Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V.

ABSTRACT:

Background

DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning.

Results

A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions.

Conclusions

The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus in

SUBMITTER: Baumann T 

PROVIDER: S-EPMC3856533 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature