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A Mutagenesis Assay for Reporter Gene Screening Using Partially Degenerate Oligonucleotides of the Tandems NNT and

Huifen Xu1, Cuilan Zhou2, Andy K Zhang1

  • 1Department of Molecular Diagnostics and Biopharmaceutics, College of Pharmaceutical Science, Soochow University, Suzhou 215123, China.

Biomed Research International
|July 14, 2015
PubMed

Insights

This study introduces a new mutagenesis assay using tandem oligonucleotides to assess protein mutation tolerance. The zeocin resistance gene (ZeoR) reporter demonstrated high mutability, proving useful for biotechnology applications.

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Protein Engineering

Background:

  • Protein mutability is crucial for biotechnology and pharmaceutical development.
  • Assessing a protein's tolerance to mutations is essential for drug discovery and protein engineering.

Purpose of the Study:

  • To develop and validate a novel mutagenesis assay for evaluating protein mutability.
  • To assess the mutability of the ZeoR (zeocin resistance gene) reporter using the new assay.

Main Methods:

  • Utilized a novel mutagenesis assay employing tandem NNT and NNC oligonucleotides.
  • The assay was designed to prevent nonsense mutations and allow flexible insertion size control.
  • Employed the ZeoR gene as a reporter to test the assay's efficacy.

Main Results:

  • The novel assay successfully tested the mutability of a candidate gene.
  • The ZeoR reporter gene exhibited a high tolerance for mutagenesis.
  • The assay design avoids nonsense mutations and offers flexibility in insertion size.

Conclusions:

  • The developed tandem oligonucleotide mutagenesis assay is effective for assessing protein mutability.
  • The ZeoR gene is a suitable reporter for high-tolerance mutagenesis studies.
  • This assay provides a flexible and reliable method for mutation tolerance testing in protein engineering.

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