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single embryo PCR

Chris Jones & Deirdre Sumpter 4christopher.jones at nashville.com
Tue Jul 28 23:18:57 EST 1998


> In article <35B4BD4A.D73 at shum.huji.ac.il>, meravc at shum.huji.ac.il wrote:
> 
> > Does anybody happen to fortuitously have a reference to a protocol or 
> > an article describing single embryo PCR used to map a gene to a 
> > deficiency/deletion?

In article <m.champagne-2207981547570001 at ocarina.cellbio.duke.edu>,
m.champagne at cellbio.duke.edu wrote:

> if you need to find out if your gene is taken out by a specific deficiency
> stock, i would recommend doing the polytene squash technique in which
> you hybridize a digoxygenin probe.  Even though our lab is quite good at
> a variety of PCR techniques, it took me a while to figure how to get the
> conditions right to get consistent product out of my PCR controls (low DNA
> yield, false positives, false negatives...).
> 
> The end result you will get out of your single PCRs will not be as convincing
> either, it will be a statistical decision (1/4 of all embryos you tested
> shouldn't have your product...).  With the polytenes, the result is clear and
> obvious, as long as your deficiency is big enough to be mapped physically
> (ie:  has anyone defined the limits...  60E3-E7?).

FWIW, I've had just the problems you described trying to do just this:
determine whether a given deletion takes out my gene of interest. Controls
were okay, but one of the two deletion lines gives a high level of "dud"
eggs, which won't amplify with either control or experimental primers.

I've now put both deletions over balancers expressing beta-gal, but
staining so far has been spotty. This may or may not work out, so I may
also turn to squashes (or just skip it entirely). So, Merav, you might
want to try something like the beta-gal trick, or yellow or whatever, just
so you can reliably separate your homozygous-deletion eggs from the
others.

Chris Jones

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