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 (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.

Evaluation of small-subunit rRNA touchdown polymerase chain reaction for direct detection of Entamoeba histolytica in human pus samples from patients with amoebic liver abscess

“…[14] Another investigator, Zumarraga et al had also developed a rapid and sensitive TD-PCR for the detection of Mycobacterium bovis in bovine samples by targeting the IS6110 element. [15] Fietto et al, described the construction of cDNA libraries using TD-PCR [16] and Horra et al, also compared single and TD-PCR protocols for detecting Pneumocystis jirovecii DNA in paraffi n-embedded lung tissue samples and suggested the enhancing effect of TD-PCR. [17] We have standardized TD-PCR and compared its results with conventional PCR using the same set of primers that hybridizes with the SSU rRNA gene and generates a product size of 166 bp.…”

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“…Although touchdown PCR has been used successfully to help overcome some of the uncertainties associated with the thermal amplification of microbial nucleic acid targets [19][20][21][22], its use in this study has extended its role further and in so doing brought closer the goal of undertaking molecular diagnostics in a routine setting. Previously its main impact has been seen where multiplexing [23,24] or degenerate primers have been needed [25][26][27] and where the problems of choosing correct annealing temperatures are at their most difficult.…”

Mapping Transposon Insertion Sites by Touchdown PCR and Hybrid Degenerate Primers

“…These primers were originally used in a touchdown PCR method for the construction of cDNA libraries using Schistosoma mansoni mRNA (13). Examples of consensus-degenerate hybrid primers APYBAF1 and HIB17 used in this work are presented in Table 1.…”

“…It is based on touchdown PCR (12,13) using a pair of primers where one is a hybrid consensus-degenerate oligonucleotide and the other is a sequence-specific primer that only anneals to one of the strands of the inserted marker gene, with its 3 end pointing toward the unknown genomic sequence. The resulting PCR products are sequenced using solely the specific primer.…”