CONFORMATION-SENSITIVE GEL-ELECTROPHORESIS FOR RAPID DETECTION OF SINGLE-BASE DIFFERENCES IN DOUBLE-STRANDED PCR PRODUCTS AND DNA FRAGMENTS - EVIDENCE FOR SOLVENT-INDUCED BENDS IN DNA HETERODUPLEXES
CONFORMATION-SENSITIVE GEL-ELECTROPHORESIS FOR RAPID DETECTION OF SINGLE-BASE DIFFERENCES IN DOUBLE-STRANDED PCR PRODUCTS AND DNA FRAGMENTS - EVIDENCE FOR SOLVENT-INDUCED BENDS IN DNA HETERODUPLEXES
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DOI：
10.1073/pnas.90.21.10325
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发表时间：
1993-11-01
影响因子：
11.1
通讯作者：
PROCKOP, DJ
中科院分区：
文献类型：
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作者：
GANGULY, A;ROCK, MJ;PROCKOP, DJ
最近已经开发了几种技术来检测含有一条野生型DNA链和一条突变DNA链的DNA异源双链中的单碱基错配。在这里，我们测试的假设，一个适当的系统，轻度变性溶剂可以放大的趋势，单碱基错配产生构象变化，如弯曲的双螺旋，从而增加差异迁移的DNA异源双链和homoduplexes在凝胶电泳。异源双链体和同源双链体的最佳分离是用在10%乙二醇/15%甲酰胺/Tris-牛磺酸缓冲液中聚合的标准6%聚丙烯酰胺凝胶获得的。正如溶剂诱导弯曲假说所预测的那样，当乙二醇或甲酰胺的浓度增加时，差异迁移减少。此外，异源双链体一端50 bp内的单碱基错配不会产生差异迁移。在一系列PCR产物中的68个单碱基错配中，有60个在大约59个不同的序列背景中被检测到。未检测到的8个错配在PCR产物最近端的50 bp内或在分离的高解链温度域中。因此，可以提前预测其中可能难以检测错配的末端区域和序列背景。该方法可以应用于200-800 bp的任何PCR产物，并且不需要特殊的设备或样品制备。
Several techniques have recently been developed to detect single-base mismatches in DNA heteroduplexes that contain one strand of wild-type and one strand of mutated DNA. Here we tested the hypothesis that an appropriate system of mildly denaturing solvents can amplify the tendency of single-base mismatches to produce conformational changes, such as bends in the double helix, and thereby increase the differential migration of DNA heteroduplexes and homoduplexes during gel electrophoresis. The best separations of heteroduplexes and homoduplexes were obtained with a standard 6% polyacrylamide gel polymerized in 10% ethylene glycol/15% formamide/Tris-taurine buffer. As predicted by the hypothesis of solvent-induced bends, when the concentration of either ethylene glycol or formamide was increased, the differential migration decreased. Also, single-base mismatches within 50 bp of one end of a heteroduplex did not produce differential migration. Sixty of 68 single-base mismatches in a series of PCR products were detected in some 59 different sequence contexts. The eight mismatches not detected were either within 50 bp of the nearest end of the PCR product or in isolated high-melting-temperature domains. Therefore, it was possible to predict in advance the end regions and sequence contexts in which mismatches may be difficult to detect. The procedure can be applied to any PCR products of 200-800 bp and requires no special equipment or preparation of samples.