non-radioactive PCR probes

Dom Spinella dspinella at chugaibio.com
Fri May 2 10:07:38 EST 1997


Dr. Duncan Clark writes:
> Hi Folks,
> 
> Can anyone give me some idea what sort of ratio of biotin-dUTP to dTTP
> to use in a PCR for preparing a non-radioactive labelled probe. Any
> ideas on Cy3 or Cy5-dCTP to dCTP ratios or Cy3 or Cy5-dUTP to dTTP
> ratios would also be very helpful. Amersham couldn't really help on the
> Cy dyes other than to say try and it see.
> 
> Many thanks
> 
> Duncan 

Duncan:  I have had good results with PCR-generated biotinylated probes
using 0.2mM dATP, dCTP, and dGTP, 0.19 mM dTTP and 0.01 mM biotin-dUTP
(final reaction concentrations).  I don't know about the Cy dyes --
judging from experience with Taq cycle sequencing with dye terminators,
each dye is different with respect to the efficiency with which a
dye-labeled nucleotide is incorporated into a nascent chain by Taq
polymerase. Maybe the 5% ratio that works for biotin is a good place to
start, but I suspect you'll just have to empirically determine the best
ratio for the dye and nucleotide you are using for labeling.  Cheers. --
D.G. Spinella



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