One-Tube-Only Standardized Site-Directed Mutagenesis: An Alternative Approach to Generate Amino Acid Substitution Collections.

Janire Mingo,Olaia Aurtenetxe,Laura Amo,Jan T G Schepens,Jesús M Cortés,Wiljan J A J Hendriks,Asier Erramuzpe,Ibai Diez,Rafael Pulido,Sandra Luna

doi:10.1371/journal.pone.0160972

Abstract

Site-directed mutagenesis (SDM) is a powerful tool to create defined collections of protein variants for experimental and clinical purposes, but effectiveness is compromised when a large number of mutations is required. We present here a one-tube-only standardized SDM approach that generates comprehensive collections of amino acid substitution variants, including scanning- and single site-multiple mutations. The approach combines unified mutagenic primer design with the mixing of multiple distinct primer pairs and/or plasmid templates to increase the yield of a single inverse-PCR mutagenesis reaction. Also, a user-friendly program for automatic design of standardized primers for Ala-scanning mutagenesis is made available. Experimental results were compared with a modeling approach together with stochastic simulation data. For single site-multiple mutagenesis purposes and for simultaneous mutagenesis in different plasmid backgrounds, combination of primer sets and/or plasmid templates in a single reaction tube yielded the distinct mutations in a stochastic fashion. For scanning mutagenesis, we found that a combination of overlapping primer sets in a single PCR reaction allowed the yield of different individual mutations, although this yield did not necessarily follow a stochastic trend. Double mutants were generated when the overlap of primer pairs was below 60%. Our results illustrate that one-tube-only SDM effectively reduces the number of reactions required in large-scale mutagenesis strategies, facilitating the generation of comprehensive collections of protein variants suitable for functional analysis.

Highlights

The assessment of gene nucleotide sequences with experimental and clinical purposes has improved with an exponential progress in the last decade
We performed a series of site-directed mutagenesis (SDM) experiments aimed to increase the yield and effectiveness of the mutagenesis process when large numbers of amino acid substitutions are wanted
These mutagenesis experiments were aimed at cDNAs encoding PTEN and PTPRZ-B, two proteins involved in human disease and of interest for comprehensive mutational analysis

Summary

Introduction

The assessment of gene nucleotide sequences with experimental and clinical purposes has improved with an exponential progress in the last decade. Randomized mutagenesis, by error-prone PCR or random-generated oligonucleotide libraries, has been extensively used to generate protein variants with a desired function [8,9] This unbiased manner, fails in obtaining comprehensive yet defined collections of mutations. Some other modifications of the DpnI-based SDM protocol include the use of three primers and a one-step gradient PCR [22], the running of two separate PCR reactions for each mutation [23], the enzymatic assembly of overlapping PCR products [24], or the running of two sequential PCR reactions using so-called megaprimers in addition to the mutagenic primers [25,26] This may improve mutagenic efficiency and increases costs and methodological complexity. We present a simple one-tube SDM protocol that, by combining in a customized manner different templates and/or distinct mutagenic primer sets with fixed lengths, efficiently generates comprehensive collections of mutant cDNAs suitable for protein functional scanning

Materials and Methods

Results and Discussion

Conclusions