"Run-off" synthesis and application of defined single-stranded DNA hybridization probes. |
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| Authors: | M Stürzl W K Roth |
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| Institution: | Department of Virus Research, Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany. |
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| Abstract: | A simple and efficient method for synthesizing radioactively labeled single-stranded DNA hybridization probes with Thermus aquaticus (Taq) DNA polymerase is described. This is done in a "run-off" polymerization with repeated cycles of denaturation, annealing, and extension. It leads to high yields of a single-stranded DNA of defined length (up to 5000 nt), which is labeled to a high specific activity (1.3 x 10(8) cpm/micrograms DNA). These hybridization probes are equally sensitive as nick-translated DNA probes, but strand specific. This was tested by slot blot hybridization with in vitro-transcribed target RNAs and by Northern blotting. The use of single-stranded DNA hybridization probes combines the benefits of DNA stability and single-strand RNA probes. |
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