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      tomcunning
      Participant

      My question pertains to using Kunkel mutagenesis to create a library of sequences, where the codon(s) for all 20 amino acids are incorporated at a designated hotspot or hotspots (e.g, a codon in the CDR of an antibody). The general Kunkel protocol for site directed mutagenesis for changing codon A to codon B, I understand but not this random mutagenesis bit.

      How are degenerate codons (NNS) in the primers of Kunkel random mutatgenesis for library creation used to incorporate random mutations, coding for all other 19 amino acids, at selected "hotspot(s)"?

      Is this taking advantage of error-prone PCR, or some other strategy to increase the error-rate of the polymerase?
      Or, are a mixture of different primers used (not just two), each set coding for a different amino acid residue at the targeted hotspot(s)?

      Many thanks, in advance.

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