One-step generation of error-prone PCR libraries using Gateway® technology

One-step generation of error-prone PCR libraries using Gateway® technology

BACKGROUND 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. RESULTS We describe a method for making epPCR l...

Error-prone PCR and effective generation of gene variant libraries for directed evolution.

Any single-enzyme directed evolution strategy has two fundamental requirements: the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very l...

One-step random mutagenesis by error-prone rolling circle amplification.

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plas...

A general model of error-prone PCR.

In this paper, we generalize a previously-described model of the error-prone polymerase chain reaction (PCR) reaction to conditions of arbitrarily variable amplification efficiency and initial population size. Generalisation of the model to these conditions improves the correspondence to observed and expected behaviours of PCR, and restricts the extent to which the model may explore sequence sp...

One-Step Reverse Transcription PCR (RT-PCR). Fatemeh Mehrpooyan*