exon enrichment by PCR for resequencing

[vasvale]

Hello all

I would like to enrich exons from a pool of genes to follow up with next gen resequencing.

I'd appreciate your suggestions on how to approach the design of primers. I could make small amplicons (how small would vary with platform?) and attach the adaptor sequences to the specific primers; or make large amplicons and fragment afterward. I'm not sure as yet if I'll use a 454 or other platform. In either case what would be the best course? I'd appreciate if you could point out papers on similar approach.

Thanks a lot

Vasvale

[swbarnes2]

The only system I know is Solexa, but I'm pretty sure that putting the adaptors on the PCR is a bad idea in any system. You will only get reads from the adaptors, so you would only get reads from the ends of your amplicons, and that's almost certainly not what you want.

So you do PCR, then you shred randomly, so that the adaptors go on in the middle of your amplicons.

There are site out there where you can give a large chunk of DNA, and it will design primers of overlapping apmplicons, but I don't know of a program publically available that will design only for exons.

You could do RNA, maybe some kind of pull down for the genes you want, then you'd only have exons.

Some exons can be quite small, so you are going to have to pad your exons with genomic DNA in a lot of cases. But having a more uniform size of PCR products might make the PCr mroe even anyway, so that's not really a problem.

[vasvale]

It has been done for 454 in HLA typing. So I guess for other platforms would need to fragment so I may just as well make big amplicons including more exons.