Restriction enzyme-free Cloning

In contrast to conventional cloning strategies, Restriction enzyme-free cloning (also called Restriction-free cloning) involves no treatment of DNA fragments to restriction enzymes. Since it does not rely on restriction endonucleases, lack of suitable restriction sites is not a problem.   However, such cloning uses a special way to prepare plasmid vectors which may be laborious. In addition, lack of directionality of the inserted DNA fragment may be a problem, therefore, it requires screening of many clones for a right orientation of the DNA fragment. Restriction-free cloning is very useful to clone PCR-amplified DNA fragments. 

Some restriction-free cloning strategies are…..

  • TA Cloning
  • GatewayR technology (Invitrogen)
  • heterostagger cloning
  • uracil DNA glycosylase
  • ligation-independent cloning such as TOPO cloning


  • Zhou & Gomez-Sanchez, 2000. Universal TA cloning. Curr Issues Mol Biol. 2(1), 1-7. PMID-11464915; Full-Text Links: caister (download PDF)
  • Felfoldi et al., 1997. Direct ligation of human CD4 polymerase chain reaction fragment into vectors at specific restriction sites with positional heterostagger cloning. Anal Biochem. 253(2), 275-277. PMID-9367518; Full Text Link: Sciencedirect
  • Rashtchian et al., 1992. Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning. Anal Biochem. 206 (1), 91-97. PMID-1456447; Full Text Link: Sciencedirect
  • Aslanidis & de Jong, 1990. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18(20), 6069-6074. PMID-2235490; Full Text Link: academic.oup (download PDF), PMC332407

Was this post helpful?

Author: admin

1 thought on “Restriction enzyme-free Cloning

Leave a Reply

Your email address will not be published. Required fields are marked *