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

Lendl Tan, Emily J. Strong, Kyra Woods, Nicholas P. West

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


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.

Original languageEnglish (US)
Article numbere5146
Issue number6
StatePublished - 2018
Externally publishedYes


  • HAC
  • High efficiency
  • High fidelity
  • Homologous alignment cloning
  • Ligation-independant
  • Low cost
  • Molecular cloning
  • Rapid
  • Simple

ASJC Scopus subject areas

  • General Neuroscience
  • General Biochemistry, Genetics and Molecular Biology
  • General Agricultural and Biological Sciences


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