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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 a-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.