Partial purification of a recombinase activity from human placenta and assay of recombination using rec-blot.
We have designed an assay for the detection of recombination intermediates. The assay can also be used for screening for recombinases. In this assay, the nonradioactive plasmid was immobilized on a nylon membrane and the radioactive plasmid counterpart was allowed to recombine in the presence of a DNA binding protein fraction from human placenta. After incubation for 30 min at 30 degrees C, the membrane was washed extensively and dried. The recombination was visualised by autoradiography and quantitated by counting the corresponding areas of the membrane. Recombination was confirmed by transformation using pRDK plasmids and by gel electrophoresis. These results indicate the presence of recombinase activity in the DNA binding protein fraction of human placenta.
Jeyaseelan
R
R
Cancer Biology Division, School of Biological Sciences, Madurai Kamaraj University, India.
Nagashunmugam
T
T
Shanmugam
G
G
D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010920:Placenta / Q000201:enzymology*; D010957:Plasmids; D011247:Pregnancy; D011995:Recombination, Genetic
D004254:DNA Nucleotidyltransferases