PCR Primer Information

Primer composition

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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