Cellular RNA extraction - Sequence-selectivity - (Jan/27/2006 )
Trying to have a brainstorm on a current project I'm working on. Previously I've used in vitro techniques to determine sequence-selective DNA binding of small molecules but now I've been asked to move into a cellular context.
Basically I need an assay which will allow me to determine where on a gene a certain drug has bound. Not so easy inside a nucleus. There are techniques such as in vivo footprinting which might be useful (though I know little about it) but I was hoping some people here might have other, more feasible suggestions.
One thought I had was that I could look at attenuated RNA transcript production (these drugs are covalent binders) and try and determine where the transcription has stopped. However, I've no idea about how to pull out the RNA for a particular gene (i.e., not extract total RNA), especially when it's not full length.
Please, any help would be most appreciated.
My thoughts on this are similar to a promoter bashing technique but in a different way. You might try after exposing your cultures to do a simple DNA extraction from your cells. Using the sequence of the gene (assuming it is available) perform PCR on the samples. The variation on the PCR is to change your reverse primer, shortening the area of the gene with each new primer. If your compound is bulky enough it will inhibit polymerase activity. This could be a good screening of your gene to determine locations of binding. If you see some inhibition with certain sets of primers you can then go on to be more detailed in the areas that might be affected.
My other question is in your in vitro experiments do you have any idea of the sequences in which these compounds are binding to because you could perform a Blast search to identify similar sequences in your gene or other genes of interest.
Thanks for the advice.
It sounds like it could turn out to be a rather lengthy process though. Also there's little guarantee of getting the PCR to work efficiently for each primer set without serious optimisation. In addition this assay may well have to be stepped up to medium to high-throughput to accommodate additional drugs.
A question regarding PCR from genomic DNA template. Presumably this involves amplification of both intronic and exonic sequences and can therefore lead to the amplification of extremely large pieces of DNA (possibly megabases). Obviously, walking along something so long with varying reverse primers is not feasible - and surely the PCR is damn hard to achieve as well.
As for BLAST - this isn't possible as we are trying to determine consensus sequences from prima facie evidence.