I would like to mutate a 226 bp fragment of dsDNA so that instead
of being about 50% GC, it becomes 75% GC. There appears to be
no good chemical way to do this large scale mutagenesis (compar-
able to, say, causing 100% C->T mutations in DNA with
bisulphite), and nifty PCR ways do't seem sloppy enough to do the
job (e.g. using biased proportions of nucleotides in PCR reactions
encouraged to misincorporate, e.g. in the presence of Mn, etc.) .
Any advice on making giant oligos? I'm thinking of ligating together
several large "mutated" oligos that cover the 226 bp region. The
oligos would be designed so that the 1st one corresponds to one
(top) strand of the fragment from position 1 to 50, the 2nd one
to the complementary (bottom) strand from position 40 to 90;
the third one would correspond again to the top strand from
position 80 to 130, etc. The oligos would be incubated together
so that the 3' end overlaps can anneal, and then they would be
primer extended and the nick left where the extension product
and the next primer meet up would be ligated. (Then maybe PCR
to amplify up the correct product if it is rare before cloning). Voila?
Anyone think this would not work? (Anyone think this would work?)
It would be great to make just 2 oligos, but what do you think is
the longest practical length to make oligos (e.g. with respect to yield
- I shouldn't need a lot, should we? Purity - would PAGE do it?).
If it isn't reasonable to make giant oligos, how about 5 or 6
50 or 60-mers? How much overlap would be reasonable? Should
assembly be done in steps, or should I let entropy rule the day,
and toss all participants into a cocktail, and sort out by size later?
Any references I should be reading whilst plotting this project?
Any comments? Thanks!