Rapid filter assay for the detection of DNA polymerase activity: direct identification of the gene for the DNA polymerase from Thermus aquaticus.
A method for rapid identification of DNA polymerase activity employing an activated DNA substrate covalently bound to nitrocellulose membranes is described. Samples containing DNA polymerase are spotted and the membranes are incubated in an appropriate polymerization buffer containing radioactively labelled dNTPs. By autoradiography of the dried filters, DNA polymerase activity can be directly identified. The method can be used for fast and large-scale screening of chromosomal expression libraries for heterologous DNA polymerases characterized by activity optima different from those of the host organisms. We have identified the gene of the thermostable DNA polymerase from Thermus aquaticus in an expression library of Escherichia coli.
Sagner
G
G
Department of Genetics, Boehringer Mannheim GmbH, Penzberg, F.R.G.
Rüger
R
R
Kessler
C
C
D002462:Cell Membrane / Q000201:enzymology; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D005798:Genes, Bacterial; D010957:Plasmids; D013698:Templates, Genetic; D013824:Thermus / Q000201:enzymology / Q000235:genetics*
D004259:DNA-Directed DNA Polymerase