My transcripts come from an annotated transcriptome of a non-model plant. I would like to look for population variation in the sequence for transcription factors related to drought tolerance. I thought of doing this with PCR and Sanger sequencing. It would be helpful to sequence across an exon. I thought I could identify flanking regions that could be relatively conserved, one of which would be (part of) the transcript in question. How can I approach this with BLAST and other bioinformatic tools? If possible, I would like to avoid a lengthy cloning exercise.
Hi,
I think that the best way to understand which regions are conserved is using blastp to found closely related sequences. Than you can download them in fasta format and use a multi-aligner tool, as Clustal or Muscle. After that visualize the alignment in Mega, for example, in order to identify the conserved regions.
Usually, I used PerlPrimer to design primers in target regions.
Best
You could find the fasta files of that gene from several related plants, upload into BLAST and use that to create an alignment. You choose the "align two or more sequences" option for a nucleotide BLAST. Should be able to design primers using BLAST tools once you know what region(s) you think you would want.
With your transcript sequence, you can do a simple blast search, but limiting the database to only include everything within the same family (or genus if it is within a very diverse group) of your organism. From the output, pick a couple of top hits particularly if they are well-annotated model sequences, and make a multiple alignment of those and your transcript with MUSCLE or any other alignment tool. Based on the alignment, you should be able to find any highly conserved region for primer design. Hope this helps.
Primer-BLAST was developed at NCBI to help users make primers that are specific to intended PCR target. It uses Primer3 to design PCR primers and then uses BLAST and global alignment algorithm to screen primers against user-selected database in order to avoid primer pairs (all combinations including forward-reverse primer pair, forward-forward as well as reverse-reverse pairs) that can cause non-specific amplifications.
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
Would be better if you had the genome sequencing of the target genes. If You decide to go on with transcripts from related species, have in mind that your primer, that you selected trough BLAST, will amplify the selected region for all the sequences in the subject genome/transcriptome. First do an alignment of the selected sequence with other hits (by blasting) and choose the least conserved part, this will assure that you are constructing your primer only for the sequence you choose. For 3' and 5' UTR region analysis (I'm assuming that is there you want verify transcription factors on the promter region) you should design a primer (primer 3 does the job) for any kind of RACE/Genome walking technique. After Genome walking proceed with sanger sequencing.
Cheers
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