Polymerase Chain Reaction and Error-Prone PCR

PCR is normally used to increase the concentration of a given DNA sequence for analysis. For this primary use, the reagent concentrations and other conditions are carefully controlled to avoid the introduction of mistakes (mutations) during the copying process. 

In this reaction, a DNA sequence (template) up to 10,000 bases is used. DNA polymerase from a thermophilic bacterium is used. For example Taq-polymerase 

Error-prone PCR uses altered reaction conditions in order to increase the rate of production of copying errors. Taq-polymerase, when used in vitro, has an intrinsic error rate of about one noncomplementary nucleotide for every 10,000-100,000 bases (an error rate of 1 in 104 or 10s). In Table 8.2, conditions are described to increase the rate of mutations, where the error rate is increased by increasing the concentration of divalent cations (Mg2+ or Mn2+) and using increasingly unbalanced initial concentrations of nucleotides. Note that the error rate is tuned to between 2 and 15 errors for every 1000 base pairs, an increase of one to three orders of magnitude over the in vitro Taq-polymerase mutation rate. 

Some protocols generate random DNA mutations, but random mutations do not introduce a random incorporation of new amino acids in the expressed protein, which is the target molecule to be improved by directed evolution. During the translation process, every consecutive 3-base sequence (codon) codes for an amino acid or for a stop codon, where protein translation ends. Leucine and arginine are coded by six different codons, whereas only one codon exists for tryptophan. Most other amino acids are coded by two to four codons. The implications of multiple codons 

TABLE 8.2. Random Mutagenesis of Gene-Sized Fragments Using Error-Prone PCRa 

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