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. 1982 Dec;20(2):139-44.
doi: 10.1016/0378-1119(82)90032-4.

Sensitive detection of RNA using strand-specific M13 probes

D M Brown, J Frampton, P Goelet, J Karn

Sensitive detection of RNA using strand-specific M13 probes

D M Brown et al. Gene. 1982 Dec.

Abstract

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.

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