Differential PCR based on 3' mismatch

John Ladasky ladasky at leland.Stanford.EDU
Thu Jun 12 00:36:44 EST 1997

In article <9706128660.AA866090364 at smtpgwy.agric.nsw.gov.au>,
Deborah Hailstones <deborah.hailstones at SMTPGWY.AGRIC.NSW.GOV.AU> wrote:
>     Dear all,
>     We have been trying to use PCR to differentiate between two closely 
>     related viroids, which differ from eachother by only a few 
>     nucleotides.  We have designed PCR primers to these regions, placing 
>     the differences at the 3' ends of the primers, to try to distinguish 
>     between the viroids.  Sounds great on paper but the primers don't 
>     actually seem to distinguish between them at annealing temp Tm - 5 
>     oC.... it seems very odd that there is no specificity. Surely this 
>     runs against the accepted wisdom of how PCR works?  Does any one out 
>     there have experience wiht this sort of assay? we are at a loss to 
>     explain it...

	I've designed a few of these allele-specific PCR's.  False posi-
tives are a problem when you only have a single mismatch between the primer
and the template.  Taq polymerase *can* extend single-base mismatches --
that's how errors are incorporated in PCR.  Remember, the mismatch exten-
sion need only occur in the first cycle in order to generate a problem.
Then when the reverse reaction occurs in the next cycle, the new template
*is* a perfect match for your primer and will extend without hindrance.

	There are two things I do in order to obtain clean allele-specific
PCR.  First, I choose the allele-specific primer so that the terminal penta-
nucleotide is the more AT-rich of the two.  The idea is to have a particu-
larly unstable 3' end that requires a perfect match for specificity.  Obvi-
ously, you only have two choices for your primer's 3' end -- forward or re-
verse, ending at the polymorphic nucleotide.  

	Remember too that some mismatches extend better than others and are
hard to test.  For example, if you want your primer to end in C and you 
are probing a C/A polymorphic site, discrimination is poor.  The C-primer,
T-template (template is the reverse strand) mismatch extends relatively well.
If you are not assaying a heterozygous sample, one option here would be to
design the primer that ends in A instead. The A-primer, G-template mismatch
extends quite poorly.  Another choice would be to use the reverse primer,
ending in G and probing a G/T polymorphism.  

	The second thing I do is to change one nucleotide in the penulti-
mate pentanucleotide so that it will mismatch *both* templates.  In other
words, if my allele-specific primer is a 20-mer, I will deliberately mis-
match one position between nucleotides 15 to 19.  This has the effect of
lowering the 3' end stability even further.  Pay careful attention to the
possibility of primer dimers and hairpins.  If possible, kill two birds with
one stone.  When you introduce the deliberate mismatch, choose it so that it
disrupts the dimer/hairpin.

	Useful references include:

	1. Newton CR et. al., NAR 17:2503 [1989]

	2. Wu DY et. al., PNAS 86:2757 [1989]

	3. Cha RA et. al., PCR Methods and Applications 2:14 [1992]

	4. _PCR_Strategies_, ed. Michael Innis, D.H. Gelfland, J.J. Sninsky,
	   Academic Press, San Diego, CA [1995]  Chapter 2.

	Good luck!

Unique ID : Ladasky, John Joseph Jr.
Title     : BA Biochemistry, U.C. Berkeley, 1989  (Ph.D. perhaps 1998???)
Location  : Stanford University, Dept. of Structural Biology
Keywords  : immunology, music, running, Green

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