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

Abstract

An oligonucleotide labeling system was developed that can produce radiolabeled 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 the Klenow fragment of E. coli DNA polymerase to add α-32P-dA residues to the 3'-end of an oligonucleotide in a primer-extension reaction. Unlike other methods of radioactive tailing (e.g., terminal transferase), a single species is produced of both known length and known specific activity. The reaction is efficient, and over 90% of probe molecules are routinely labeled. Using this method of labeling, an oligonucleotide was shown to be tenfold more sensitive in detecting target DNA sequences in a dot blot hybridization assay, compared to the same oligonucleotide labeled using polynucleotide kinase. Northern blots of Schizosaccharomyces pombe RNA were probed with an oligonucleotide specific for intron 1 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.