Polynucleotide kinase: Functional purification and use in the direct kinetic measurement of single- and double-strand cleavages of DNA by restriction and other endonucleases of limited action

Localization on the viral genome and nucleotide sequence of the gene coding for the two major polypeptides of the Hepatitis B surface antigen (HBs Ag)

“…Preparation of 5' 32P labeled DNA fragments 10 to 20 picomoles of Eco HBV DNA were fully hydrolysed by different restriction enzymes according to the conditions recommended by the manufacturer. DNA fragments were dephosphoylated by alkaline phosphatase which was then inactivated by alkaline treatment and the DNA was precipitated with ethanol (20). After redissolution in spermidine buffer, DNA was labeled at its 5'ends with y3 p ATP (NEN : 3,000 Ci/mM) and polynucleotide kinase (21).…”

Gene-sized pieces produced by digestion of linear duplex DNA with mung bean nuclease

“…Mung bean nuclease was isolated and purified to homogeneity as described . Polynucleotide kinase was isolated in a form which was devoid of any interfering activities (Kroeker and Laskowski, 1977). Alkaline phosphatase (Sigma) was further purified by the method of Weiss et al (1968).…”

“…(1968) for labeling the 5'-phosphorylated termini of DNA with polynucleotide kinase after treatment with alkaline phosphatase at either 25 (to determine double-strand cleavages) or 65 °C (to determine nicks plus double-strand cleavages) was modified to provide an accurate and convenient procedure for measuring the limited endonucleolytic cleavages of native DNA. The procedure (Kroeker and Laskowski, 1977) takes advantage of the rapid diffusion of unreacted [32P]ATP from agarose pellets which retain the large, terminally labeled DNA products. Acid-soluble products, which were <2% of the total DNA, did not interfere with the end-labeling reaction.…”