Generation of Blunt Ends after Restriction Digestion

Dominik Haudenschild, February 1995, at Genzyme

BASIC PROTOCOL: Repairing 3' or 5' Overhanging Ends to Generate Blunt Ends

For many cloning experiments, it is necessary to convert the ends generated by restriction endonucleases into blunt ends (UNIT 3.16).

1. In a 20-ul reaction, digest 0.1 to 4 ug DNA with a restriction endonuclease.

DNA can be treated with exonucleases such as Bal 31, l exonuclease, exonuclease III, or endonucleases such as S1 or mung bean nuclease if desired.

2. Add 1 ul of 0.5 mM each dNTP.

It is unnecessary to inactivate the restriction endonuclease, to change buffers, or to repurify the DNA prior to adding the Klenow fragment.

3. Add 1 to 5 U of the Klenow fragment and incubate at 30∞C for 15 min.

Repair of 5' extensions is carried out by polymerase activity, whereas repair of 3' extensions is carried out by 3' to 5' exonuclease activity. Due to the relative inactivity of exonuclease, this method is not desirable in cases where extensive repair of overhanging 3' ends is required. In such situations, T4 DNA polymerase (a much more expensive enzyme) or native T7 DNA polymerase are better choices.

4. Stop the reaction by heating to 75 C for 10 min or by adding 1 ul of 0.5 M EDTA.

For restriction fragments produced by cleavage with different endonucleases, it is possible to repair one end selectively. This is done by cleaving with enzyme 1, repairing the ends, inactivating the Klenow fragment by heat (75 C for 10 min), and cleaving with enzyme 2.