Detection and Localization of Base Changes in RNA Using a Chemical Cleavage Method

The detection of base changes in DNA and RNA is of central importance in genetic research. Mismatched cytosines and thymines in heteroduplex DNA molecules show increased chemical reactivity with hydroxylamine and osmium tetroxide, respectively, and the DNA can then be specifically cleaved at the modified nucleotides. We show here that mismatched cytosines and thymines can be detected and located directly in RNA: DNA heteroduplex molecules. In order to detect guanosine and adenosine base changes the complementary cDNA strand must be analyzed. In addition, the sensitivity of the technique can be increased by employing the polymerase chain reaction. To test the fidelity of this method a number of known or predicted mutations were analyzed. These include single point mutations in the human collagen alpha 1(I) and rat phenylalanine hydroxylase mRNA, two engineered point mutations in a mouse collagen alpha 1(I) mRNA, and a deletion in a human collagen alpha 2(I) mRNA. All known base changes were detected and correctly localized. In addition, the predicted base changes were confirmed.