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

TL;DR

This paper introduces a new mutagenesis assay using specific DNA sequences to test how well a gene can tolerate mutations, which is important for biotechnology and drug development.

Contribution

The novel mutagenesis assay uses NNT and NNC oligonucleotides to safely test gene mutability without causing harmful mutations.

Findings

The NNT and NNC oligonucleotides prevent nonsense mutations and allow flexible insertion sizes.

The ZeoR gene was confirmed to be highly tolerant to mutagenesis using this new assay.

Abstract

Not all proteins are tolerable to mutations. Whether a specific protein can be a mutable target is of importance in the biotechnology and pharmaceutical industry. This study reported a novel mutagenesis assay using tandem NNT and NNC oligonucleotides to test the mutability of a candidate gene. These two tandem oligonucleotides avoid the risk of forming nonsense mutations and render flexibility of truncating or expanding the insertion size. As a reporter gene, ZeoR (zeocin resistance gene) was confirmed to have a high tolerance for mutagenesis by this new assay.

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