I intend to analyse the effect of an unstable intron element in exon splicing. For this, I'm planning to include the flanking exons (say 3rd and 4th exons of a gene) by PCR amplifying them from genomic DNA and cloning them into a mini gene vector.
Would it be wrong to assume that in the control experiment, the transcription will start from the 1st nucleotide of the exon (e.g..3rd exon) if I include 300 nucleotides upstream? And similarly that the 4th exon will be spliced correctly if I include 300 nucleotides downstream?
Hi,
First of all the start of the transcription is defined by your promoter. Different promoter will have different transcription start site. So you better check it for your promoter.
Second for the splicing. Basically for an exon to be spliced it needs the binding of U1 and U2 in upstream intron and some say also U1 binding in downstream intron. So to make it easier to be sure that an exon would be spliced usually we put two neighboring exons one upstream and one downstream. So it doesn't matter how many nucleotides you add it depends on U1 and U2 binding sites on the intron.
Hope it helps!
Dear Gargi Nanda
In your experiment control the transcription should start from the first base that you clone (300 bp upstream the third exon) and should end to the last base (300 bp downstream the fourth exon)because you have no splicing donor for the third exon and no splicing acceptor for the fourth exon. The intron (between third and fourth exons) you are interested to study, will be spliced.
Since you are interested to a possible intron retention (if I have well understood), I would clone the genomic region containing the third and fourth exons with the regulated intron from the first base of third exon to the last base of fourth exon. In this manner you will exclude elements that are probably just involved in the splicing of introns upstream and downstream to the third and fourth exons.
However, elements involved in splicing regulation are not ever predictable. You should try.
Good luck !
Thank you so much Rudolfo and Reymond. Your Suggestions really helps in my designing my exp.
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