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Updated: May 14, 2026

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
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Published on: February 5, 2019

Protein engineering: single or multiple site-directed mutagenesis.

Pei-Chung Hsieh1, Romualdas Vaisvila

  • 1New England Biolabs, Inc., Ipswich, MA, USA. hsieh@neb.com

Methods in Molecular Biology (Clifton, N.J.)
|February 21, 2013
PubMed
Summary

We present two streamlined protocols for site-directed mutagenesis to study protein structure and function. These methods, using inverse PCR and Gibson Assembly, efficiently introduce single or multiple mutations, saving time and reagents.

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Area of Science:

  • Molecular Biology
  • Protein Engineering

Background:

  • Site-directed mutagenesis is crucial for understanding protein structure-function relationships.
  • Traditional methods can be time-consuming and reagent-intensive.

Purpose of the Study:

  • To provide simplified and expedited protocols for site-directed mutagenesis.
  • To enable efficient introduction of single and multiple mutations in genes of interest.

Main Methods:

  • Single mutations: Inverse PCR with mutagenic primers and Phusion(®) DNA Polymerase.
  • Multiple mutations: One-step PCR with mutagenic primers followed by Gibson Assembly™ Master Mix.

Main Results:

  • The inverse PCR method efficiently introduces point mutations, insertions, or deletions.
  • The Gibson Assembly method allows simultaneous multiple nucleotide changes, saving time and reagents.

Conclusions:

  • These optimized protocols simplify and accelerate the process of protein mutagenesis.
  • The methods offer efficient and cost-effective alternatives for molecular biology research.