    Homologous alignment cloning;    HAC;   

DOI  :  10.7717/peerj.5146

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

Homologous alignment cloning (HAC) is a rapid method of molecular cloning that facilitates low-cost, highly efficient cloning of polymerase chain reaction products into any plasmid vector in approximately 2 min. HAC facilitates insert integration due to a sequence alignment strategy, by way of short, vector-specific homology tails appended to insert during amplification. Simultaneous exposure of single-stranded fragment ends, utilising the 3′→5′ exonuclease activity of T4 DNA polymerase, creates overlapping homologous DNA on each molecule. The exonuclease activity of T4 polymerase is quenched simply by the addition of EDTA and a simple annealing step ensures high yield and high fidelity vector formation. The resultant recombinant plasmids are transformed into standard E. coli cloning strains and screened via established methods as necessary. HAC exploits reagents commonly found in molecular research laboratories and achieves efficiencies that exceed conventional cloning methods, including another ligation-independent method we tested. HAC is also suitable for combining multiple fragments in a single reaction, thus extending its flexibility.

【 授权许可】

CC BY