Modified parallel strategies for preparation of heteroduplex plasmids for in vitro mismatch repair assays

We present efficient and reproducible parallel strategies for preparing large quantities of pure heteroduplex plasmids containing defined mismatches. The strategies described involve the use of synthetic oligonucleotides, the commercially available pGEM-T plasmid, and nicking enzymes to prepare prerequisite ssDNA. Alternatively, bacterial packaging cell lines containing an engineered phagemid construct to produce ssDNA without the need of a helper phage were utilized, hence providing added flexibility and choice. These integrated approaches help to construct different mismatch substrates of choice in large quantities, thus enhancing the usability of mismatch repair assays and extending their range and accessibility to wider research groups.