I have an annotated transcriptome and would like to develop PCR primers for particular transcribed genes. My species is a non-model plant. Can I use BLAST or another tool to identify potential PCR primer sequences? Or more generally, is there a workable bioinformatics approach to identifying potential PCR primers for transcripts arising from a non-model organism? Any literature refs would be great too.
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$\begingroup$ Aren't classic primer design tools such as ncbi.nlm.nih.gov/tools/primer-blast enough? Is there something special here I am missing? $\endgroup$– terdon ♦Jul 10, 2018 at 11:53
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$\begingroup$ I guess there are limitations when designing primers within smaller transcripts. $\endgroup$– Peter PearmanJul 10, 2018 at 12:52
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1 Answer
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It depends if you want to amplify your target from genomic DNA or cDNA.
Depending on intron size, primers that anneal to different exons may not be able to amplify across the intron(s), and if you're unlucky enough to design a primer that spans a splice junction, you'll definitely be stuffed if you're working with genomic DNA. I would try blastn or translate to an amino acid sequence and use tblastn to see if you can identify the intron/exon structure in a related species. If you don't have a genome sequence available for anything close, I think your only option is to target as small an amplicon as possible and hope for the best.
If you are working with cDNA, any standard primer design tool should do
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$\begingroup$ So taking the blastn approach, one would have to blast against reference genomes (of vascular plants in this case) to try to get the intron structure? Or would you blast on something else? $\endgroup$ Jul 10, 2018 at 15:41
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$\begingroup$ I'd be tempted to just blast against the full nr database and see what comes up. Unless you know specifically what the closest reference is $\endgroup$ Jul 10, 2018 at 15:59