title: Use of high specific activity StarFire 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 alpha-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.
authors.affiliation: Integrated DNA Technologies, Coralville, IA, USA. mbehlke@idtdna.com
authors.forename: M A
authors.initials: MA
authors.lastname: Behlke
authors.forename: S A
authors.initials: SA
authors.lastname: Dames
authors.forename: W H
authors.initials: WH
authors.lastname: McDonald
authors.forename: K L
authors.initials: KL
authors.lastname: Gould
authors.forename: E J
authors.initials: EJ
authors.lastname: Devor
authors.forename: J A
authors.initials: JA
authors.lastname: Walder
mesh_terms: D015345:Oligonucleotide Probes; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism*; D012568:Schizosaccharomyces / Q000235:genetics*
chemical_list: D015345:Oligonucleotide Probes; D012322:RNA Precursors; D012331:RNA, Fungal