Primer dimer-really serious?

NJ 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:

> hi,
> 
> 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?
> 
> thanks,
> 
> linan
> 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

NNNNNNNNNNNNNNNNNNCATG------------------>
<-----------------GTACNNNNNNNNNNNNNNNNNN

or

2. when both primers have sequences of several nuculeoties complementary to
each other at their 3' ends, like

NNNNNNNNNNNNNNNNNNATCG------------------>
<-----------------tagcnnnnnnnnnnnnnnnnnnn

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.
--
NJ




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