Cloning Isoforms - (Nov/03/2004 )
The gene I am interested in has 3 isoforms that we wish to clone the cDNAs.
Isoform 1 has the same sequence as isoform 2, but isoform 2 has an insert in the middle making the cDNA about 200 bases longer. We are using primers to amplify isoform 2 and they also amplify isoform 1 (there is nothing unique with isoform 1). When we do a RT-PCR, we get two bands on the gel of the correct sizes corresponding to both isoforms. However if we isolate isoform 2 from the gel and re-PCR for cloning it amplifys as the size of isoform 1.
We have also tried direct cloning of the RT-PCR product and we only see isoform 1 in the clones.
Any advice on how to clone isoform 2 would be greatly appreciated.
I know its quite an old post and probably you dont read this message any more at all.
But we are trying to do something very similar like you desribed (trying to clone isoforms from cDNA). So if your experiment worked out in the meantime, I would be very interested in how you did it at least and would really appreciate some tips !
Hoping for some reply ...