Rapid localization of point mutations in PCR products by chemical (HOT) modification.
Our studies of mutational mechanisms in mammalian cells use the AS52 Chinese hamster ovary cell line. AS52 mutants can be selected as 6-thioguanine resistant colonies and mutations are studied at a chromosomally integrated gpt locus. Mutant gpt sequences are amplified using the polymerase chain reaction (PCR) to distinguish deletions from putative point mutations. PCR is efficiently performed from a few thousand lysed cells or from isolated genomic DNA. Amplified mutant PCR fragments carrying putative point mutations are further characterized by localizing the site of the mutation using chemical modification. A heteroduplex molecule consisting of one wild-type and one mutant DNA strand is generated. A base mismatch will be produced at the site of the mutation. Mismatched cytosine or thymine residues are sensitive to modification by hydroxylamine or osmium tetroxide, respectively. The modified DNA heteroduplex is then sensitive to piperidine cleavage. If one strand is 32P-end labeled, then the cleavage product can be separated on a denaturing acrylamide sequencing gel and visualized using autoradiography. Thus, the site of a mutation can be localized to a specific region of the gene, thereby simplifying the DNA sequence analysis and facilitating the rapid generation of mutational sequence spectra.
Tindall
K R
KR
Experimental Carcinogenesis and Mutagenesis Branch, National Institue of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Whitaker
R A
RA
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004277:DNA, Single-Stranded / Q000737:chemistry; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009993:Osmium Tetroxide / Q000737:chemistry; D010430:Pentosyltransferases / Q000235:genetics; D016133:Polymerase Chain Reaction
D004277:DNA, Single-Stranded; D006898:Hydroxylamines; D019811:Hydroxylamine; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D009993:Osmium Tetroxide