Nhe1 Restriction digestion - (Jan/26/2006 )
could anyone please help. I am genotyping the PSA gene using Nhe1 which is supposed to show AA 300bp , AG 150, 300 bp and GG 150 bp bands. I'm confused with the AG band .why the 150 bp band is always lighter than the 300 bp band which is more prominent. should i consider it as AG or AA in that case. I have not got any 300 bp bands even after running 50 DNA samples and Hardy-Weiberg Equilibrium should have 25%AA, 50% AG and 25% GG genotypes in normal samples.
I am not sure what you mean by AA, AG and GG but the 150bp band is expected to be lighter than the 300. Since it was 1/2 the base pairs than the 300bp strand you would expect it to bind less EtBr molecules and thus appear dimmer. That is perfectly normal.
Seems like i am not the only one who is facing this problem, as my current research also involves the polymorphism at the PSA gene. I managed to get my first 300bp banding pattern (AA genotype) at my 45th sample. I'm also not sure wad could be the reasons on its low frequency... Most of my other samples are also of a GG genotype... wad about yours?
I know nothing about the PSA gene but allele frequencies rarely exist as you have described. Often one allele/ genotype is very common. The frequency differs between species and ethnicity(human). I've worked on polymorphisms from a pharmacogenetic point of view that become redundant because of low frequencies i.e. most of the population are one genotype.
From a methodological point of view it might be worth sequencing a few to check that enzyme shows specificity
hope this helps