Purifying dsDNA from ssDNA after a PCR reaction - (Aug/15/2009 )
The model organism I use has a unique genome structure. It has 2 functionally distinct nuclei. In one of the nuclei, the MAC, it has gene-sized chromosomes. These gene-sized chromosomes are also amplified hundreds and sometimes thousands of times. I am looking at the 5' UTR regions of specific MAC chromosomes.
Previous analysis has determined a conserved motif in many different MAC chromosomes. I also have the coding sequence of the gene of the chromosome I am studying. What I need is between the sequence between the 5' telomere and the coding sequence.
My idea is to use a forward primer that binds to the telomere end and another primer that binds to the coding region. The problem is that I will get a lot of products that is of many different sizes as the telomere primer will bind to all the different MAC chromosomes. They will all be ssDNA of different lenghts and look like a smear on the gel. The MAC chromosomes range from 1kb to 5kb in lenght. The PCR product that I want will be dsDNA. It will have a higher molecular weight than the average ssDNA so it will run slower on the gel, but it will still be in the large size regions. The problem will be that the PCR product that I want will be in an area of other PCR products
Is there a way to purify dsDNA from ssDNA? Is there a enzyme the degrades ssDNA but leaves dsDNA alone?
I have not tried the PCR reaction yet so I'm not sure it will even work, but I wanted to get the entire procedure though out before I started this method.
Exonuclease1 seems to do the job (click).
Wait, some thoughts...
You want to do a PCR, why do you think you'll end up with ssDNA? Sure, your Telomere primer will bind to all telomeres, but you've got a reverse primer that is gene specific. So you'll end up with loads of gene specific dsDNA, that you can clone or directly sequence. Or did I get something wrong? Do you have multiple copies of your gene?
Sorry, if I'm confused...
I agree. I see no source of ssDNA in these reactions. You may also be able to construct a telomere primer which will bind to the first repeat of the telomere sequence. If your telomere repeat is something like ggggtttttttt, you could use a primer aaaaaaaccccb, where is the b is a degenerate non-a base mixture.