Choosing NNK or NNS as a degenerate primer for saturation mutagenesis

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Instead of the NNK codon for saturation mutagenesis, one can use the NNS codon. Is one a better choice? Maybe. K represents G or T, while S represents G or C. The sixteen codons that contain G in the last position are identical in both choices of degenerate codon. The remaining sixteen codons are NNT for NNK degenerate codon, but are NNC for NNS degenerate codon. These sixteen codons are synonymous, so they encode the same amino acids. They do differ at the level of nucleotides - the NNS-containing primer has a higher GC content. While the NNK degenerate primer contains five rare codons, it includes synonymous non-rare codons. In contrast, the NNS degenerate primer lacks a synonymous non-rare codon for arginine, so variants with this amino acid substitution may express poorly. (This potential problem only occurs in yeast; no problems predicted for expression in E. coli.) The melting temperature of the NNS degenerate primer will be slightly higher than the NNK degenerate primer, which, depending on the partner primer, may be an advantage. Choosing between NNK and NNS may also minimize formation of primer hairpins or primer dimers.

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This page contains a single entry by rjk published on June 4, 2013 12:19 AM.

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