3'-end labeling of DNA with [alpha-32P]cordycepin-5'-triphosphate.
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.
D001287:Attachment Sites, Microbiological; D001483:Base Sequence; D004254:DNA Nucleotidyltransferases; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000378:metabolism*; D004270:DNA, Circular; D003838:Deoxyadenine Nucleotides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008722:Methods; D010761:Phosphorus Radioisotopes; D010957:Plasmids
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D004270:DNA, Circular; D003838:Deoxyadenine Nucleotides; D010761:Phosphorus Radioisotopes; C011746:3'-deoxyadenosine 5'-triphosphate; D004254:DNA Nucleotidyltransferases