heterologous hybridisation (some principles)

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Mon Dec 19 13:26:48 EST 1994


Marc Van De Craen writes:

> I need to do hybridisations with probes of about 100 bp and 20 bp on 
> heterologous target dna's.  The expected HOMOLOGY between the probes and the 
> target is about 60%.  I realise that the 20 bp probe probably won't work but 
> there has to be a nice PROTOCOL for the 100 bp probe.  

Beyond what's in the standard manuals I can give you two suggestions.

1. Typical hybridization conditions call for hybridization under moderate
   stringency (down to about 75% identity) and then refine the stringency
   with the wash steps.  To detect 60% identity, you'll have to adjust
   stringency of both the hybridization and the washes. 
2. To label a fragment of only 100 bp, it may help to self ligate it
   first to make a concatemer.  I know this helps with nick translation;
   and I would guess it will help with random hexamer priming.

> I have to do 
> this by colony hybridisation,  so I will have a lot DNA on my blots (HYBOND N 
> nylon membranes (AMERSHAM)).  As a possitive control I will use the homologous 
> target.   Is it possible to discriminate the homologous target and the 
> heterologous target just by using two consecutive washing steps?  

Yes; just don't let the filters dry out inbetween.  Your major problem will
be in discriminating the low stringency signal from random matching.  You 
may be happier if you have two probes from different places in the sequence
so that you can use being double positive as a criterion for a meaningful
signal.

Hope this helps.
Steve Hardies, Dept. of Biochemistry, Univ. of Texas HSC at San Antonio
Hardies at thorin.uthscsa.edu
 



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