The user may choose where in relation to the start and stop codons the primers are located. The default option will find primers in the first and last 35 basepairs of the DNA sequence entered. The user may define exact endpoints to start and stop the primer, thus the default option will allow amplification of a region whose endpoints are in the first and last 35 basepairs, while choosing exact enpoints will cause all primers evaluated to start and end with exactly the same sequence. Minimum, optimum, and maximum values are able to be user customixed. One method for accounting for this problem was developed by Hillier and Green, PCR Method.
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.