Generation of a Polymerase Chain Reaction Renewable Source of Subtractive cDNA

Abstract

Differential (+/-) first-strand cDNA screening methods identify clones corresponding to mRNAs that are expressed at a higher level in one of a pair of phenotypically different cells. This approach is limited by the fact that screening of libraries with labeled first-strand cDNAs synthesized from unfractionated mRNA can detect clones containing sequences representing approx 0.1% or more of the complexity of mRNA (i.e., mRNAs present at greater than about 200 copies per cell since a typical mammalian cell line contains approx 250,000 mRNAs).