DNA cloning - (May/09/2009 )
I need your advice in DNA cloning.
How am I going to clone my DNA (include intronic site) for further protein expression study? My population study show 4bp deletion in intron4 of my gene is one of the significant risk factor for a genetic disease. I cant do cDNA cloning as it is in intron site. What should I do in order to characterize this deletion?
Thanks in advance.
If it is a risk factor- is it just a statistical association? If so why do you need to clone it?
I need to characterize it at RNA or protein level to see whether this deletion has anything to do with protein expression?
feiliang on May 11 2009, 03:32 AM said:
to look for gene expression you need to run a Q-PCR in order to compare people bearing the insertion against people not bearing it.....so no need to clone it
Second problem if your mutation is altering a splicing site, I guess you need to clone your gene and see if it is spliced correctly or you are getting extra bands...look at the mini-gene or something over internet I guess there is a technique to look for aberrant splicing sites
If you have cells from people or your mutation is polymorphic in population you should consider an RNA extraction and amplification to look for different splicing in people with the insertion/deletion.
So cloning your gene doesn't make sense unless you know which technique you are going to use
Cloning an intron for expression studies also won't work, the constructs use bacterial expression style promoters etc, meaning that everything after the ATG is expressed, even when you transfect it into human cells.