Reactivity of potassium permanganate and tetraethylammonium chloride with mismatched bases and a simple mutation detection protocol
Reactivity of potassium permanganate and tetraethylammonium chloride with mismatched bases and a simple mutation detection protocol
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DOI：
10.1093/nar/27.8.1866
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发表时间：
1999-04-15
影响因子：
14.9
通讯作者：
Cotton, RGH
中科院分区：
文献类型：
--
作者：
Lambrinakos, A;Humphrey, KE;Cotton, RGH
许多突变检测技术依赖于DNA异源双链体中错配碱基对的识别。高锰酸钾与四乙基氯化铵（TEAC）的组合能够化学修饰错配的胸苷残基，然后可以通过用哌啶处理在该点切割DNA链。高锰酸钾（KMnO 4）在TEAC对错配的反应性进行了研究，在29个不同的突变，代表58个错配碱基对和116个错配碱基。所有不匹配的胸苷残基被KMnO 4/TEAC修饰，其中大多数表现出较强的反应性。高锰酸钾/TEAC也能够修改许多错配的鸟苷和胞苷残基，以及匹配的鸟苷，胞苷和胸苷残基相邻，或附近，错配碱基对。以前的技术使用四氧化锇（OsO 4）修饰错配的胸苷残基已被限制的明显缺乏反应性的三分之一的所有T/G错配，KMnO 4/TEAC显示没有这样的现象。在这个系列中，所有29个突变检测KMnO 4/TEAC处理。最新开发的单管错配化学裂解法在单管中通过KMnO 4/TEAC和羟胺（NH 2 OH）检测胸苷和胞苷错配，而无需在两个反应之间进行清理步骤。这种技术节省了时间和材料，而不会破坏任何反应的灵敏度和效率。
Many mutation detection techniques rely upon recognition of mismatched base pairs in DNA heteroduplexes. Potassium permanganate in combination with tetraethylammonium chloride (TEAC) is capable of chemically modifying mismatched thymidine residues, The DNA strand can then be cleaved at that point by treatment with piperidine. The reactivity of potassium permanganate (KMnO4) in TEAC toward mismatches was investigated in 29 different mutations, representing 58 mismatched base pairs and 116 mismatched bases. All mismatched thymidine residues were modified by KMnO4/TEAC with the majority of these showing strong reactivity. KMnO4/TEAC was also able to modify many mismatched guanosine and cytidine residues, as well as matched guanosine, cytidine and thymidine residues adjacent to, or nearby, mismatched base pairs. Previous techniques using osmium tetroxide (OsO4) to modify mismatched thymidine residues have been limited by the apparent lack of reactivity of a third of all T/G mismatches, KMnO4/TEAC showed no such phenomenon. In this series, all 29 mutations were detected by KMnO4/TEAC treatment. The latest development of the Single Tube Chemical Cleavage of Mismatch Method detects both thymidine and cytidine mismatches by KMnO4/TEAC and hydroxylamine (NH2OH) in a single tube without a clean-up step in between the two reactions. This technique saves time and material without disrupting the sensitivity and efficiency of either reaction.