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Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
Published on: March 31, 2022
Cytogenetic challenge assays for assessment of DNA repair capacities
William W Au1, Salama A Salama
1Department of Preventative Medicine and Community Health, The University of Texas Medical Branch, Galveston, TX, USA.
Abstract:
Different challenge assays have been used to investigate cellular responses following exposure to DNA damaging agents. Our protocol uses X- or gamma-rays or ultraviolet light to challenge cells to repair the induced damage, and chromosome aberrations as a biomarker to indicate DNA repair proficiency. The assay was used successfully to demonstrate base- and nucleotide-excision repair deficiency in certain polymorphic DNA repair genes, namely XRCC1 751Gln and XPD 312Asn, respectively. In addition, populations with elevated exposure to certain environmental mutagenic agents-cigarette smokers, pesticide sprayers, and residents who lived near uranium mining and milling sites-showed DNA repair deficiency. Because expression of chromosome aberrations is associated with a significantly increased incidence of both cancer morbidity and mortality, the challenge assay may be useful in predicting cancer risk. The protocol for the assay is straightforward and the data have practical applications.
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Cells are regularly exposed to mutagens—factors in the environment that can damage DNA and generate mutations. UV radiation is one of the most common mutagens and is estimated to introduce a significant number of changes in DNA. These include bends or kinks in the structure, which can block DNA replication or transcription. If these errors are not fixed, the damage can cause mutations, which in turn can result in cancer or disease depending on which sequences are...
