How error prone PCR can be run?

Error-prone PCR. Error prone PCR is a method by which random mutants maybe inserted into any piece of DNA. 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.

What is error prone DNA?

Error-prone DNA polymerases are characterized by probabilities of base substitution or frameshift mutations ranging from 10−3 to 7.5 · 10−1 in an intact DNA, whereas the spontaneous mutagenesis rate per replicated nucleotide varies between 10−10 and 10−12.

How do you do random mutagenesis?

2.1. Random mutagenesis can also be accomplished by insertion or deletion of nucleotides from a target gene sequence. Random insertion or deletion leads to a net change in the length of the gene of interest, opening a new realm of diversity that cannot be reached by point mutation alone.

How are error prone PCR libraries used in directed evolution?

Error-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway ® technology allows constructing epPCR libraries virtually devoid of any background (i.e., of insert-free plasmid), but requires two steps: the BP and the LR reactions and the associated E. coli cell transformations and plasmid purifications.

How is error prone PCR used in mutagenesis?

The 130loop+190helix or 190helix+220loop in hemagglutinin gene of influenza A/California/04/09 (H1N1) virus are amplified by error-prone PCR (epPCR) by using a GeneMorph II Random Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) (step 1). epPCR products are then used as primers in the site-directed mutagenesis (step 2).

How does the PCR protocol reduce mutational bias?

This protocol reduces mutational bias often associated with error-prone PCR methods and allows the experimenter to control the degree of mutagenesis by controlling the number of gene-doubling events that occur in the PCR reaction.

How is a random library of PCR mutants generated?

A random library of mutants generated by error-prone PCR (epPCR) and/or DNA shuffling [ 2] is screened for variant proteins more soluble than the wild-type ( wt) protein. To that end, the mutated DNA sequences may be expressed as fusion proteins with a C-terminal “solubility reporter” such as the green fluorescent protein (GFP) [ 3 ].