i already have 5 sequence genes that have high similarity with each other, so i need design primer for each one for qpcr to identify the expression pattern , please does anyone help me to do this an important step.? thanks in advance
-first of all be sure that you know your coding regions. not all mRNA sequences are translating to protein.
- check for most specific area for each gene for your species. generally last %40 of coding sequences are having most diversity.
- be sure Tm values should be very close to each other. max 1 deg. diffirence works well.
- Dont go with long pcr products. 100-200 bp is better.
- GC amount should be lower than %60 but higher than %35 for avoiding nonspecific products.
- Try to design all your primers with same tm (or as close as it can be) for running in one plate.
- and the best way of doing that pcr, look for KASP (KAST or CAST in diffirent companies) method. using a polymerase with proof reading ability helps a lot. put the sequence diffirences to the 3' region of each primer. In my phd thesis i was working with SNP oligos and it worked quite well.
After you follow the Deniz Kom suggestions, you should use some softwares to predict if the sequences of your primers will not produce the primer dimer interactions. Gene runner, Genious are softwares that you can use for.
Deniz Kom and Vinicious Nunes provided the best of it. In addition, you may try to be careful about selecting specific nucleotides at the 3' ends of primer. It is better not to have more than two G or C out of last five nucleotides in 3' end. At least I have seen it works in my case. Again, check for hetero- and homo-dimer formation tendency of primers. It is best to keep del-G value within -6 kJ. Furthermore, I reckon, sample source is also important whether it would be human sample, plant or micro-organism.
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