Gene copy number usig PCR
wgallin at gpu.srv.ualberta.ca
Wed Oct 11 11:27:16 EST 2000
Yes, in theory, but in practice there are some problems.
Using quantitative PCR to amplify a known amount of genomic DNA and
known amounts of cloned target will yield the number with minimal math
(I'll leave the obvious analysis to you).
The problem is that the genomic DNA and the plasmid DNA may not amplify
with the same exponent, usually because a whole genomic DNA prep will
have some/different inhibitors that the plasmid won't have.
To control for this you have to PCR dilutions of the plasmid, dilutions
of the template and combinations of the two. If you don't get a simple
sum of signal strength in the mixtures you have two options. 1) repurify
the genomic DNA and try it again or 2) Calculate a correction factor
based on the calculable depression of signal from the plasmid in the mixture.
Rodney Earl Pettway wrote:
> Is there a way to determine gene copy number by using a PCR based
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