PCR to make labeled probes

FrohlichM frohlichm at starbase1.caltech.edu
Tue Feb 1 02:53:34 EST 1994


In article <2ijnba$goj at ysics.physics.sunysb.edu>, S. Gupta
<Sugupta at ccmail.sunysb.edu> writes:

> Has anyone tried to make a 32P labeled DNA probe to do a library screen? 
> The size I am trying to amplify will be around 100bp.  Any suggestions
> will be appreciated.

You can make extremely hot probes if you do PCR in a very small volume with
one hot and three cold dNTPs.  If the total volume is small enough you do not
need to dilute the fourth, labeled, dNTP with cold stuff; the concentration
you get from the 32P labeled material is adequate.

We've used this very successfully for library screening and to probe Southern
blots.  I've gotten decent bands with exposures as short as two hours.

This is published now (Balakrishnan, R., M. W. Frohlich, P. T. Rahaim, K.
Bachman & R. R. Yocum.  1993.  J. Biological Chem. 268(33): 24792-24795). 
Here is the recipe from that paper:

Probe was made in a 10 microliter reaction containing the following reagents:

5.0 microliters of alpha 32P dATP stock solution (3000 Ci/mmol; Du Pont NEN
     NEG-012H; final dATP concentration 1.7 micromolar); 
1.5 microliter of dTTP, dCTP, dGTP mix, containing each at 125 micromolar 
1.0 microliter of Perkin-Elmer-Cetus PCR buffer I (equivalent to buffer II
plus
     15mM MgCl2); 
1.0 microliter of mixed primers, containing each at 10 micromolar; 
0.5 microliter of Taq polymerase mix, diluted to 1/10 concentration of stock 
     (0.25 units)
1.0 microliter of template solution, containing about 0.1 ng of template  
      (Template was PCR product from a previous (cold) amplification.)

The reaction was covered with 10 microliters of oil and run with the same
program we used on standard-scale amplifications.

We amplified products of 83 and ca. 500 bp.  For the 83 bp product we used
degenerate primers based on the amino acid sequence coded by the gene we
wanted to clone.  About 3/4 of the radioactivity was incorporated into the
PCR product.  We had previously done trial runs with cold dNTPs at the same
concentrations, volumes, etc. and visualized the products on agarose gels. 
We saw distinct bands of full-length products and a pronounced streak of
smaller partial products.  All of these, including the partial-length
products, should bind to the intended target.  The PCR protocols book by
Innis, et al, says that PCR can be done with dNTP concentrations as low as 2
micromolar, so we expected this method would work.

Two caveats: the PCR products should be diluted as soon as possible after the
PCR reaction is finished (by putting it through a sephadex column, for
example) so the PCR products aren't damaged by storage in the small volume
containing highly concentrated radioisotope.  Also, these probes will break
apart into short fragments more quickly than probes of lower specific
radioactivity, because these have such a high proportion of labeled
phosphates.  Don't expect to get any use out of probes that are a few weeks
old.

Good luck in probings!

Michael Frohlich




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