Hi Alexander,

I can't speak to AAV vectors exactly, but I have worked on challenging GC-rich templates before. At the end of the day, site-directed mutagenesis is just a PCR reaction with a funky primer, thus the standard terms and conditions of PCR apply. This means that strategies that work for other high GC content template-based PCR should theoretically work, including: 1) using a higher melting temperature to melt out secondary structures better (be careful not to denature your enzyme though, some polymerases can handle very high T, some can't), 2) using longer extension step to give the polymerase time to overcome its stalling on the GC rich region (one time I had to use 12 minute extension, but then it worked beautifully!), 3) adjusting the Mg2+ concentration, 4) adding adjuvants such as DMSO. I've attached a little article on some of these strategies. 

For PCRing through AAV backbone, the same points as for the site-directed mutagenesis apply here. You may have to modify some conditions, but in theory you should be able to get it to work. Of course, make sure to sequence verify your vector after changes to make sure you haven't introduced any errors before proceeding to downstream applications.

Best of luck!

http://bitesizebio.com/24002/problems-amplifying-gc-rich-regions-problem-solved/

Hello Alexander - I performed such mutations on AAV vectors (AAV-GFP-shRNA) from stratagene without any problems using the appropriate Taq polymerase (High Fidelity/Proof reading Taq) and standard PCR conditions with DMSO supplement. The mutated vectors were used to knockdown gene expression in vivo and they worked perfectly fine. Don't worry give it a try.

Best of luck

I personally avoid methods involving long PCRs (like Stratagene kits). The LTRs will be hard to sequence. My favorite trick to remove restriction site is to cut it and insert a short compatible oligo, designed so that the restriction site is not recreated after the ligation. On the oligo you can put a few other restrictions sites if you want.

As for the PCR-based cloning, as long as you can sequence everything, it's fine. I never tried Gibson Assembly. Some sequences can give hard time during PCR attempts. My advice: use PCR only when unavoidable and make restriction enzymes your friends.

Cheers,

Lech

Years ago I tried site directed mutagenesis on AAV plasmids with out much success. Newer enzymes may work better now but the TRs were the problem before I believe.

Hi Alexander,

I once tried to do site-directed mutagenesis on an AAV2-ITR containing plasmid (using the "Quikchange II" kit), which failed but worked very smoothly on the basically same plasmid that lacked ITRs. So I indeed believe that mutant strand replication is an issue with ITR-containing plasmids! Nevertheless, good luck!