Use of high specific activity StarFire (TM) oligonucleotide probes to visualize low-abundance pre-mRNA splicing intermediates in S-pombe

An oligonucleotide labeling system was developed that can produce radiolabe led hybridization probes with tenfold or more higher specific activity than is obtained by traditional 5'-end-labeling with polynucleotide kinase. Yet the system is as rapid and simple as kinase labeling. The reaction uses th e Klenow fragment off. coli DNA polymerase to add alpha-P-32-dA residues to the 3'-end of an oligonucleotide in a primer-extension reaction. Unlike ot her methods of radioactive tailing (e.g, terminal transferase), a single sp ecies is produced of both known length and known specific activity. The rea ction is efficient, and over 90% of probe molecules are routinely labeled. Using this method of labeling, an oligonucleotide was shown to be tenfold m ore sensitive in detecting target DNA sequences in a dot blot hybridization assay, compared to the same oligonucleotide labeled using polynucleotide k inase. Northern blots of Schizosaccharomyces pombe RNA were probed with an oligonucleotide specific for intron I of the tf2d gene, a TATA-box binding transcription factor Kinase-labeled tf2d probe detected only unspliced RNA, while the same oligonucleotide labeled using the new method detected both unspliced tf2d RNA and rare pre-mRNA splicing intermediates.