competitive PCR

tweissen at my-deja.com tweissen at my-deja.com
Fri Jul 28 04:00:48 EST 2000


No I haven't tried it and rather wouldn't :-).
If you want to use competition as a way to quantitate
the initial amount of target copies in your sample you
will have to make sure that the competitor has similar
amplification efficiency, yet can easily be discriminated
from the target fragment.
Probably the best way of doing so is by introducing a
restriction site via a point mutation into the original
amplimer, but quantitative interpretation of the banding
patterns can be complicated (I can find you some
literature if you are interested in the details).
Nevertheless you will end up trying to
coamplify TWO difficult templates rather than one,
meaning that yields (and therefore sensitivity)
will be quite low!

Thomas


Thomas Weissensteiner
Edward Jenner Institute for Vaccine Research
Compton RG20 7NN, U.K.
T.:   0044 1653 577900 x3931
Fax: 0044 1635 577901
Edward Jenner Institute: http://www.jenner.ac.uk
PCR Jump Station: http://www.highveld.com/pcr.html

In article <8lpa2f$dak$1 at nnrp1.deja.com>,
  Andreas Firzinger <a_firzinger at my-deja.com> wrote:
> Hello,
>
> does anyone of you have experiences with competitive PCR of
> long DNA-fragments (such as 7000 to 8000 basepairs). Usually it
> is not easy to obtain such long fragments by PCR, so I
> assume that a competitive PCR with fragments of 7kBp and 8kBp
> would be rather difficult. Maybe someone of you already
> tried it and could tell me if she/he managed it and how.
>
> Thanks in advance for your hints,
>
> Andreas :)
>
> Sent via Deja.com http://www.deja.com/
> Before you buy.
>


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