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functional analysis of intron mutations - (Dec/07/2009 )

Hi all,

could anyone advise me, if exists any assay for functional analysis of intron mutation - I need to find out if intron mutation has influence on splicing. I know there are some assays for promoter mutation (luciferase reporter) and dont know if it possible to use it for intron mutation.


Thanks!

-krst-

hi,
I have done the assays for intronic mutation.....first let me know where ur muation lies....near the splice site or deep intronic.Also use some prediction software like Netgene2 to check whether it affects or not.

-HMG-

Hi,
I have mutations near splice site, I used prediction programs but now I have to find out if it could be verified by experiment
I found some minigene assays, but I dont understand it well, for example- if it can be used for any length of intron ...


thanks for any advice!

-krst-

hi,

yes u r right we can use minigene constructs to evaluate the effect of intronic mutations near splice site.I have done it. just clone the twoexons and the intervening intron in a mammalian expression vector-i did in pcDNA3.1. sequence confirm it. transfect the cells with wild type and mutant construct and prepare RNA after 36hrs.then do a RT and then PCR on this cDNA using vector specific primers.......for pcDNA3.1-primer pair is T7F and BGHR.run them on gel for wild type transcript size and that of predicted mutant. It works perfect and clean result.

if u still need help...write to me...i will upload a paper for u which has all these.

-HMG-

Hi,

I will be very thankful for the paper!
Still a lot of questions...can you clone exons and the intron of any length? For deep intronic mutation, can you use it too?

thanks a lot!!!

-krst-

Hi,

i am attaching the paper.have a look.

yes u can clone two exons and their intron-this is called minigene and for splicing it uses cell's machinery. u can even do with deep intronic mutations but with modifications. i had cloned 2.7kb intron along with the exons.no problems.

which univ r u in?

-HMG-

i have a problem in uploading.....

this is the paper
Splicing in action: assessing disease causing sequence changes. D Baralle, M Baralle.J Med Genet 2005;42:737748. doi: 10.1136/jmg.2004.029538

-HMG-

Thanks!!!

Im not in a university, Im working in a hospital, mostly doing molecular diagnostics - mainly DNA, RNA sequencing and sometimes a little of "research" .

-krst-