Genome-wide detection of unknown subtle mutations in bacteria by combination of MutS and RDA.
We propose a procedure for detecting unknown, subtle DNA changes throughout the entire bacterial genome by a combination of MutS and RDA. Current techniques detect subtle mutations after PCR amplification of the target regions, so the mutation detection is done between amplified PCR fragments. In this paper, genome-wide subtle mutation scanning in bacteria was performed by combining the MutS and RDA techniques. Our strategy for cloning a small mutation region is composed of two steps: an enrichment of fragments containing subtle mutations using MutS, followed by an RDA subtraction procedure for further enrichment. We successfully identified small mutations such as a four-base insertion, a two-base insertion, and transition mutations in bacteria.
Gotoh
K
K
Kazusa DNA Research Institute, Kisarazu, Chiba, Japan. koshichi@kazusa.or.jp
Hata
M
M
Miyajima
M
M
Yokota
H
H
D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics*; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015723:Gene Library; D016680:Genome, Bacterial; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D051719:MutS DNA Mismatch-Binding Protein; D016254:Mutagenesis, Insertional; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D019914:Taq Polymerase; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli