Detection and mapping of mismatched base pairs in DNA molecules by atomic force microscopy.
Attempts were made to apply atomic force microscopy (AFM) imaging to the detection and mapping of the sites of base substitutions in DNA molecules. In essence, DNA fragments to be examined for possible base substitutions were mixed with an equal amount of a corresponding DNA standard and subjected to heat denaturation and subsequent annealing. The reassociated DNA was incubated with MutS protein, a protein that recognizes and binds to mismatched base pairs in duplex DNA. Bound MutS protein molecules were then detected by AFM and their positions along the DNA molecules were determined by calculating the distance from one of the DNA termini, which had been tagged with a biotin-avidin complex. Base substitutions present in DNA molecules >1 kb were effectively detected by this procedure, and the positions determined were in good agreement with the actual mutation sites. This method is quite simple, has virtually no limitations on the size of DNA fragments to be examined and requires only a very small amount of DNA sample.
Tanigawa
M
M
Joint Research Center for Atom Technology, 1-1-4 Higashi, Tsukuba, Ibaraki 305-0046, Japan.
Gotoh
M
M
Machida
M
M
Okada
T
T
Oishi
M
M
D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins / Q000378:metabolism / Q000648:ultrastructure; D020137:Base Pair Mismatch; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D018625:Microscopy, Atomic Force; D051719:MutS DNA Mismatch-Binding Protein
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli