Primers that contain a skewed AT/GC ratio can fail to give high
specificity, or yield primers that are in other ways not well
behaved. The user is allowed to enter minimum, optimal, and maximum
values for the percentage of basepairs which are either G or C.
Primer melting temperature
Melting temperature heavily influence the results of PCR. This
utility calculates the Tm of an oligonucleotide by the nearest
neighbor method (Borer, et al. J. Mol., Biol. 86; 843-53, 1974),
(Rychlik, et al. NAR, 18; 6409-12, 1990), and Breslauer, et al. PNAS,
83; 3746-50, 1986. Minimum, optimum, and maximum values are able to
be user customixed.
Primer Annealing
Primers also tend to dimerize and anneal to themselves, this can
present significant problems in using PCR. One method for accounting
for this problem was developed by Hillier and Green, PCR
Method. Applic., 1; 124-8, 1991. Maximum values annealing between
primers are able to be set by the user.
Ranking the pairs of primers
Primers are assigned a value based on their features and based on the
user defined preferences. The best pair of primers is defined to be
the pair of primers with the lowest score. This score is calculated
in the following way: +1 per 10% difference from the optimal GC
percentage, +1 per degree Celsius difference from the optimal Tm, +1
per 5 units of annealing at the end of primers, +1 per 10 units of
annealing in the middle of primers, and +1 per 2 basepairs difference
from the optimal length.
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