Despite the number of mutation detection systems currently available, the enzyme mismatch cleavage (EMC) method of mutation detection is one of a few techniques capable of quickly detecting and locating a single nucleotide mutation within kilobase stretches of DNA. EMC is analogous to the chemical cleavage of mismatch (CCM) technique in that it cleaves heteroduplex DNA at base pair mismatches however it does not require toxic chemicals or multiple precipitation steps. By melting and re-annealing wild-type (WT) and mutant DNA in the same tube, heteroduplexes are formed. EMC exploits the activity of a bacteriophage T4 enzyme, endonuclease VII that cleaves heteroduplex DNA at single base pair mismatches and deletions/insertions. The cleavage products can then be visualised by autoradiography after gel electrophoresis (1). The manner in which the heteroduplexes are formed can however, result in a large percentage of the denatured DNA re-annealing to form homoduplex, rather than heteroduplex, DNA. Whilst T4 endonuclease VII has a higher activity on mismatched DNA than on Watson-Crick paired DNA, it nevertheless cleaves homoduplex DNA at a low but significant rate. Therefore the high level of homoduplex DNA in solution acts a source of 'background cleavage'. This background cleavage decreases the signal-to-noise ratio which may lead to a mutation not being detected and we consider this to be the only major drawback to the technique. Here we describe a simple adaptation to the EMC method that significantly enhances the signal-to-noise ratjo by removing the radiolabelled homoduplex DNA.
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