Sensitive detection of RNA using strand-specific M13 probes.
We have extended the method of Hu and Messing (Gene 17 (1982) 271-277) to prepare highly radioactive M13 probes suitable for use in RNA-DNA hybridization experiments. Single strands of M13 DNA carrying cloned sequences are rendered partially double-stranded by primed synthesis using a synthetic oligonucleotide primer complementary to a region 5' to the cloning site. The newly synthesized radioactive complementary strand is then covalently cross-linked to the M13 phage DNA by UV irradiation in the presence of 4,5,8-trimethylpsoralen (trioxsalen). Since the cross-linked probe is stable to heat denaturation, and the region of cloned sequence is kept single-stranded, these complexes may be used as strand-specific hybridization probes to detect RNA sequences under conditions which would denature DNA-DNA duplexes.
D001483:Base Sequence; D003090:Coliphages / Q000187:drug effects / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D009843:Oligoribonucleotides / Q000032:analysis; D012313:RNA / Q000235:genetics*; D014307:Trioxsalen / Q000494:pharmacology
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009843:Oligoribonucleotides; D012313:RNA; D014307:Trioxsalen