primer design to discriminate sequences in intron-exon MSA

Question

[Anand Rao]

I seek advice in the form a bioinformatics strategy to design primers to distinguish 10 sequences from one another.

These 10 sequences are genic sequences, and across which their respective exonic sequences are highly conserved, and so is the number of exons. Their non-exonic sequences, as expected, are not always as highly conserved.

The Multiple Sequence Alignment of these 10 genes that I have generated factor in the rules of splicing at intron-exon junctions.

My goal is to design primers that anneal to a common conserved region across these 10 sequences. But the amplicon that is generated across these should be different enough in size or sequence or both, so that I can figure out which sequence was the PCR template from among 10 possible ones. Better yet if the primers can be used for template discrimination during both qPCR (with gDNA template) and q RT-PCR (with mRNA template).

Since I have ~ 3000 such alignments, I want and need to automate this, and not squint looking at them onscreen! I could follow a purely MSA parsing approach, but I wonder if newer tools used for completely different purposes (e.g. genome mappers etc) can be co-opted for my task. Thanks, folks!

Answer 1

[Kevin Blighe]

The last time that I checked, the primer design process (if you want to do it right) ought to be done manually. Primers are used in many different types of technologies but many of them have specificity to more than just the target region of interest, which leads to problems in many downstream bioinformatics applications.

Assuming that you know the exact sequences that you want to target, I suggest that you follow a standard operating procedure that I wrote at the University of Leicester a few years ago: https://www2.le.ac.uk/centres/cancer/internal-information/procedures/sops/sop646v01.pdf

The key tool is Primer3 (http://primer3.ut.ee/ ), which is still used to this day by many researchers. In my SOP, I allude to another design tool, Primer Express, which is licensed and not available (neither is it required for your purposes as it's mainly used for real-time qPCR where a probe is also used).

Hope that this helps.

Kevin

[Anand Rao]

Assuming that you know the exact sequences that you want to target

Actually I do not, and eyeballing 3000 alignments to identify primer annealing sites is not feasible.

While I appreciate your inputs, I am looking for suggestions to identify the primer annealing regions 1st - that will also maximize template discrimination. So that is the more interesting component of my bioinformatic task at hand. Any ideas?

[Kevin Blighe]

Apologies, I somehow missed the fact that you have ~3,000 alignments!

Primique ( https://www.ncbi.nlm.nih.gov/pubmed/17910777 ), written in Python, is a program that may be of interest for what you intend to do.

Other than that, I'm hopeful that others have suggestions.

[Anand Rao]

Thanks Kevin. No need to apologize, especially when you are helping :) And help you did: some digging revealed an author of Primique to also be the author of GeMprospector = Jakob Fredslund. I've e-mailed him seeking pointers about which tools that can help me. I'll update this post when I hear back from him and/or successfully implement those idea(s). Cheers!

Answer 2

[Amruta]

Kevin I saw this post from you and followed to your SOP link: https://www2.le.ac.uk/centres/cancer/internal-information/procedures/sops/sop646v01.pdf Unfortunately it is no longer accessible and does not work.

WIll you mind sharing another link that works? Thank you