genotyping SCID and Nu/Nu mice

David de Graaf degraaf at genome.wi.mit.edu
Thu Jan 29 18:28:44 EST 1998


In article <Pine.SOL.3.95.980129213453.10276B-100000 at Hermes.nki.nl>, Jorg
Kirberg <kirberg at nki.nl> wrote:

> On Thu, 29 Jan 1998, David de Graaf wrote:
> > 
> > If one base is missing, wouldn't this be the ideal place to use LCR? Even
> > plain PCR with the deletion as the last base should work without a hitch.
> > The product can then be tiny. At the same time, a positive control can be
> > amplified from a region close by to make sure that there is nothing wrong
> > with the DNA prep.
> > 
> Dear David,
> yes, you are right about that. But I did't know how robust screening
> this way would be to identify the heterozygous ones.
> 
> jorg

Well, one thing you could do is to design two oligos of different lenght
or with different tags, one matching the deletion and one matching
wildtype. If the lenght difference is minimal, competition should be
minimal. It does depend on whether hyb conditions or secondary structures
are very different for the two oligos. give them a length difference of 3
bp and you should be able to pick it up on a high percentage special
agaroser gel (the brandname escapes me). This is basically a modification
of allele specific PCR, a routine technique.

David



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