Mapping random mutagenesis

[Peter Wang]

Benjamin Braun (bbraun at ucla.edu) wrote:
>I am thinking of a project that would entail random mutagenesis of
>a 300-1500 bp promoter fragment.  My question is, is there any way
>to identify where a mutation lies to within a few base pairs short
>of sequencing the construct?  That way I could assess an entire
>population of mutants in one reaction - to assess the randomness
>of mutation, and after a genetic selection to look for clusters of
>mutations.  For example, I was considering using RNase protection
>against a library of mutants.  In an ideal mutagenesis I would get
>cleavage at each base with equal frequency, but hot spots would
>show greater cleavage frequency, and therefore darker bands.
>Linker insertion would be great, but I don't know of an easy way 
>to get good distribution of the linkers.
>
>Any ideas out there?  I'd love to hear some!

Benjamin:

My disclaimer for the ideas below is that I don't have any personal 
experience with the methods, but have been looking into them for uses 
similar to what you have in mind (mapping mutations in selected 
populations of DNAs).

Similar to your idea of RNAse protection (RP) of a selected population, 
you might look into chemical cleavage of mismatches (CCM) which probably 
would work better than RP for what you have in mind.  You can look for 
reviews by Richard G. H. Cotton (I think the original reference is PNAS 
85:4397 (1988), but there is probably more recent stuff too).  
Heteroduplexes between the wildtype DNA and mutant DNA (made by PCR) are 
cleaved by chemical reagents like hydroxylamine that specifically attack 
unpaired bases, and the location of the mutation can be estimated by 
sizing the fragments on a denaturing gel (DNAs are radioactively 
labeled).

A very elegant way to use CCM, if you have access to an ABI automated 
sequencer, is fluorescence-assisted mismatch analysis (FAMA) (PNAS 
91:1873 1994).  I heard a very nice talk by Tommy Meo about this and he 
would be very helpful if you were interested, I think.

Similarly, there are a couple of papers on using enzymes to cleave at 
mismatches (Nature Genetics 9:177 (1995) and PNAS 92:87 (1995)), though 
I think CCM is the gold standard.

Cheers,
- Peter

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Peter Wang, M.D., Ph.D.
MRC Centre for Protein Engineering,
Hills Road, Cambridge, CB2 2QH, England

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