Webtag: a new web tool providing tags/anchors for RT-PCR experiments with prokaryotes

The use of a relational database, in conjunction with a series of scripts written in PHP and Perl, allows the user to rapidly obtain tags that are: 1) suitable for a specific organism, and 2) compatible with other oligonucleotides to be used in the experimental procedures.This new web tool allows scientists to easily and rapidly obtain suitable tags for RT-PCR experiments, and is available at http://www.egs.uu.se/software/webtag/ webcite.In order to better understand different aspects of metabolism it is important to study the underlying transcriptional profile. A key factor to assemble such profiles is the ability to obtain good gene expression data. For that purpose, reverse transcription polymerase chain reaction (RT-PCR) [1] became the method of choice more than a decade ago [2,3].RT-PCR allows exponential amplification of even a very low copy number mRNA. Because of its high sensitivity, the process is very vulnerable to DNA contamination.Unfortunately, no RNA extraction method can guarantee the absolute absence of DNA in any given sample, ultimately leading to amplification, during PCR, of both cDNA and contaminating genomic DNA [4-11].The effects of DNA contamination can be overcome using techniques like oligo d(A) selection, intron spanning primer design, DNase I treatment or restriction endonuclease digestion [7,8,11]. However, these procedures can be time consuming, expensive and contribute to RNA degradation. Moreover, in the particular case of prokaryotes, oligo d(A) selection and intron spanning primer design are not applicable solutions.One proposed strategy leading to the specific amplification of cDNA involves the use of anchors/tags [4,9,12]. As illustrated in Figure 1, during reverse transcription of mRNA, a unique tag positioned at the 5'end of the gene specific primer is included in the cDNA. This tag will later be used as primer targeting the second cDNA strand. Application of this strategy, like in the cases of RS-PCR [4] and (EXACT) RT-PCR [9]