The principle underlying this method resembles that in ribonuclease A cleavage. The DNA heteroduplex is formed from a mixture of wild-type and mutant DNA heating and annealing. Mismatched T bases of the heteroduplex are modified by osmium tetroxide and mismatched C bases are modified by hydroxylamine. The modified strands of DNA are cleaved at the site of the modification by incubation with piperidine. Fragments are subsequently separated on denaturing polyacrylamide gels and identified by autoradiography. By labeling the antisense strand, adenosine and guanosine mismatches are also detected. This method is capable of detecting at least 95% of mismatches when only the wild type is labeled and 100% when both strands are labeled. PCR products of up to 2 kilobases in length have been studied successfully.52,70 The precise location and the nature of the mutation can also be determined from the size of the band and the cleaving agent. The primary disadvantage of this method is the use of highly mutagenic and explosive chemicals that are unsuitable for a routine clinical laboratory and limit the potential for automation.
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