Use of Degenerate Primers and Touchdown PCR for Construction of cDNA Libraries. (June 2002)

• Record Type: Journal Article
• Authors: Fietto, Juliana L.R.; DeMarco, Ricardo; Verjovski-Almeida, Sergio
• Is Part Of: Biotechniques. Volume 32:Number 6(2002)
• Page Start: 1404
• Page End: 1411
• Publication Date: 2002-06
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/02326rr01
• Languages: English
• ISSNs: 0736-6205

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-throughput approach described here is likely to help in the discovery of expressed genes in any complex organism.