Enzymatic Mismatch Cleavage EMC

This strategy exploits the ability of bacteriophage enzymes (resolvases) to recognize and cleave DNA containing unpaired bases. Resolvases can be thought of simply as restriction enzymes that cut DNA only in the presence of mutation. Heteroduplexes created by heat denaturation and renaturation of PCR products are cleaved enzymatically, and subsequently separated by denaturing or non-denaturing polyacrylamide gel electrophoresis. Resolvases that have been used with this technique include T4 endonuclease VII and T7 endonuclease.71_73 Mutations were detected in DNA fragments between 88 and 940 base pairs71 and up to 1.5 kilobases in length.72 An automated version of this technique claims to have improved earlier versions by eliminating the need for sample purification, shortening hybridization time, and increasing the signal-to-noise ratio.73

Only a few systematic studies have been conducted with this technique and further evaluation of its reliability is needed. It has been observed that some mutations were poorly recognized by resolvases. Thus, while cleavage of small deletions (1_3 base pairs) is detected, only 13 of 14 point mutations representing all possible nucleotide exchanges were detected.71 In a second report, only 3 of 4 small deletions and 17 of 18 point mutations were detected.72 Mutations that are poorly recognized may result in incomplete DNA digestion, and the occurrence of nonspecific bands complicates interpretation of the electrophoretic patterns obtained. Another problem with EMC is that homozygous mutant samples escape detection, but at the same time, non-specific cleavage of homoduplexes is observed where none is expected. As the efficiency and reliability of enzymatic mismatch cleavage remains to be proven, this technique is not yet considered acceptable as a strategy to screen for variants in DNA.

Was this article helpful?

0 0

Post a comment