"Run-off" synthesis and application of defined single-stranded DNA hybridization probes.
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.
Department of Virus Research, Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
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D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D015342:DNA Probes / Q000138:chemical synthesis*; D004277:DNA, Single-Stranded / Q000138:chemical synthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003845:Deoxycytosine Nucleotides; D004587:Electrophoresis, Agar Gel; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes; D010982:Platelet-Derived Growth Factor / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D019914:Taq Polymerase
D015342:DNA Probes; D004277:DNA, Single-Stranded; D003845:Deoxycytosine Nucleotides; D010761:Phosphorus Radioisotopes; D010982:Platelet-Derived Growth Factor; C024107:2'-deoxycytidine 5'-triphosphate; D012313:RNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase