Radio-labeling of the PstI restriction fragments and improvement in the sequencing procedure.
A new and simple method has been developed for efficient labeling of the protruding 3' ends of DNA fragments generated by class-II restriction enzymes. This new method utilizes a synthetic oligodeoxynucleotide 5'-GTGCA-3', which is complementary to the protruding 3' end of the fragment and hybridizes with it in such a manner that the end of the restriction fragment will now have a protruding, one-nucleotide (nt)-long 5' end. This then serves as a template to incorporate just one nt at the 3' end of the restriction fragment. This procedure is easy to use, highly efficient (labeling at 80% of the theoretical incorporation) and generates labeled fragments that are suitable for sequencing. We have also improved the nt sequencing procedure of Bencini et al. [Biotechniques 2 (1984) 4-5] by replacing 'A greater than C' reaction with 'G' and 'C' specific reactions and employing butanol precipitation of DNA.
D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; D009838:Oligodeoxyribonucleotides
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004262:DNA Restriction Enzymes; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific