change not to hot
nvahsen at notmail.com
Wed Oct 2 13:19:44 EST 2002
first I wouldn't be too suspicious about any primer with a hairpin. It
may still work better than you expect, or at least it should work
after hotstart (because after hotstart there will be no more chance
for the harpin to form if you have stringent annealing conditions).
Second: Get yourself MeltCalc anyway (http:www.meltcalc.com). This
software is freeware for non-commercial use. It calculates exact
primer Tms even considering mismatches. For description see Schütz and
von Ahsen (1999): Spreadsheet software for thermodynamic melting point
prediction of oligonucleotide hybridization with and without
mismatches. Biotechniques 27:1218-1224
You can then calculate the Tm for the mismatched primer and add as
many upstream bases as you need to reach your favorite Tm.
In the software use 0.5 µM Oligo concentration and 200 mM sodium
equivalents for approximating general PCR conditions. For a further
discussion see von Ahsen,N; Wittwer,CT und Schütz,E (2001):
Oligonucleotide melting temperatures under PCR conditions: nearest-
neighbor corrections for Mg(2+), deoxynucleotide triphosphate, and
dimethyl sulfoxide concentrations with comparison to alternative
empirical formulas. Clin Chem 47:1956-1961
The reference may also be interesting for all those that still believe
the Wallace rule (2*AT+4*GC=Tm) is good for something. A much better
is given there.
With best regards
University Hospital Goettingen
Dept. Klinische Chemie/Clinical Chemistry
Dr. med. Nicolas von Ahsen
On 2 Oct 2002 08:02:22 -0700, bonehammer73 at yahoo.com (Bonehammer)
>Hello everyone. I wonder if anyone could help me with an issue.
>I am designing a set of primers to work in a multiplex PCR. So far, I
>have managed to keep all four primers with a good GC/AT balance,
>similar Tm, etc., but one of the primers has a strong tendency to form
>3'- primer dimers (we're talking 8 bp). I have managed to overcome
>this by introducing a mismatch (G instead of T) but what impact will
>this have on the annealing temperature of the primer? Should I add one
>or more bases, and if so, to which side? The mismatch is at the sixth
>base counting from the 5', and the primer is 25 bases long.
>Thanks in advance - I'd hate to order a primer that has no chances of
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