The utility of restriction endonucleases as a tool in molecular biology is in large part due to the high degree of specificity with which they cleave well-characterized DNA recognition sequences. The specificity of restriction endonucleases is not absolute, yet many commonly used assays of biological phenomena and contemporary molecular biology techniques rely on the premise that restriction enzymes will cleave only perfect cognate recognition sites. In vitro, mispaired heteroduplex DNAs are commonly formed, especially following PCR amplification. A recent study into the Cleavage of Mispaired Heteroduplex DNA Substrates by Numerous Restriction Enzymes investigated a panel of restriction endonucleases to determine their ability to cleave mispaired heteroduplex DNA substrates. Two straightforward, non-radioactive assays were used to evaluate mispaired heteroduplex DNA cleavage: a PCR amplification method and an oligonucleotide-based assay. These assays demonstrated that most restriction endonucleases are capable of site-specific double-strand cleavage with heteroduplex mispaired DNA substrates, however, certain mispaired substrates do effectively abrogate cleavage to undetectable levels. These data are consistent with mispaired substrate cleavage previously reported for EcoRI and extend the curren knowledge of mispaired heteroduplex substrate cleavage to 13 additional enzymes.

from Langhans and Palladino in Current Issues in Molecular Biology

Further reading: Cleavage of Mispaired Heteroduplex DNA Substrates

Labels: DNA, DNA cleavage, PCR, restriction enzymes