Tips How to increase your chances of returning primer resultsĭue to the way Geneious Prime and Primer3 communicate, it is worth simply increasing the number of pairs to generate if you are not returning any primers. Off-target primer pairs that result in amplicon sizes larger than that indicated in the fifth dropdown are considered as target primer candidates due to the decreasing efficiency of PCR as the amplicon size increases. Primer candidates with at least as many mismatches as indicated in the fourth dropdown are considered unlikely to bind to the off-target sequence(s) and will be accepted as primer candidates for the target sequence. Binding at the 3' end is required for initial annealing to the template. The third dropdown indicates the location of the mismatches. The value in the second dropdown is a subset of the first dropdown and gives more weight to the location of the mismatches, due to annealing chemistry. Primers with less mismatches than those entered in the first dropdown are considered to be likely to bind to off-target sequence(s) and excluded as primer candidates for the target sequence. If you are not sure what options to use, we suggest using the default options and tweaking from there. Any primers that are considered to have off-target binding are not returned as a primer annotation. The next section determines what you consider to be off-target binding. Configure your off-target binding settings Primers can be tested against a single sequence (including Alignments), list of sequences, or a folder containing multiple sequences.Ģ. Under Specificity Testing check the checkbox and Choose the off-target sequence(s) to test against. To design your target-specific primers, select your target sequence then choose Primers > Design New Primers. Select the sequence(s) you want to off-target test against How to use the Specificity Testing tool: 1. Primers are then designed (using Primer3) on areas of low similarity according to the specified criteria. The Specificity Testing tool uses Geneious BLAST to detect and exclude areas of high similarity between your target and the selected off-target database. Primer Specificity Testing enables you to design target specific primers and test their specificity against an off-target sequence(s) in a single step.
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