I need to design primers to amplify and quantify cDNA targets in a qPCR assay, the cDNA of which has been reverse transcribed from isolated RNA from pig tissues. However the mRNA sequences for most of the biomarkers I wish to analyse show up as "predicted" on the NCBI database, therefore I cannot know for sure that if I design a primer from this sequence on whether it will bind to the actual sequence for the genes I wish to study.
Does anyone have any experience in designing primers from predicted sequences? Any advice or help anyone can offer me will be truly appreciated.
Many thanks in advance.
Cheers,Adam
I hope this link be useful for you.
http://www.scienceforums.net/topic/49272-how-useful-are-predicted-sequences-in-ncbi/
I would sequence the DNA first and get the correct sequence. I would not run a qPCR with primers that are "approximate", this only creates problems later on. If your sequences are all unknown, then make an alignment of some closely related sequences first. This will allow you to judge where your sequences are most likely conserved and where they tend to vary. Next, order degenerated primers where you include different bases at those positions that are variable. Then use a rather high concentration of primers for your PCR and an annealing temperature that allows some mismatch.
That's how I would proceed.
Many thanks for your answers everyone. Dr Velavan it's good to hear from you! I hope things have been going well for the group back at Tuebingen.
Dominik may I ask if there is a certain supplier from whom is best to order degenerated primers? Could there also be a way to check whether your degenerated primers are at risk of primer-dimers, self-annealing e.t.c?
Thanks once again.
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