phosphorus-radioisotopes and 3--deoxyadenosine-5--triphosphate

This protocol is used to label RNA molecules (in vitro-synthesized or in vivo-purified RNA molecules) that have free 3'-hydroxyl termini. The reaction is performed in 10 min using yeast poly(A) polymerase and 3'-deoxyadenosine 5'-[α-(32)P]triphosphate (cordycepin 5'-[α-(32)P]triphosphate), a chain-terminating nucleotide. At the end of the procedure, the reaction is desalted by gel filtration to remove any unincorporated nucleotides.

Topics: Deoxyadenine Nucleotides; Molecular Biology; Phosphorus Radioisotopes; Polynucleotide Adenylyltransferase; RNA; Saccharomyces cerevisiae; Staining and Labeling; Time Factors

Cordycepin-5'-triphosphate (3'-deoxyadenosine-5'-triphosphate) can be incorporated into the 3'-ends of DNA fragments using terminal deoxynucleotidyl transferase from calf thymus (Bollum, 1974). Because cordycepin-5'-monophosphate lacks a 3'-OH group, only a single residue is incorporated. Furthermore, DNA molecules that contain cordycepin-5'-monophosphate at their 3'-ends become resistant to hydrolysis by exonucleases that require free 3'-OH ends. As an alternative to 5'-end labeling of complementary DNA strands, we have used [32P]cordycepin-5'-triphosphate labeling of 3'-ends to confirm the nucleotide sequence of a HhaI-endonuclease-generated pTU4-plasmid DNA fragment that contains several hot spots for insertions of the transposable genetic element Tn3. 3'-End labeling with [32P] cordycepin-5'-triphosphate has also proved useful in determining the sequence of the pTU4 DNA in the vicinity of a strategically located SstII endonuclease cleavage site in the replication region of the plasmid.

Topics: Attachment Sites, Microbiological; Base Sequence; Deoxyadenine Nucleotides; DNA Nucleotidyltransferases; DNA Transposable Elements; DNA, Bacterial; DNA, Circular; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Methods; Phosphorus Radioisotopes; Plasmids