Problem PCR amplifying gene promoter regions - (Jul/31/2006 )
I am trying to clone 7 promoters from 7 genes. I've designed primers on the basis of the "ensembl" sequence, with several primers combinations. I mean I've designed at least 3 primers forward and 3 reverse for each gene. Of course I first analyzed the 5' region thanks to on line programs that helped me find the putative promoter regions. now the question is: Why I do not have amplification product? I manage to have only in one gene and only with a set of primers. In all other cases I got nothing. though if I use the "In silico PCR" (with my primers)I got the right product. Where am I wrong? Is polimorfism in the 5' regions so frequent? Which is a good genomic PCR amplification program? I usually denature for 5 minutes without enzyme and then add Taq and start PCR...
thanks in advance!, Venusia
....Just an additional information, the genes I am trying to clone are human genes. The DNA I am using in my PCR is human DNA extracted from periferal blood with a kit... thanks...
Many promoters have high GC regions and making it quite difficult to amplify and some of them impossible to clone. There r some enzymes to amplify GC rich sequences. Try with these enzymes.
If you want to amplify GC-rich sequences you can add DMSO (5%) and/or Betaine (final concentration 1M). Some polymerase-kits also contain a premixed solution for enhancing. Usually it's DMSO.
i had very good results using betaine (1-2.5M). another thing you can do (i had good results also in some cases) is to digest your gDNA with a couple of restriction enzymes which do not cut within the fragment you try to amplify: i think this makes denaturation easier.