A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).
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.
Limsirichaikul
Siripan
S
Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, 852-8523 Japan.
Niimi
Atsuko
A
Fawcett
Heather
H
Lehmann
Alan
A
Yamashita
Shunichi
S
Ogi
Tomoo
T
D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D004260:DNA Repair; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000528:radiation effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D008856:Microscopy, Fluorescence; D013936:Thymidine / Q000378:metabolism; D014316:Tritium; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism
D014316:Tritium; D004247:DNA; C031086:5-ethynyl-2'-deoxyuridine; D013936:Thymidine; D003857:Deoxyuridine