Use of degenerate primers and touchdown PCR for construction of cDNA libraries.
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 (ie., 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.
Fietto
Juliana L R
JL
Universidade de São Paulo, Brasil.
DeMarco
Ricardo
R
Verjovski-Almeida
Sergio
S
D000818:Animals; D017931:DNA Primers; D018076:DNA, Complementary; D020224:Expressed Sequence Tags; D015723:Gene Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D012550:Schistosoma mansoni / Q000235:genetics; D012997:Solvents; D013696:Temperature
D017931:DNA Primers; D018076:DNA, Complementary; D012997:Solvents