A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of Ethynyl deoxyuridine (EdU)

Limsirichaikul, Siripan, Niimi, Atsuko, Fawcett, Heather, Lehmann, Alan, Yamashita, Shunichi and Ogi, Tomoo (2009) A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of Ethynyl deoxyuridine (EdU). Nucleic Acids Research, 37 (4). ISSN 0305-1048

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Abstract

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed unscheduled DNA synthesis (UDS), is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

Item Type: Article
Additional Information: In Endnotes Ref: GDSC269
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH0301 Biology
Related URLs:
Depositing User: Gee Wheatley
Date Deposited: 23 Feb 2009
Last Modified: 07 Mar 2017 10:52
URI: http://sro.sussex.ac.uk/id/eprint/2115

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