Determining restriction digest efficiency using the SNaPshot single-base extension method and CE

Incomplete restriction endonuclease digestion may cause complications during particular applications, where any undigested product may interfere with genotyping accuracy, cloning efficiency or PCR amplification. In some instances it may be useful to measure the amount of undigested DNA remaining in the sample, allowing redigestion or elimination of unsuitable samples. A single-base extension SNP genotyping method (SNaPshot, Applied Biosytems) was adapted to interrogate restriction sites and their digestibility. The digested DNA was amplified by nested PCR to specifically amplify sections containing the restriction sites of interest. Single interrogation primers, terminating immediately 5' of the cleavage site, were used in conjunction with the SNaPshot multiplex system and CE to detect intact, amplifiable product remaining in the digest. This method detected as little as 10 pg of intact DNA, within a larger proportion of digested DNA. Across two different restriction enzymes, the amount remaining undigested was found to be dependent on the amount of DNA, ranging from 0.08% with 200 ng to 1.25% with 600 ng.