Efficient synthesis of 32P-labeled single-stranded DNA probes using linear PCR; application of the method for analysis of strand-specific DNA repair.
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.
Ruven
H J
HJ
MGC Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden, The Netherlands.
Seelen
C M
CM
Lohman
P H
PH
Mullenders
L H
LH
van Zeeland
A A
AA
D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D015342:DNA Probes / Q000096:biosynthesis*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000096:biosynthesis*; D006801:Humans; D008969:Molecular Sequence Data; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011740:Pyrimidine Dimers / Q000032:analysis; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004277:DNA, Single-Stranded; D010761:Phosphorus Radioisotopes; D011740:Pyrimidine Dimers; D004247:DNA