Directly fishing out subtle mutations in genomic DNA with histidine-tagged Thermus thermophilus MutS

Abstract

Tth MutS, a mismatch repair protein from Thermus thermophilus, was reported to effectively recognize all eight possible types of base pair mismatches and insertions or deletions up to three base pairs at a wide temperature range up to 60 °C. Here a procedure for directly fishing out subtle unknown mutations in bacterial genome with Tth MutS was described. Wild type genomic DNA and mutant one were mixed, digested with restriction enzymes, denatured and re-annealed. Hetero-duplex DNA carrying mispaired bases were bound to Tth MutS and recovered through Ni-NTA His-Bind ® Resin. The recovered DNA was cloned into plasmids, producing a mini-library with inserts of the mutated regions. Further DNA sequencing and genetic complementation demonstrated that the method was extremely efficient in fishing out the mutations from total genomic DNA. Using this method, the mutations existed in a Psedomonas aeruginosa mutant strain were screened, indicating that A/G transitions at nt 181 and nt 314 in chloramphenicol acetyltransferase (catB7) gene conferred this strain with a high chloramphenicol dosage resistant. Compared with those reported previously, this protocol can screen the mixed mutations more easily.