Asymmetric PCR puzzle

B.L.Cohen gbga13 at udcf.gla.ac.uk
Wed May 14 09:27:07 EST 1997


I would appreciate suggestions that might enable me to conceptualize the
following puzzle and hence perhaps find a way around the problem.

We often use asymmetric PCR to produce single-stranded (SS) templates for
sequencing, old hat though that approach may be. With a number of genes
and varied primer pairs it has generally worked well, though occasionally
we encounter a taxon/gene/primer combination in which one strand (let's
say Watson) can be made readily under standard conditions, but the other
strand (Crick) is not synthesized.  

The problem cannot be due to priming site variation, because the SS-PCRs
are always done from a double-stranded (DS) PCR product that has been
purified either by ultrafiltration or by silica spin-column binding and
further DS amplification from that input DNA works efficiently (T-anneal =
55C; T-ext = 72C. Extension time not limiting, 25 cycles. Nucleotides,
Polymerase, etc. not limiting). Thus, when both primers are present in
equimolar concentrations, amplification proceeds in a perfectly standard
fashion.

When the concentration of one primer is reduced to zero in graded steps,
synthesis of one (Watson) restriction endonuclease-sensitive (DS) product
declines in proportion to the reduction in the minority primer and
synthesis of the desired restriction endonuclease-resistant (SS) product
occurs. The problem arises when trying to make the other strand:- DS
synthesis declines as before as minority primer is reduced from equimolar,
but SS product _does not_ appear. The majority primer in these
circumstances is an 18-mer, 50% G+C, T-anneal = 54 C by the GC=4, TA=2
rule. Its 3' bases are GGC, so it ought to bind well at that critical
point.

Wha could account for this result?

We have thought about possible self-complementary secondary structure in
the template strand (which could obviously differ between Watson & Crick),
but it is hard to see how this could block SS synthesis and yet have no
apparent effect on DS synthesis. Moreover, in asymmetric amplification,
the copy-number of the two strands should never be less favourable to
non-self complementary, secondary structure-breaking interactions than in
DS amplification, and as SS accumulates non-self complementary ineractions
should be favoured by mass-action, restoring configuration of the template
towards what obtains during DS amplifications.

We have also tested many of the variables, including some that might
affect a secondary structure interaction, so far without relevant effect.
Amongst variables that have failed to permit synthesis of Crick are:
raising T-extension to 80C; increasing number of cycles to 40 (at 72 or
80C); decreasing T-anneal to 45C; increasing concentration of polymerase;
adding glycerol, formamide or DMSO separately or combined at varying
concentrations up to obviously inhibitory levels.

Ideas that will help the design of realistic hypothesis-based experiments
will be appreciated. Please DO NOT suggest changing primers! Nor
alternative sequencing strategies!

Thank you.

-- 
Bernie Cohen                   Phone (+44) (0)141 339 8855 ext. 5103/5101
Molecular Genetics              Fax               330 5994
University of Glasgow
56 Dumbarton Rd,
Glasgow G11 6NU
Scotland, UK.



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