Error prone PCR
Error prone PCR is a method by which random mutants maybe inserted into any piece of DNA. The technique is based on the well founded PCR (polymerase chain reaction), which is a standard technique in many molecular biology laboratories. Normally the replication of DNA by the polymerase is extremely specific,The difference in error prone PCR is that the fidelity of the Taq DNA polymerase is modulated by alteration of the composition of the reaction buffer. In these conditions, the polymerase makes mistakes in the base paring during DNA synthesis that results in the introduction of errors in the newly synthesized complementary DNA strand. By carefully controlling the buffer composition the frequency of mis-incorporation of nucleotide bases, and therefore the number of errors introduced into the sequence may be regulated. In directed evolution experiments, the substitution frequency is normally controlled at around 1 - 3 base pair substitutions per kilobase of DNA.
For the technique to work properly, it is important to use a Taq DNA polymerase which does not have proof-reading ability. This proof-reading, or auto-correction of nucleotide sequence, is a property that is found in many commercially available Taq DNA polymerases. However, use of a proof-reading DNA polymerase is used in a error prone PCR reaction will result in the automatic correction of the mismatched nucleotides, and any mutations that were introduced during the reaction will be lost.