PCR labelling of probes
Charlie Wright Genetics
cw117 at mole.bio.cam.ac.uk
Tue May 30 07:21:58 EST 1995
dbarton at acer.gen.tcd.ie (Dr David E Barton) writes:
>Sandra Munro <smunro at cs.mcgill.ca> wrote:
>>Another favour - can someone provide me with a method of P32-labelling a
>>DNA probe by PCR, or a reference for the method?
> PCR labelling with 32P doesn't work very well, in my experience. I believe
> that this is because the Taq has a higher Km for dNTPs than say Klenow, so
> it doesn't cope well with the very low concentrations of labelled dNTP found
> in a labeling reaction.
> \|/ ____ \|/ | David Barton
I'm not sure about the concentration's effect, but I do know that the
protocols I have used (with variable success) for DIG-labelling probes
with PCR overcome this problem by using DIG-dUTP at a typical labelled
nucleotide concentration and then balancing the concentrations with dTTP in
the labelling mix. So you have dATP, dCTP, dGTP at standard PCR
concentrations, dTTP at about 2/3 of this, and DIG-dUTP at about 1/3 this.
It seems to work alright with DIG single stranded preparation.
Caveat: DIG-strands make very poor templates, so it is not very useful
for labelling both strands in a two-primer reaction, yield drops
dramatically, and is effectively a linnear amplification (much like the
C. R. Wright Dept. of Genetics
+44 (0)1223 333970 telephone Univ. of Cambridge
+44 (0)1223 333992 telefax Downing Street, Cambs.
cw117 at mole.bio.cam.ac.uk CB2 3EH, England
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