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Note however that no computer program or rule-of-thumb assessment can accurately predict either success or failure of a primer. A primer that seems marginal may perform well, while another that appears to be flawless may not work at all. Avoid obvious problems, design the best primers you can, but in a pinch if you have few options, just try a few candidate primers, regardless of potential flaws.
V. Verify the site-specificity of the primer. Perform a sequence homology search (e.g. dot-plot homology comparison) through all known template sequence to check for alternative priming sites. Discard any primers that display 'significant' tendancy to bind to such sites. We can provide only rough guidelines as to what is 'significant'. Avoid primers where alternative sites are present with (1) more than 90% homology to the primary site or (2) more than 7 consecutive homologous nucleotides at the 3' end or (3) abundance greater than 5-fold higher than the intended priming site.
VI. Choosing among candidate primers. If at this point you have several candidate primers, you might select one or a few that are more A-T rich at the 3' end. These tend to be slightly more specific in action, according to some investigators. You may want to use more than one primer, maximizing the likelihood of success.
If you have no candidates that survived the criteria above, then you may be forced to relax the stringency of the selection requirements. Ultimately, the test of a good primer is only in its use, and cannot be accurately predicted by these simplistic rules-of-thumb.
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