Title

Unscheduled DNA synthesis: the clinical and functional assay for global genomic DNA nucleotide excision repair

ISBN or ISSN

1064-3745

Publication Title

Methods in molecular biology

Volume

1105

Publication Date / Copyright Date

1-1-2014

First Page

511

Last Page

532

Publisher

Humana Press

DOI Number

10.1007/978-1-62703-739-6_36

Abstract

The unscheduled DNA synthesis (UDS) assay measures the ability of a cell to perform global genomic nucleotide excision repair (NER). This chapter provides instructions for the application of this technique by creating 6-4 photoproducts and pyrimidine dimers using UV-C irradiation. This procedure is designed specifically for quantification of the 6-4 photoproducts. Repair is quantified by the amount of radioactive thymidine incorporated during repair synthesis after this insult, and radioactivity is evaluated by grain counting after autoradiography. The results are used to clinically diagnose human DNA repair deficiency disorders and provide a basis for investigation of repair deficiency in human tissues or tumors. No other functional assay is available that directly measures the capacity to perform NER on the entire genome without the use of specific antibodies. Since live cells are required for this assay, explant culture techniques must be previously established. Host cell reactivation (HCR), as discussed in Chapter 37, is not an equivalent technique, as it measures only transcription-coupled repair (TCR) at active genes, a small subset of total NER.

Disciplines

Medicine and Health Sciences | Pharmacy and Pharmaceutical Sciences

Keywords

DNA Repair, DNA Replication, Genome, Human, Humans, MCF-7 Cells, Staining and Labeling, Tissue Fixation