DNA qunatification by PCR - does it work?

mariaseg at hotmail.com mariaseg at hotmail.com
Wed Nov 1 06:03:46 EST 2000


I am interested in the application of PCR for DNA quantification.

I have tried other methods to quantify DNA, but they all lack the
sensitivity and accuracy I need for a downstream application.  The type
of samples I wish to quantify are cattle DNA. My idea is to amplify
these samples with a marker that is homozygous (to avoid problems with
preferential amplification) and then run them on a ABI 310 DNA
sequencer. I then plan on using the peak heights as a direct measure of
the amount of target DNA that was present originally. Other methods such
as fluoresence while being fairly accurate are nonetheless quite
variable. Optical density readings on a spec are useless for what I am
trying to do. I also feel that PCR will provide a good indication of the
 non-degraded amplifiable DNA (which is what is important) than total
DNA.

I would be grateful for any assistance in this regard. If say 10 ug of
DNA produces a peak height of 1000 units, would 20 ng produce exactly
twice that height? What about the PCR itself, do the number of cycles
have to be regulated such that it stays in the linear phase?

Thank you for all of your assistance,

Max
________
University of Melbourne
Australia

E-mail: mariaseg at hotmail.com


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