Can restriction digestion be replaced by CHIP? - (Feb/28/2007 )
I'm dealing with very low DNA quantities and I need to shear my DNA before the bisulfite mod. as to increase the specificity of my primers. Has anybody tried CHIP to shear the DNA instead of restriction enzymes?
well... so CHIP doesn't technically shear the DNA, DNA shearing is part of the CHIP protocol, but in some protocols is done by sonication in some nuclease activity etc., and I imagine any consistent means of getting an average 500bp pieces of the genome would be suitable.
So could you sonicate?, sure, but my thing is why would you shear before bisulfite and how does that have anything to do with primer specificity and why would low DNA concentration be associated with either?
Bisulfite treated DNA is less stable than untreated and is therfore suseptible to degradation more than standard gDNA so i would think the goal should be to have "good" (unsheared) DNA because the more degradation you have the better chance you will get breaks between you primer binding sites and therefore not have amplification... so, I am confused, but may be encountering a similar problem so I would really appreciate an explaination of why this approach is being used...
thanks for your answer beccaf22. well it's a general observation from my experiments that I need to digest the DNA and then bisulfite treat otherwise I get multiple bands or smears around my expected band. A different possibility could be that the digest makes the DNA more susceptible to the modification and gives better and cleaner products ... ?
My amplicons are generally larger than 500 bp. I guess it is possible to sonicate my cell extract to get larger fragments. But it must shear my target DNA as well making it more difficult again to get a proper product ...
What is the best method (bearing in mind I have a low DNA yield) to break up the DNA like a digestion would?
I am just about as confused as beccaf22 ?!?
As you already replied, sonication might cause strand breaks in the region you want to amplify, because you cannot control where sonicaion cleaves the DNA, so I wouldn't do that (especially as you have a low DNA conc.) .
Why do you want to switch methods? The restriction digest seems to be working fine for you, or not? (or is it too expensive?)
if you are starting with minimal DNA for bisulfite treatment I suggest you ensure denaturation for conversion (as opposed to reducing the genomic complexity by shearing).
So instead of a 37C incubation in 0.3M NaOH, you can up the NaOH concentration for denaturation and/or up the denaturation temperature to 100C and then quickly add your bisulfite reagent for conversion.
Seems to work well in my hands with repeat elements in an human system.
Thanks for your suggestions,
I'll give them a go.