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PCR and Strand Displacement

Robert Preston rapr at MED.PITT.EDU
Thu Jul 28 08:01:23 EST 1994


> 
> 
> 
> According to the literature, Taq DNA polymerase does not possess strand displacement
> activity.  In other words, if the pol complex was extending on a large ss DNA
> template, which had a downstream duplex region (which is perfectly base paired) the
> enzyme would stop !
> 
> So, during PCR amplification of a large DNA fragment (>3,000 bp) at every heat
> denaturation step at 94C, are both DNA strands fully denatured and separated,
> so that primer cann anneal to its target sequence and thud yield PCR amplification ?
> 
> Or, are the two strands only partially denatured, allowing the primers access to their
> target site.  Is the this already displaced partial duplex amplifiable by Taq DNA pol ?
> 
> I would be very grateful for people's views on whether duplex DNA is completely denatured
> at every cycle in PCR or not.
> 
> Many thanks
> Mick
> 
OK, here's my several cents worth:  maybe Mick's question cuts to a core
(and I suppose there might be several different cores available) of PCR
efficiency limitations.  I imagine that no, Taq does not displace strands,
since there are technologies that rely on that fact for their success.
Also, it's kinetically impossible, especially in the last few PCR cycles
that set the overall yield, for product NOT to begin to anneal, let alone
hairpins if possible.  However, in a subsequent cycle, any partially
synthesized product, since it's long relative to primers, would stand a
good chance of reannealing and being finished, after all.  BUT, does
lack of strand displacement only apply to strands whose 5' end is tacked
down, complementary?? Will Taq displace strands whose 5' end is non-comple-
mentary, as in a limited hairpin?? I'd like to know that.

Rob Preston
rapr at med.pitt.edu




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