Beck S
Medical Research Council, Laboratory of Molecular Biology, Cambridge, England.
Anal Biochem. 1987 Aug 1;164(2):514-20. doi: 10.1016/0003-2697(87)90526-4.
A sensitive, colorimetric method for visualizing the band pattern of DNA sequencing reaction is described. The enzymatic incorporation of radioactive nucleotides commonly used for the band detection is replaced by biotin conjugated to the 5'-terminus of a synthetic oligonucleotide by chemical synthesis. The oligonucleotide so labeled is used as a primer for dideoxy DNA sequencing in a primer extension reaction. The products of the sequencing reactions are analyzed on a denaturing polyacrylamide gel using the direct blotting electrophoresis technique. This technique makes it possible to transfer the band pattern during the electrophoresis onto an immobilizing matrix, on which it is made visible by an enzymatic reaction in less than 3 h. This biotin-based detection method is so sensitive that the sequencing reactions can be performed under the same conditions and concentrations as those for the radioactive detection.
本文描述了一种灵敏的比色法，用于可视化DNA测序反应的条带模式。用于条带检测的常用放射性核苷酸的酶促掺入被通过化学合成与合成寡核苷酸5'端共轭的生物素所取代。如此标记的寡核苷酸在引物延伸反应中用作双脱氧DNA测序的引物。使用直接印迹电泳技术在变性聚丙烯酰胺凝胶上分析测序反应的产物。该技术使得在电泳过程中将条带模式转移到固定基质上成为可能，在该基质上通过酶促反应在不到3小时内使其可见。这种基于生物素的检测方法非常灵敏，以至于测序反应可以在与放射性检测相同的条件和浓度下进行。
