High-Stringency Subtraction for the Identification of Differentially Regulated cDNA Clones

Abstract

The technique of high-stringency subtraction described here facilitates subtractive hybridizations between directional cDNA libraries constructed in λ ZAP® II cloning vectors and represents an improvement on earlier methods for the subtraction of entire cDNA libraries. High-stringency subtraction is designed to eliminate the subtraction of differentially expressed cDNAs, which show similarity to constitutive sequences by the incorporation of a novel high-stringency wash step. This method also allows the size-selection of target cDNAs and incorporates an improved procedure for the synthesis of driver DNA used in subtractions.