weird rflp-pcr - (May/26/2011 )

I am trying to detect a SNP using a restriction enzyme. When I do the PCR I always get a bright band when the sample is homozygous for the wild type allele and a relatively weak band when the sample is homozygous for the other allele. DNA was extracted from venous. I have no contamination with neither the wild type PCR product nor with a homozygous template dna for the wild (common) allele. Where does this stupid discrimination power come from? It's really funny. For heterozygous samples I almost get no band for the mutant allele.

This is really frustrating. No contamination is there because I get very clean bands following PCR. This also happened for all the samples (50 different samples)

You are going to have to explain a bit better I think, I have no idea exactly what you are asking?

(also, a clean band is not an indication of no contamination!)

leelee on Fri May 27 05:57:26 2011 said:

Thanks for your reply.

I'll explain further. I extracted genomic DNA from blood. I amplify a 198 bp fragment to detect a SNP (wild type allele frequency is 78% and mutant type alelle frequency is 22%). Digesting with the restriction enzyme should yield a 177 bp fragment in case of homozygous for the wild type allele, and not cut should happen in case of the mutant allele (the resulting band should therefore be 198 bp). Everything is fine up to this point and I am actually getting either one of these two bands or both in case of heterozygous genotype. However, for heterozygous genotype the 198 bp is 6 times less intense than the 177 bp fragment. Also for samples homozygous for the mutant allele the band intensity is also less than the intensity of the undigested band which is of the same length.

Now I couldn't explain it in terms of contamination because I never get a 177 bp band for homozygous for the mutant allele (the 198 bp fragment)
I couldn't explain it in terms of star activity for the restriction enzyme because the 177 bp fragment is always bright.

By the way the protocol I am using has been developed in-house and it is based on mismatch RFLP (meaning that I introduced the restriction site close to the 3' end of the primer)
The incubation temperature is 55 C and the restriction enzyme is BsaJI.

It's getting me crazy because sometimes the 198 bp is not visible in heterozygous. in theory the two fragments should be of equal intensity because heterozygous means 50% for each.

I never had a problem with another SNP for which allele frequencies for the wild type and mutant type were 47% and 53%, respectively. So It feels like allele frequency is affecting the intensity of the bands!!! isn't that crazy?

By the way this problem happens with all samples (50 sample) . there is no way all these are contaminated. They are stored in aliquots and the only one using them is me.

Hmmm this is indeed puzzling. I agree with you that contamination doesn't seem to be the problem (sorry, didn't quite know what you meant the first post). I am stumped sorry!