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Nucleic Acids Research, 2000, Vol. 28, No. 9 E38-e38
© 2000 Oxford University Press

Detection and mapping of mismatched base pairs in DNA molecules by atomic force microscopy

Masato Tanigawa, Masanori Gotoh1, Masayuki Machida2,*, Takao Okada and Michio Oishi3

Joint Research Center for Atom Technology, 1-1-4 Higashi, Tsukuba, Ibaraki 305-0046, Japan, 1Amersham Pharmacia Biotech K.K., 3-25-1 Hyakunin-cho, Shinjuku-ku, Tokyo 169-0097, Japan, 2National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan and 3Kazusa DNA Research Institute, 1532-3 Yanauchino, Kisarazu, Chiba 292-0812, Japan

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.

* To whom correspondence should be addressed. Tel: +81 298 61 6214; Fax: +81 298 61 6240; Email: machida@nibh.go.jp


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