Chemiluminescent Detection of Sequential DNA Hybridizations to High-Density, Filter-Arrayed cDNA Libraries: A Subtraction Method for Novel Gene Discovery

Abstract

A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotinbased random priming method followed by a streptavidin/alkaline phosphatase/CDPStarTM detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid highdensity filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently “deplexed” into individual components for specific probe localizations.