Quantitation of genes and transcripts by saturation hybridization in urea solutions using strand-selected probes

Abstract

This report describes methods for quantifying specific sequences in preparations of single-stranded or double-stranded nucleic acids. We use saturating amounts of hybrid-selected, strand-specific probes and perform hybridizations in urea solutions. Hybrids (RNA-DNA or DNA-DNA) are then analyzed by either of two methods. The first employs standard S1 nuclease digestion, precipitation of resistant material on glass fiber filters, and assay by liquid scintillation counting. This method is generally chosen in the assay of rare transcripts in total nucleic acid extracts as well as preparations of polyadenylated RNA. The second employs the separation of excess probe from probe-target hybrids by gel electrophoresis, recovery of hybrids on ion-exchange paper, and determination of cpm bound by liquid scintillation counting. This method is of particular value in the assay of double-stranded sequences and the quantitation of restriction fragment length polymorphisms. Whereas both methods are accurate over ranges of target abundance representing at least several orders of magnitude, the latter (“NA45 assay”) is most sensitive, and the selection of M13-bound or unbound DNA fragments by this method can be exploited in a variety of other applicatons.