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Error Prone PCR
Error-prone Polymerase Chain Reaction (EP-PCR) is a simple in vitro procedure used for the creation of mutagenic DNA sequences. Error-prone PCR is commonly applied for the directed evolution of proteins, being a step used to synthesize mutant libraries. EP-PCR works through a myriad of similar means to direct point mutations at a higher-than-normal rate into a target DNA sequence during replication. Increasing the normal mutation rate can be done by the utilization of: mutagenic (overhanging) primers, improper reaction conditions, or error-prone polymerases.

History
Directed evolution and its practices has lead to many advancements in the field of protein design across medicine, industry, and academia. Medical benefits brought forth from the directed evolution of proteins includes: the development of pharmaceuticals and vaccines, stabilizing antibody fragments, increasing the responsiveness of azidothymidine (AZT) for HIV treatment, and many more enzymatic alterations. Error-prone PCR was first used purposefully for directed evolution experiments in the late 1980s. It was developed as a furtherance of the wealth of understanding classical geneticists and researchers unearthed discovering PCR. Since it's original use, many modifications and alternative methods have been created, however error-prone PCR still remains a powerful tool.

Methodology
Error-prone PCR relies on the traditional in-vitro polymerase chain reaction protocol with slight differences. The major difference between traditional PCR and EP-PCR is that the later introduces many more mutations into the sequence. The fidelity of replication can be altered by a number of methodological differences, including:


 * 1) Mutagenic (overhanging) primers
 * 2) Low fidelity DNA Polymerase
 * 3) Mutagenic reaction conditions



Mutagenic Primers
Error-prone PCR can be done in vitro through multiple methodologies, one methodology uses mutagenic (overhanging) primers to deliver mutagenesis to normal PCR. Mutagenic primers are RNA oligomers that are able to prime the target sequence but leave a single-stranded overhang. The lack of a template sequence across from the mutagenic primers allows for DNA polymerase to elongate the primer, which is in itself a mutation. Normal polymerase chain reaction following the addition of mutagenic primers serves to amplify the resulting mutants.

Low Fidelity Polymerase
Another method of evoking mutagenesis is by using an error-prone DNA polymerase. An error-prone DNA polymerase differs from a ‘normal’ DNA polymerase in either its catalytic activity or enzyme kinetics. EP-polymerases may lack exonuclease activity causing them to inherently have a higher rate of mutation or they may have low substrate-specificity allowing more haphazard reactions to occur during synthesis. A polymerase without exonuclease activity is unable to excise bases that are mistakenly incorporated during synthesis and low substrate specificity would result in a promiscuous DNA polymerase with a higher inherent rate of mutation than high substrate specificity polymerases.

Mutagenic Reaction Conditions
Another method of forcing errors into normal PCR is by creating reaction conditions that favor mutagenesis. One way is by altering the levels of magnesium or manganese available in the reaction, commonly done by manipulations of magnesium chloride or manganese chloride. This is able to directly effect the efficacy of the DNA polymerase complex, as the divalent cations play essential roles in nucleotide transfer and base excision. In solution, divalent cations are able to bind to oxygens of the phosphate backbone of dNTPs with high affinity in solution. Increasing the cation concentrations decreases PCR fidelity by making it more difficult for DNA polymerase to bind to incoming dNTPs, decreasing the base selectivity, and promoting erroneous nucleotide incorporation.

The fidelity can also be decreased by unbalancing the concentrations of the dNTP substrates. In vivo, the ribonucleotide reductase as well as the mono- and diphosphate kinase enzymes help serve to balance the pool of dNTP substrate. Being as PCR happens without these substrate concentration stabilizing enzymes the dNTP levels are more susceptible to fluctuation. Decreasing the levels of one dNTP diminishes its incorporation into newly replicated strands, where the corresponding other pyrimidine or purine will supplement.