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; C M Seelen; P H Lohman; L H Mullenders; A A van Zeeland

doi:10.1016/0921-8777(94)90018-3

Efficient synthesis of 32P-labeled single-stranded DNA probes using linear PCR; application of the method for analysis of strand-specific DNA repair

Mutat Res. 1994 Sep;315(2):189-95. doi: 10.1016/0921-8777(94)90018-3.

Authors

H J Ruven¹, C M Seelen, P H Lohman, L H Mullenders, A A van Zeeland

Affiliation

¹ MGC Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, The Netherlands.

PMID: 7521000
DOI: 10.1016/0921-8777(94)90018-3

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Base Sequence
DNA / analysis*
DNA Probes / biosynthesis*
DNA Repair*
DNA, Single-Stranded / biosynthesis*
Humans
Molecular Sequence Data
Phosphorus Radioisotopes
Polymerase Chain Reaction / methods*
Pyrimidine Dimers / analysis
Sensitivity and Specificity

Substances

DNA Probes
DNA, Single-Stranded
Phosphorus Radioisotopes
Pyrimidine Dimers
DNA