qPCR primer design - (May/11/2009 )
Basic question regarding designing primers for use in amplifying cDNA using qPCR:
I believe you are supposed to design primers using different exons, or that are close to intronic splice sites, something to that effect, in order to rule out genomic DNA contaimination...
If I have a gene sequence from NCBI (+50,000bp), how does one go about figuring out the best way to design a primers? Is there a program that can be used to determine the best locations, what is the best approach?
This is probably common sense but your help would be appreciated, thanks.
Get the cDNA or coding sequence and identify the exon boundaries. Design primers that will give you a product of 100-200 bp and span an intron that is big enough so that PCR won't work.
Note there are free web based databases of working qPCR primers that you should probably look at first - someone has already done the design work.
Thanks bob1, I had heard of Primer Bank but did not have much luck, do you mind giving me the names of ones you work with?
bob1 on May 11 2009, 06:15 PM said:
Note there are free web based databases of working qPCR primers that you should probably look at first - someone has already done the design work.
dna_nerd on May 11 2009, 08:53 PM said:
bob1 on May 11 2009, 06:15 PM said:
Note there are free web based databases of working qPCR primers that you should probably look at first - someone has already done the design work.
Hi
There's a Primer3 software programme which you can use for primer designing. just give in ur cDNA sequence. As mentioned by Bob the amplicon size is generally kept around 100-150 bp. But make sure that these primers are specific to your gene on NCBI primer blast
I have not seen any gene with 50k mRNA sequence. You need to retrieve the mRNA or cDNA sequence for primer design. NCBI assigns refseq to each gene and refseq represents the most correct and complete version of the gene's transcript. In terms of which region to design primer, in theory any region (UTR, coding) will do, but I prefer regions closer to the 3' end. Also you should check whether your gene has isoforms and whether you want your primers to cover or not to cover all of them, which determines where you want to design your primers.
Consideration of template secondary structures is important in designing primers, especially in qPCR.
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