Hello,
I have designed a specific primer for my Gene, when I blast this primer, it just finds the mRNA of the gene, but in nr blast, I have found some CDSs that are not relevant to my Gene. Do these CDSs cause problem in real time PCR? the length of the CDSs approximately are 200 nucleotides.
PCR depends on the proximity and orientation of 2 primers. The genome is large and there are gene families and pseudogenes with similar sequences so you expect to find similar sequences in many places but if the reverse primer is only found close to the forward primer in one place then the pcr should be specific
I am not sure why would you run a blast when you are designing a primer, and above all for real time PCR. There are online tools to help you decide which is the best primer, I am sure you can get advice in all of them. I do think ncbi may offer advice to design primers for real time PCR.
https://www.genscript.com/tools/pcr-primers-designer
https://molbiol-tools.ca/PCR.htm
https://www.researchgate.net/?ref=logo&_sg=QkrcaIQZXgIvZRuGBgUqkghocBdVtPfzkzwXY9fM-to0sOvDEW-yISMMPrIygKTsXXzIhtiDiaPU_Go
Take a look if it helps at least suggest the online programs will do the rest. (maybe exclude the cds?). Try and look for the advice on the ncbi
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