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Who is wrong with primer design? [long]

John Dixon jpcd100 at mole.bio.cam.ac.uk
Tue Aug 1 14:44:24 EST 2000

A while back Oswaldo (oswaldo at iats.csic.es) asked about the confusion
over whether primers for PCR should be made with a 3' GC clamp.

To recap:

On the one hand:

(from http://www.eppendorfsi.com/PCR_appl_primer.html) 

> (include)... a G or C residue at the 3' end of primers. This ³GC Clamp²
> helps to ensure correct binding at the 3' due to the stronger hydrogen
> bonding of G/C residues. It also helps to improve the efficiency of the
> reaction by minimizing any ³breathing² that might occur. 

And on the other:

(from "Theories and Formulas" of Net Primer"s help (Netprimer,
Biosoft's international)

> An ideal primer has a stable 5'end and an unstable 3' end. If the
> primer has a stable 3' end, it will bond to a site which is
> complementary to it other than the target with its 5' end hanging off
> the edge...[nice terminology BTW]...Stability of the 5' termini allows for efficient bonding of the
> primer to the target site. This stable 5' region is called the GC
> Clamp. It ensures adequate binding of the primer to the template...
> Notice that the 3' end should not be very stable and the 5' end should
> have a strong GC clamp. 

I remember Oligo (Mol Bio Insights) saying this in its manual too. So I
did a search of a few of the likely sources of info, to see whether
there was a consensus opinion. Below are abridged summaries of what I
could find, while putting off actually doing any PCRs.


U Nebraska Medical Centre:

> 45-55% G+C
> Include G/C Clamp at the 3' End 1-2 G/C nucleotides. The 3' end of the
> primer should be G or C

U Capetown: http://www.uct.ac.za/microbiology/pcroptim.htm

> 50-60% (G+C); 
> Primers should end (3') in a G or C, or CG or GC: this prevents
> "breathing" of ends and increases efficiency of priming; 
> Runs of three or more Cs or Gs at the 3'-ends of primers may promote
> mispriming at G or C-rich sequences (because of stability of
> annealing), and should be avoided. 


Clontech: http://www.clontech.com/techinfo/manuals/index.html#g

> GC-content of 40­60%
> No more than three G¹s and C¹s in the last six positions at
> the 3' end of the primer

Eppendorf: http://www.eppendorfsi.com/PCR_appl_primer.html

> 45% and 55% GC.
> No PolyG or Poly C stretches that can promote non-specific annealing.
> Poly A and Poly T stretches are also to be avoided as these will
> "breath" and open up stretches of the primer-template complex. This can
> lower the efficiency of amplification. Polypyrimidine (T, C) and
> polypurine (A, G) stretches should also be avoided. Ideally the primer
> will have a near random mix of nucleotides a 50% GC content...It is
> well established that the 3' terminal position in PCR primers is
> essential for the control of mis-priming [ref]. Inclusion of a G or C
> residue at the 3' end of primers... helps to ensure correct binding at
> the 3' end due to the stronger hydrogen bonding of G/C residues. It
> also helps to improve the efficiency of the reaction by minimizing any
> "breathing" that might occur. 

Invitrogen: http://www.invitrogen.com/pdf_manuals/pcroptim_man.pdf

> 50 to 60% GC
> avoid runs of of 3 G's or C's at the 3´ end

Life Technologies: http://www.lifetech.com/

> 40% to 60% GC
> Avoid a GC-rich 3´ end.
> Design primers with G or C residues in the 5´ and central regions.


> 40-60% G+C   
> Avoid three G or C nucleotides in a row near the 3´-end of the primer

Roche: http://biochem.roche.com/prodinfo_fst.htm?/pcr/

> 40­60% G/C.
> Have a balanced distribution of G/C and A/T rich domains.

Lastly, here is my personal favourite (for entertainment not education,
as it summarizes all the above confusion nicely):

Alkami: http://www.alkami.com/primers/refprmr.htm

who, to be fair, first disclaim:

> The guidelines listed below are from a variety of origins and the
> ranges listed are the extreme values from all sources. 

but then produce...

> Primer Ends: 
> Start and end primers with 1-2 purine bases. 


> Try to avoid Gs and Cs at the 3' ends 

err...so that leaves 3' A's then...

...and finish with...

> The primer should be able to form "G/C" clamps 

...at an unspecified location, presumably the middle. 

Well, thanks. That's really cleared it up then. 

Good luck, Oswaldo.

JD (no affils to any of above, obviously. all errors my own etc. etc.)

P.S. FWIW, my preferred offerings to the PCR gods are primers with a
single G or C 3', FWIW. Seems to keep'em happy most phases of the moon.

John Dixon                    Lab 44 (1223) 334131
Wellcome/CRC Institute        Fax 44 (1223) 334089
Cambridge University
United Kingdom CB2 1QR       e-m: jpcd100 at mole.bio.cam.ac.uk

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