Dahl H H, Lamande S R, Cotton R G, Bateman J F
Murdoch Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.
Anal Biochem. 1989 Dec;183(2):263-8. doi: 10.1016/0003-2697(89)90477-6.
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.
DNA和RNA中碱基变化的检测在基因研究中至关重要。异源双链DNA分子中错配的胞嘧啶和胸腺嘧啶分别对羟胺和四氧化锇表现出更高的化学反应活性，然后DNA可在修饰的核苷酸处被特异性切割。我们在此表明，错配的胞嘧啶和胸腺嘧啶可直接在RNA:DNA异源双链分子中被检测和定位。为了检测鸟嘌呤和腺嘌呤碱基变化，必须分析互补的cDNA链。此外，通过采用聚合酶链反应可提高该技术的灵敏度。为了测试该方法的保真度，分析了一些已知或预测的突变。这些包括人胶原蛋白α1(I)和大鼠苯丙氨酸羟化酶mRNA中的单点突变、小鼠胶原蛋白α1(I)mRNA中的两个工程化点突变以及人胶原蛋白α2(I)mRNA中的一个缺失。所有已知的碱基变化均被检测到并正确定位。此外，预测的碱基变化也得到了证实。
