PCR Mutagenesis

micprf at lure.latrobe.edu.au micprf at lure.latrobe.edu.au
Mon Nov 30 08:41:59 EST 1992

In article <mxdq3j#@lynx.unm.edu>, bhjelle at carina.unm.edu () writes:
> I encountered an impressively high level of mutagenesis (14 
> errors out of 411bp) in amplifying a segment of DNA from
> a plasmid clone. All mutations involved T or A in some
> manner. 11 were point mutations (transitions and
> transversions), but 3 were 1 bp insertions or deletions.

I also found high rates of random mutagenesis in a PCR cloning -
it was a nested inverse PCR cloning from Dictyostelium genomic DNA.
I had about 1 mutation in 300-400 bases, usually a T->C base substitution
as I recall. I had relatively high MgCl2 (~3.5 mM from memory) and
assumed this was the reason as I think I recall reading somewhere
that Taq fidelity can decrease at high magnesium. I also thought
it could be useful for deliverate mutagenesis ... seems like others
have thought the same way. In one case I had a stretch of 15 T's
converted into a run of 19 T's. I thought this could have arisen
from partial strand separation and rehybridization with a 4 bp
loop in the stretch of Ts during a cycle. I was using 30 cycles.
Anyone else find similar things? Paul Fisher (Micro Dept. La Trobe Uni).

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