exons alternatively spliced - How to detect which variant is present? (Apr/10/2006 )
If the first two exons of a gene are non-coding and alternatively spliced. How can I detect that?
How can I detect the presence of one or the other variant.
Is it possible to do it by sequencing the exon amplified by PCR?
You can use primers flanking your alternative exons and detect isoforms by RT-PCR (where RT = Reverse Transcriptase). You can also perform direct sequencing of your PCR product.
I will sequence because I already did the PCR and the sequence reaction.
The biological significance of this alternative splicing is unknown.
what kind of assay could I do to test it?
Usually alternative splicing in the 5' UTR is thought to affect the translation of the mRNA while the 3' UTR is thought to affect message stability... while either is still a possibility I would test whether transcripts are in vitro translated at the same rate... start with in vitro systems (make the mRNA and then translate with a reticulocyte lysate or something like that... for species specific you can make expression vectors for both forms and then assay in a cell line from your species... You may be able to accomplish this by replacing the coding region with luciferase to assay (assuming interaction with the coding region of your gene is not important for the effect)... From there you can do deletions etc to find out what seqs are important etc if you want...
you could try a reporter system where you couple your sequence upstream of a reporter (say GFP or Luciferase), transfect and then use either FACS (flow cytometry) or luciferase assay to determine whether your sequences effect the translatability of the reporter.....