REACTIVITY OF CYTOSINE AND THYMINE IN SINGLE-BASE-PAIR MISMATCHES WITH HYDROXYLAMINE AND OSMIUM-TETROXIDE AND ITS APPLICATION TO THE STUDY OF MUTATIONS
REACTIVITY OF CYTOSINE AND THYMINE IN SINGLE-BASE-PAIR MISMATCHES WITH HYDROXYLAMINE AND OSMIUM-TETROXIDE AND ITS APPLICATION TO THE STUDY OF MUTATIONS
复制标题
DOI：
10.1073/pnas.85.12.4397
复制
发表时间：
1988-06-01
影响因子：
11.1
通讯作者：
CAMPBELL, RD
中科院分区：
文献类型：
--
作者：
COTTON, RGH;RODRIGUES, NR;CAMPBELL, RD
研究了胸腺嘧啶(T)与胞嘧啶、鸟嘌呤和胸腺嘧啶碱基不匹配时的化学反应性，以及胞嘧啶(C)与胸腺嘧啶、腺嘌呤和胞嘧啶不匹配时的化学反应性。含有这种错配碱基对的异双链DNA首先与四氧化三铯(对于T和C错配)或羟胺(对于C错配)孵育，然后与哌啶孵育以在修改的错配碱基处切割DNA。用含有不匹配的T或C的内部标记的链来研究这种裂解产物，这样就可以用凝胶电泳法检测DNA裂解，从而检测到反应活性。确定了总共13T和21C错配的切割(通过两侧至少三个正确配对的碱基)单碱基对错配。所有研究的T或C错配都被切割。通过使用包含T或C单碱基对错配的末端标记DNA探针和有限切割条件，我们能够表明切割是在序列分析预测的碱基上进行的，并且这种方法可以很容易地检测到一段DNA的错配。该程序可以通过使用正义和反义探针来检测所有单碱基对错配，从而可以用于识别突变的碱基及其在异源双链中的位置。
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 cleavate 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.