Editor's Choice for February 2016

Environmental and Molecular Mutagenesis Editor's Choice - February 2016

The Editor's Choice for February is "Mutational Analysis with Random DNA Identifiers (MARDI) Catalogs Pig-a Mutations in Heterogeneous Pools of CD-48-Deficient T Cells Derived from DMBA-Treated Rats" by Javier R. Revollo, Nathaniel M. Crabtree, Mason G. Pearce, M. Monserrat Pacheco-Martinez, and Vasily N. Dobrovolsky.

Clone-by-clone Sanger sequencing is the most common method for identifying mutations in a reporter gene. The method relies on isolating individual mutant cells, allowing them to multiply and grow into a clone over a period of days, and sequencing the reporter gene in each clone separately. This is a time-consuming and labor-intensive approach that 1) does not work well for slow or poorly growing cells and 2) may not identify the full range of mutations in a given sample. In this article a new method for identifying mutations in reporter genes, MARDI (Mutation Analysis with Random DNA Identifiers), overcomes these weaknesses while maintaining a low error rate. As a proof of concept, pools of Pig-a mutant (CD48-negative) T cells were isolated by flow cytometry from rats treated with the powerful mutagen 7,12-dimethylbenz[a]anthracene (DMBA), and their total RNA was extracted. Pig-a cDNA then was synthesized, barcoded with short random oligonucleotide tags to uniquely identify parental DNA copies, and PCR-amplified for Next Generation Sequencing analysis. The types of mutations detected by MARDI correlated strongly with those obtained by clone-by-clone Sanger sequencing, and MARDI was able to identify additional mutations as it analyzed many more mutant cells. In summary, this report describes MARDI as a rapid high-fidelity approach for identifying gene mutations in reporter genes.  In the age of bioinformatics, research needs the development of technically straightforward and rapid methods to identify mutations in genes. This work “Mutational Analysis with Random DNA Identifiers (MARDI) Catalogs Pig-a Mutations in Heterogeneous Pools of CD-48-Deficient T Cells Derived from DMBA-Treated Rats” stands to make drug-inducible mutagenesis analysis available to wider audience of researchers and is why the entry has been named the Environmental and Molecular Mutagenesis February 2016 Editor’s Choice article. Environ. Mol. Mutagen. 2016.  © 2015 Wiley Periodicals Inc.  

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