Efficient synthesis of 32P-labeled single-stranded DNA probes using linear PCR; application of the method for analysis of strand-specific DNA repair - PubMed (original) (raw)
Efficient synthesis of 32P-labeled single-stranded DNA probes using linear PCR; application of the method for analysis of strand-specific DNA repair
H J Ruven et al. Mutat Res. 1994 Sep.
Abstract
We have developed a rapid method to synthesize radioactively labeled single-stranded DNA probes suitable for strand-specific analysis of single copy genes on Southern blot. Linear PCR with 10 microCi alpha 32P-dATP (3000 Ci/mmol) as the only dATP source enabled us to generate strand-specific DNA probes with high specific activity. The probes synthesized by this method have higher specific activities and the same strand specificity compared to the end-labeled single-stranded DNA probes obtained from single-stranded M13mp18/19 vectors. Application of the method for strand-specific analysis of ultraviolet-induced DNA lesions in defined DNA sequences significantly improved the hybridization signal.