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HotStarTaq PCR Handbook

primers can be used. These are actually mixtures of several primers whose sequences differ at the positions that correspond to the uncertainties in the template sequence. Hot-start PCR using HotStarTaq DNA Polymerase often improves the specificity of PCR amplifications that employ degenerate primers by reducing the formation of nonspecific PCR products and primerdimers. Table 8 gives recommendations for further optimizing PCR using degenerate primers. Table 9 shows the codon redundancy of each amino acid.

Table 8. Guidelines for design and use of degenerate primers

Sequence: Avoid degeneracy in the 3 nucleotides at the 3' end.
If possible, use Met- or Trp-encoding triplets at the 3' end.
To increase primertemplate binding efficiency, reduce degeneracy by allowing mismatches between the primer and template, especially towards the 5' end (but not at the 3' end).
Try to design primers with less than 4-fold degeneracy at any given position. Concentration: Begin PCR with a primer concentration of 0.2 M.
In case of poor PCR efficiency, increase primer concentrations in increments of 0.25 M until satisfactory results are obtained.
Table 9. Codon redundancy


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