Duplicate regions in site-directed mutagenesis
hzhen at freeuk.com
Sat Dec 4 14:16:29 EST 2004
Paul Shinn wrote:
> I'm fixing several cDNA clones with point mutations using
> site directed mutagenesis. I'm using the Stratagene
> QuickChange protocol but using KOD HiFi polymerase
> rather than Pfu Ultra because I can get almost 100%
> product every time with KOD. After PCR I do a DpnI digest.
> All the clones I sequence have the point mutation fixed.
> However, a high percentage of the clones have the mutated
> region duplicated--exact copy of the primers used. It
> looks like a tandem region.
> I've seen only a little talk of this happening and why, but
> no real solutions to the problem. I synthesize the oligos
> myself and desalt them prior to use. Of the 4 clones I pick
> for each "fixit" reaction, all of them will have this duplication.
> KOD is very fast and for a 6kb plasmid, my extension time
> is only 40 seconds. After 23 cycles I get tons of product.
> However, I see the same problem with Pfu, too.
I have never seen this problem, and I have done numerous mutagenesis
using the Quikchange protocol. Are there sequences on the oligos that
may prime to each other, and do you do a hot start? Check the
duplicated sequence to see if the sequences are completely duplicated
or only partially duplicated, if it is the latter, then I'd think that
the oligos are annealing to each other.
> Any ideas? Thanks, Paul
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