Before PCR-based amplification can be carried out, a pair of primers that selectively bind to the target DNA sequence has to be designed. Although the process of designing primers is relatively straightforward, there are often PCR specificity issues leading to either no PCR amplicon, or mis-priming which results in the wrong amplicon being amplified. Frequently, these errors are due to mismatch errors between the primer and the target DNA template. Hence not only is primer uniqueness critical, but ensuring that there are no close homologues of the primer after introducing mismatches is equally important.

SynaHybridise is a high speed web-based tool which analyses the specificity of primer sequences by progressive introduction of mismatches. Ideally, the primer would be complementary only to the target sequence, even if one or two mismatches are introduced. A poor primer would have multiple template matches after the introduction of mismatches.

As an example, two primers for an α–Thalassemia PCR using the Human Genome as a template were designed.

To analyse the specificity of these primers, please follow the steps below:

Step 1 of 2 To verify primer sequence 1, click here and submit query.

The result shows that primer sequence 1 is specific, with only one possible binding site in the genome, even after the introduction of 2, 3 or 4 mismatches.

Step 2 of 2 To verify primer sequence 2, click here and submit query.

The result shows that primer sequence 2 reported 3 hits to other locations on the Human Genome when 3 mismatches were introduced. Hence it can be concluded that primer sequence 1 is more likely to result in the desired amplicon compared to primer sequence 2 which is likely to lead to multiple false PCR amplicons from other locations on the genome.