'Run-off' polymerization with digoxigenin labelled nucleotides creates highly sensitive and strand specific DNA...

In this paper the in vitro synthesis and application of non-radioactively labelled strand specific DNA probes is described. The probe is labelled by incorporation of nucleotides with the hapten digoxigenin into single-stranded DNA during a 'run-off' reaction catalyzed by Thermus aquaticus (Taq) DNA-polymerase. The 'run-off' reaction requires a linearized plasmid template and one primer binding site at a defined distance from the restriction site. Single-stranded DNA can be synthesized during repeated cycles of denaturation, annealing, and extension. The conditions for the incorporation of digoxigenin-11-dUTP (dig-11-dUTP) during polymerization were optimized to generate strand specific DNA hybridization probes up to a length of 5000 nt. The strand specificity is demonstrated by a dot-blot, with in vitro-transcribed target RNA of c-sis. The sensitivity of the probe was tested in a Northern blot, and found to be identical to a probe radiolabelled by nick-translation (specific activity 6.5 x 10(8) cpm micrograms-1). The resolution of the signals and speed of development was even superior compared to the radiolabelled probe.