Need colorimetric assay to detect PCR products.

John R. Thompson jrt at intac.com
Mon Aug 7 22:23:08 EST 1995


ijmehr at merle.acns.nwu.edu (Ian J. Mehr) wrote:

>I am searching for colorimetric methods of detection for PCR products. 
>The assay need not be specific, as in DNA sequence, just a simple positive
>or negative result relating to the presence or absence of product.  This
>will facilitate the screening of thousands of PCR reactions I will be
>performing in search of a negative result.  It would be much easier (not
>to mention less time consuming) to place the reactions in a microtiter
>dish and have it screened by a plate reader, or visually if possible, than
>to screen every sample by agarose gel electrophoresis.  Any ideas would be
>appreciated.

>Ian J. Mehr
>ijm at nwu.edu

Biotin label one of the primers and the label the other primer with either
digoxigenin or fluorescein.  The PCR product is recovered on strepavidin
coated plates via the biotinylated primer. Then product is detected via
direct fluorescein fluorescence (if sensitive enough) or indirectly using
Anti-fluorescein (or Anti-digoxigenin) antibody conjugated with Alk. Phos.
or peroxidase and the appropriate chromogenic or chemiluminescent
substrate.

There's a reference to this but I don't have it handy.

Yours, 
John R. Thompson
Merck Research Labs






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