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Nucleic Acids Research Advance Access published online on January 29, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp023
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

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

Siripan Limsirichaikul1,2, Atsuko Niimi1,3, Heather Fawcett3, Alan Lehmann3, Shunichi Yamashita1 and Tomoo Ogi1,*

1Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 852-8523 Japan, 2Department of Biopharmacy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, 73000 Thailand and 3Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ, UK

*To whom correspondence should be addressed. Tel: +81 95 819 7103; Fax: +81 95 819 7104; Email: togi{at}nagasaki-u.ac.jp

Received September 25, 2008. Revised January 7, 2009. Accepted January 7, 2009.

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.

The authors wish it to be known that, in their opinion, the fourth and the fifth author contributed equally to the work.


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