Mutation detection using fluorescent enzyme mismatch cleavage with T4 endonuclease VII.

Abstract

Mutation detection techniques are often limited by sensitivity, ease of use and short fragment lengths. Enzyme mismatch cleavage (EMC) is a technique capable of rapidly scanning 1 kbp fragments of DNA for mutations. It relies on the ability of a bacteriophage resolvase enzyme, T4 endonuclease VII, to cleave DNA at single base pair mismatches and small heteroduplex loops. Originally the process was performed using radioactively labeled DNA and the results analysed after denaturing polyacrylamide gel electrophoresis and autoradiography. However, access to systems capable of detecting fluorescent species migrating through a gel and the widespread availability of fluorescently tagged primers have greatly improved upon the original technique. A number of mutations were detected using fluorescent EMC and the results compared to performing the technique using radiolabeled DNA. Fluorescent EMC detected the presence, position and number of mutations in DNA fragments as large as 1 kbp. The fluorescent method was found to have advantages over the original method in its ease of use, increase in signal-to-noise ratio and the ability to multiplex samples by labeling DNA fragments with different fluorophores. This improvement on an already established method provides a sensitive, robust technique for mutation detection.