Hi dear fellows,
I would like to perform ChIP-PCR on the promoter sequences of genes, but I am not quite sure how to design primers for the promoter sequences. Can someone please help me?
My second question is: is it possible that the real-time PCR for these primers has to be performed in some particular way?
Thank you very much for help
You can use genomatix promoter analysis online tools or CLC genomic workbench software
hi Barbara Kurpisz
1. You can explore the Eukaryotic Promoter Database (EPD) https://epd.epfl.ch//index.php
2. Yes, ChIP qRT-PCR needs specific preparation which includes crosslinking, sonication, bead binding, elution etc. you can look this
https://www.sigmaaldrich.com/catalog/product/SIGMA/CHP1?lang=en®ion=IN
cheers!!!
hi,
you can use primer designing tools, such as primer 3, Try to restrict your primer pairs in between 500 -1000 bp upstream or 500 bp downstream.
For your ques 2, they should amplify in a standard procedure.
Thank you very much for your help. I will try my best to design good primers
Dear Barbara Kurpisz , the promoter sequences are often found to be GC rich so, it might be tricky to find a good region with less GC%. More GC% might lead to primer hetro or self dimers or stem-loop structures that might cause negative results in PCR. I prefer to use the IDToligoAnaLyzer website to check for those conditions. If the del G value is positive or more than -10 deg celsius those sequences are good for primer designing. i will avoid the regions where del G values are less than - 10 deg celsius. Tm preferably should be between 60 to 65 deg celsius to reduce mis-amplifications.
For qPCR, the same condition applies but, I will limit myself with 80 - 100 bp amplicon size and small primer length (17-19 bp).
Barbara Kurpisz I am not sure if you meant no information is available on promoter sequences. In that case, several strategies are there depending upon what information is available: 5' and 3' RACE strategy can be used. Another strategy is if you know the sequence of closely related promoter sequences then you can design a degenerate primer that would be able to bind to a position even though it is not an exactly complementary sequence.
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