Primer dimer-really serious?
njuni at poppy.ocn.ne.jp
Fri Oct 5 02:28:45 EST 2001
in article 200110022316.SAA27167 at delphi.bsd.uchicago.edu, Linan Chen at
lichen at delphi.bsd.uchicago.edu wrote on 10/3/01 8:16 AM:
> Sounds very stupid. It is the common sense to avoid forming dimer when
> design PCR primers. But is it really serious that two primers for pcr form
> dimer? I doubt it because the high temp in the PCR cycle will sure destroy
> the dimer. Am I right?
> Chen, Linan
> University of Chicago
I don't think so.
First, let's think about how primer-dimers are formed.
Typical primer-dimers have head-to-head configurations of primer sequences.
This can be formed either:
1. when either primer has a self-complementary sequence of several
nuculeoties at its 3' end, like
2. when both primers have sequences of several nuculeoties complementary to
each other at their 3' ends, like
Annealing of such short nuculeoties should be unstable, but still be able to
prime polymerization by chance.
Then, my understanding is as follows:
1. Before PCR reaction is brought to high temperatures enough to dissociate
mis-annealed primers, extension reaction can proceed more or less even at
low temperatures (unless hot-start).
2. Because primers are added at high concentrations in PCR reaction, there
still remains good chance that transient primer-to-primer association occurs
even at high temperatures.
3. Once primer-dimers are, if not so often, formed for above reasons by
chance, they can be nice templates for PCR, having satisfactory
primer-annealing sequences and high efficiency of amplification because of
their shortness, which can defeat amplification of correct targets.
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