Use of degenerate primers and touchdown PCR for construction of cDNA libraries

Abstract

Optimized construction of low-redundancy cDNA mini-libraries using low-stringency RT-PCR is described. cDNAs are generated using arbitrary consensus-degenerate hybrid oligonucleotide primers and nanogram amounts of Schistosoma mansoni mRNA. A number of conditions such as temperature and salt concentration are combined to create balanced, low-stringency conditions that permit a normalized amplification of a diversified, random set of sequences. On average, 350 different sequences are obtained per mini-library, which represents a significantly higher diversity of messages per library when compared to previously published conditions (i.e., 20-40 sequences/mini-library). The optimized high-through-put approach described here is likely to help in the discovery of expressed genes in any complex organism.