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Updated: May 13, 2026
The Lambda Select cII Mutation Detection System
Published on: April 26, 2018
A high-throughput and quantitative method to assess the mutagenic potential of translesion DNA synthesis
David J Taggart1, Terry L Camerlengo, Jason K Harrison
1Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, USA.
Abstract:
Cellular genomes are constantly damaged by endogenous and exogenous agents that covalently and structurally modify DNA to produce DNA lesions. Although most lesions are mended by various DNA repair pathways in vivo, a significant number of damage sites persist during genomic replication. Our understanding of the mutagenic outcomes derived from these unrepaired DNA lesions has been hindered by the low throughput of existing sequencing methods. Therefore, we have developed a cost-effective high-throughput short oligonucleotide sequencing assay that uses next-generation DNA sequencing technology for the assessment of the mutagenic profiles of translesion DNA synthesis catalyzed by any error-prone DNA polymerase. The vast amount of sequencing data produced were aligned and quantified by using our novel software. As an example, the high-throughput short oligonucleotide sequencing assay was used to analyze the types and frequencies of mutations upstream, downstream and at a site-specifically placed cis-syn thymidine-thymidine dimer generated individually by three lesion-bypass human Y-family DNA polymerases.
Insights
We developed a new sequencing method to study how DNA polymerases cause mutations when encountering DNA damage. This cost-effective assay reveals mutagenic profiles of translesion DNA synthesis, advancing our understanding of genome stability.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Cellular genomes face continuous damage from endogenous and exogenous sources, leading to DNA lesions.
- While DNA repair mechanisms are robust, some lesions persist, posing challenges during genomic replication.
- Existing low-throughput sequencing methods limit understanding of mutagenic outcomes from unrepaired DNA lesions.
Purpose of the Study:
- To develop a cost-effective, high-throughput sequencing assay for assessing mutagenic profiles.
- To analyze translesion DNA synthesis catalyzed by error-prone DNA polymerases.
- To investigate mutation types and frequencies associated with specific DNA lesions.
Main Methods:
- Developed a high-throughput short oligonucleotide sequencing assay utilizing next-generation DNA sequencing.
- Created novel software for aligning and quantifying vast amounts of sequencing data.
- Applied the assay to analyze mutations around a site-specific thymidine dimer using human Y-family DNA polymerases.
Main Results:
- The assay provides a cost-effective, high-throughput method for evaluating mutagenic profiles.
- Novel software enables efficient analysis of complex sequencing data.
- Characterized mutation types and frequencies associated with lesion bypass by specific DNA polymerases.
Conclusions:
- The developed assay significantly enhances the ability to study mutagenic outcomes of DNA damage and repair.
- This method facilitates a deeper understanding of the role of DNA polymerases in genome mutagenesis.
- Provides a powerful tool for investigating genome stability and the impact of DNA lesions.
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