PCR for radiolabelling probes

[kvernick at helix.nih.gov]

Richard P. Phipps wrote:

> I once tried labeling a PCR product (350 bp) by reamplifying (PCR) the
> fragment in the presence of CTP, GTP, TTP and 32P-ATP.  However, this 
> procedure did not appear to yield a probe which was hotter than if this 
> fragment was randomly-labeled following a standard protocol.
> Could anyone enlighten me on the advantages
> /disadvantages of these two procedures?


PCR labelling works fine, you can achieve very high specific activity, but there are are 
some important considerations which affect specificity and spp activity. A protocol was 
reported (Schowal & Sommer (1989) Anal Biochem 177,90-94) which works well but if you 
are using a PCR product as a template for labelling, you must gel-purify it first. The 
article describes why. A recent optimization of PCR labelling was reported (BRL Focus 16, 
45-48) which I haven't tried yet, but it seems carefully done. They will also sell you 
their optimized system (ahem...) for a slight fee...

Ken Vernick
kvernick at helix.nih.gov