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Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method.

Naohiro Taniguchi1, Hiroshi Murakami2

  • 1PeptiDream Inc., 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8505, Japan.

Methods in Molecular Biology (Clifton, N.J.)
|October 7, 2016
PubMed
Summary
This summary is machine-generated.

We developed MUPAC, a novel method for protein engineering, enabling precise gene mutations. This technique efficiently introduces multiple site-directed and saturation mutations for DNA assembly and cloning.

Keywords:
CloningProtein engineeringSaturation mutagenesisSite-directed mutagenesis

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Area of Science:

  • Molecular Biology
  • Protein Engineering
  • Synthetic Biology

Background:

  • Protein engineering relies on precise gene modification.
  • Introducing multiple mutations is crucial for protein function studies.

Purpose of the Study:

  • To introduce a method for multiple site-directed and saturation mutagenesis.
  • To facilitate DNA assembly for gene cloning.

Main Methods:

  • Developed a method termed MUPAC (Multiple site-directed and saturation mutagenesis).
  • Applied MUPAC to green fluorescent protein (GFP) and moloney murine leukemia virus reverse transcriptase (MMLV-RT) genes.
  • Utilized MUPAC for introducing randomized codons at five positions in the GFP gene.

Main Results:

  • Successfully introduced multiple site-directed mutations in GFP and MMLV-RT genes.
  • Demonstrated successful introduction of randomized codons at five desired positions in the GFP gene.
  • Showcased MUPAC's utility in simple DNA assembly for cloning.

Conclusions:

  • MUPAC is an effective method for introducing multiple mutations.
  • The method simplifies gene construction for protein engineering applications.
  • MUPAC facilitates DNA assembly and cloning processes.