Directly fishing out subtle mutations in genomic DNA with histidine-tagged Thermus thermophilus MutS.
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 degrees 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((R)) 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.
Wang
Jian
J
School of Life Science and Technology, Shanghai Jiaotong University, 800 Dong-Chuan Road, Shanghai 200240, China.
Liu
Jianhua
J
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020137:Base Pair Mismatch; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D024881:Drug Resistance, Bacterial; D018628:Gene Dosage; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D051719:MutS DNA Mismatch-Binding Protein; D016254:Mutagenesis, Insertional; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D017354:Point Mutation; D011550:Pseudomonas aeruginosa / Q000235:genetics; D016963:Thermus thermophilus / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009153:Mutagens; D008769:Methylnitronitrosoguanidine; D006639:Histidine; D015500:Chloramphenicol O-Acetyltransferase; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein