Nachweis und Charakterisierung von DNS-Polymerasen durch Micro-Disc-Elektrophorese

Abstract

DNA polymerase from Escherichia coli was studied using a new technique of micro-disc-electro· phoresis on polyacrylamid gel carried out in 5 μl capillary tubes. Two methods for the identification of DNA polymerase among other protein zones of crude preparations were developed: 1. The binding test in which DNA polymerase forms a complex with poly d (A — T) in the presence of Mg2⊕ ions, d ATP, and dTTP. This complex of rather high molecular weight cannot enter the separation gel and remains in the interphase between concentration and separation gel. Consequently, the appropriate zones are missing after staining with amido black 10 B in acetic acid. 2. The polymerisation test in which the separation gel contains a minute amount of poly d (A—T) primer, sufficient to induce an extensive synthesis of poly d (A—T) by DNA polymerase in the gel when incubated with d ATP and dTTP in the presence of Mg2⊕ions. The newly synthesized poly d (A— T) is stained with pyronine and can be estimated with a sensitive microdensitometer. The application of this micro-method to biochemical problems is discussed. Earlier findings of a multiplicity of E. coli DNA polymerase are confirmed.