Hello Friends, In Eukaryotas, splicing is well known phenomenon and is used by almost every cell. During RNA synthesis, introns are removed and exons are joined to make mRNA, the actual size if gene on DNA is much longer than mRNA, it seems like during RNA synthesis domains of the proteins are also removed, is it true? or in some genes, antisense copy of the gene exactly acts as mRNA without removal of any intron or just a small small inton is removed. Can you please elaborate this phenomenon.. Thank you so much..
Some genes have numerous splice variants - (e.g., VEGF; up to 9 variants, other genes many more than that) so you want to think in terms of multiple splice variant possibilities rather than just 1...
thank you for the reply, Many genes generates many of the mRNA upto 50, or even more than that.. and some just only one, is there any simple way to know that the gene is generating how many of the mRNA?
If you run PCR on total cDNA using primers that flank the entire region of interest and run a 1-5% agarose gel, you can visualize many of the variants that way... or run SYBR Green-based qPCR (using strategic priming) and check the number of different melting curves you obtain afterwards. Just a couple of options.
I agree with both of you, my mates, thanks for reply.. I have another idea in my mind, if we just make primers from the start and end of the gene and run a gradient PCR giving maximum possible extension temperature, will it help to give us all possible variants of the gene? Secondly, Grow our required organism on different conditions and then do the same thing, what are the possibilities of getting maximum active variants?
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