Cotton R G, Rodrigues N R, Campbell R D
Department of Biochemistry, University of Oxford, United Kingdom.
Proc Natl Acad Sci U S A. 1988 Jun;85(12):4397-401. doi: 10.1073/pnas.85.12.4397.
The chemical reactivity of thymine (T), when mismatched with the bases cytosine, guanine, and thymine, and of cytosine (C), when mismatched with thymine, adenine, and cytosine, has been examined. Heteroduplex DNAs containing such mismatched base pairs were first incubated with osmium tetroxide (for T and C mismatches) or hydroxylamine (for C mismatches) and then incubated with piperidine to cleave the DNA at the modified mismatched base. This cleavage was studied with an internally labeled strand containing the mismatched T or C, such that DNA cleavage and thus reactivity could be detected by gel electrophoresis. Cleavage at a total of 13 T and 21 C mismatches isolated (by at least three properly paired bases on both sides) single-base-pair mismatches was identified. All T or C mismatches studied were cleaved. By using end-labeled DNA probes containing T or C single-base-pair mismatches and conditions for limited cleavage, we were able to show that cleavage was at the base predicted by sequence analysis and that mismatches in a length of DNA could be readily detected by such an approach. This procedure may enable detection of all single-base-pair mismatches by use of sense and antisense probes and thus may be used to identify the mutated base and its position in a heteroduplex.
已对胸腺嘧啶（T）与胞嘧啶、鸟嘌呤和胸腺嘧啶错配时，以及胞嘧啶（C）与胸腺嘧啶、腺嘌呤和胞嘧啶错配时的化学反应活性进行了研究。首先将含有此类错配碱基对的异源双链DNA与四氧化锇（用于T和C错配）或羟胺（用于C错配）一起孵育，然后与哌啶一起孵育，以在修饰的错配碱基处切割DNA。使用含有错配的T或C的内部标记链研究这种切割，这样DNA切割以及反应活性就可以通过凝胶电泳检测到。在总共分离出的13个T和21个C错配（两侧至少有三个正确配对的碱基）处的切割被鉴定为单碱基对错配。所研究的所有T或C错配都被切割。通过使用含有T或C单碱基对错配的末端标记DNA探针以及有限切割的条件，我们能够表明切割发生在序列分析预测的碱基处，并且通过这种方法可以很容易地检测到一段DNA中的错配。该程序可以通过使用正义和反义探针检测所有单碱基对错配，因此可用于鉴定异源双链中突变的碱基及其位置。
