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mismatch repair analysis

An assay system for detection of single-base mismatches in a DNA fragment of up to 10 Kb in length, which depends upon an Escherichia coli repair system that detects mismatched bases between a newly synthesized (unmethylated) DNA strand and an unreplicated (methylated) strand. The unmethylated DNA strand in a hybrid of the two is nicked, digested and resynthesized on the methylated template. This system, however, does not replace insertions or loops larger than 5 bases. A mutated DNA fragment is inserted into a plasmid that contains a reporter gene, such as LacZ, which can be used to identify colonies that express the plasmid, and is then transformed into an E. coli dam- strain, which results in an unmethylated plasmid. The wild-type DNA fragment is inserted into the same plasmid in which a 5-base sequence interrupts the reporter gene; this is transformed into a dam+ strain, which fully methylates the plasmid. The two strains are grown, the plasmids isolated, mixed, denatured and reannealed. Fully methylated homoduplexes are digested with DpnI and unmethylated homoduplexes are digested with MboI. Surviving heteroduplexes, which contain both the intact and the interrupted reporter genes, are circularized, ligated and transformed into E. coli. If there is no mismatch, both strands of the plasmid will be replicated and the intact reporter gene will be expressed. However, if there is a mismatch, the non-methylated strand, along with the intact reporter gene will be digested and will not be expressed.Faham, M. and Cox, D. R. (1995) genome Res. 5, 474-482

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