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Updated: Mar 28, 2026
Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells
Published on: February 24, 2014
Mutation analysis with random DNA identifiers (MARDI) catalogs Pig-a mutations in heterogeneous pools of
Javier R Revollo1, Nathaniel M Crabtree2, Mason G Pearce1
1Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas.
Abstract:
Identification of mutations induced by xenotoxins is a common task in the field of genetic toxicology. Mutations are often detected by clonally expanding potential mutant cells and genotyping each viable clone by Sanger sequencing. Such a "clone-by-clone" approach requires significant time and effort, and sometimes is even impossible to implement. Alternative techniques for efficient mutation identification would greatly benefit both basic and regulatory genetic toxicology research. Here, we report the development of Mutation Analysis with Random DNA Identifiers (MARDI), a novel high-fidelity Next Generation Sequencing (NGS) approach that circumvents clonal expansion and directly catalogs mutations in pools of mutant cells. MARDI uses oligonucleotides carrying Random DNA Identifiers (RDIs) to tag progenitor DNA molecules before PCR amplification, enabling clustering of descendant DNA molecules and eliminating NGS- and PCR-induced sequencing artifacts. When applied to the Pig-a cDNA analysis of heterogeneous pools of CD48-deficient T cells derived from DMBA-treated rats, MARDI detected nearly all Pig-a mutations that were previously identified by conventional clone-by-clone analysis and discovered many additional ones consistent with DMBA exposure: mostly A to T transversions, with the mutated A located on the non-transcribed DNA strand.
Insights
Mutation Analysis with Random DNA Identifiers (MARDI) offers a faster, more efficient method for identifying genetic mutations. This Next Generation Sequencing approach bypasses traditional cell cloning, directly cataloging mutations in cell pools.
Area of Science:
- Genetic Toxicology
- Molecular Biology
- Genomics
Background:
- Identifying xenotoxin-induced mutations is crucial in genetic toxicology.
- Conventional clone-by-clone analysis using Sanger sequencing is time-consuming and labor-intensive.
- There is a need for more efficient mutation identification techniques.
Purpose of the Study:
- To develop a novel, high-fidelity Next Generation Sequencing (NGS) approach for efficient mutation identification.
- To introduce Mutation Analysis with Random DNA Identifiers (MARDI) as an alternative to clonal expansion.
- To catalog mutations directly in pools of mutant cells.
Main Methods:
- MARDI utilizes oligonucleotides with Random DNA Identifiers (RDIs) to tag progenitor DNA molecules.
- RDIs enable clustering of descendant DNA molecules after PCR amplification.
- This method eliminates sequencing artifacts introduced by NGS and PCR.
Main Results:
- MARDI successfully identified nearly all Pig-a mutations previously found by clone-by-clone analysis in DMBA-treated rat T cells.
- The approach discovered additional mutations consistent with DMBA exposure.
- Predominant mutations were A to T transversions on the non-transcribed DNA strand.
Conclusions:
- MARDI is a high-fidelity NGS method that circumvents clonal expansion for mutation analysis.
- This technique significantly enhances the efficiency of genetic toxicology studies.
- MARDI provides a powerful tool for both basic and regulatory genetic toxicology research.
