TY - JOUR
T1 - Chemical cleavage of heteroduplex DNA to identify mutations.
AU - Cotton, R. G.
AU - Grompe, M.
PY - 2001/5
Y1 - 2001/5
N2 - Mutation screening by the chemical-cleavage method is based on the fact that mismatched cytosine (C) and thymidine (T) are more reactive with the compounds hydroxylamine and osmium tetroxide, respectively, than are Watson and Crick-paired cytosine and thymidine bases. In this protocol, an excess of unlabeled target DNA is hybridized with labeled wild-type DNA probe and heteroduplexes are formed. One aliquot is treated with hydroxylamine, which reacts with mismatched C bases. Another aliquot is treated with osmium tetroxide, which reacts with mismatched T bases. The reactions are mixed with piperidine; the strands are then cleaved at the sites where hydroxylamine and osmium tetroxide react. Cleaved fragments are then electrophoresed and sized on polyacrylamide gels, identifying the point of cleavage (and hence the position of the mutation). Then only a small portion of the mutant gene needs to be sequenced to define a single change between two DNA sequences.
AB - Mutation screening by the chemical-cleavage method is based on the fact that mismatched cytosine (C) and thymidine (T) are more reactive with the compounds hydroxylamine and osmium tetroxide, respectively, than are Watson and Crick-paired cytosine and thymidine bases. In this protocol, an excess of unlabeled target DNA is hybridized with labeled wild-type DNA probe and heteroduplexes are formed. One aliquot is treated with hydroxylamine, which reacts with mismatched C bases. Another aliquot is treated with osmium tetroxide, which reacts with mismatched T bases. The reactions are mixed with piperidine; the strands are then cleaved at the sites where hydroxylamine and osmium tetroxide react. Cleaved fragments are then electrophoresed and sized on polyacrylamide gels, identifying the point of cleavage (and hence the position of the mutation). Then only a small portion of the mutant gene needs to be sequenced to define a single change between two DNA sequences.
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M3 - Article
C2 - 18428307
AN - SCOPUS:44049094518
SN - 1934-8266
VL - Chapter 7
SP - Unit 7.6
JO - Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.]
JF - Current protocols in human genetics / editorial board, Jonathan L. Haines ... [et al.]
ER -