Venegas J, Solari A
Departamento de Bioquímica, Facultad de Medicina, Universidad de Chile, Santiago.
Anal Biochem. 1994 Aug 15;221(1):57-60. doi: 10.1006/abio.1994.1378.
A nonradioactive method is developed to detect DNA polymerase activity after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis containing gapped DNA as template. The technique is based on the use of digoxigenin- or biotin-labeled deoxynucleotides during DNA synthesis, and their detection by means of an anti-digoxigenin antibody-alkaline phosphatase conjugate or by a streptavidin-alkaline phosphatase conjugate. The detection of the DNA polymerase catalytic subunit is achieved after incubation of the gels with colorimetric alkaline phosphatase substrates. The technique is able to detect nanogram amounts of Escherichia coli DNA polymerase I and picogram amounts of its Klenow fragment. The results with other DNA polymerases and E. coli extracts suggest that this colorimetric detection system could be used for the analysis of an extended range of DNA polymerase enzymes. The method presented in this report offers an alternative to the already described radioactive techniques for detection of DNA polymerase activity after SDS-polyacrylamide gel electrophoresis.
开发了一种非放射性方法，用于在以缺口DNA为模板的十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳后检测DNA聚合酶活性。该技术基于在DNA合成过程中使用地高辛配基或生物素标记的脱氧核苷酸，并通过抗地高辛配基抗体-碱性磷酸酶偶联物或链霉亲和素-碱性磷酸酶偶联物对其进行检测。在凝胶与比色碱性磷酸酶底物孵育后，可实现对DNA聚合酶催化亚基的检测。该技术能够检测纳克量的大肠杆菌DNA聚合酶I及其皮克量的克列诺片段。对其他DNA聚合酶和大肠杆菌提取物的检测结果表明，这种比色检测系统可用于分析更广泛的DNA聚合酶。本报告中介绍的方法为已描述的用于SDS-聚丙烯酰胺凝胶电泳后检测DNA聚合酶活性的放射性技术提供了一种替代方法。
