A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).
A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU).
复制标题
DOI：
10.1093/nar/gkp023
复制
发表时间：
2009-03
影响因子：
14.9
通讯作者：
Ogi T
中科院分区：
文献类型：
--
作者：
Limsirichaikul S;Niimi A;Fawcett H;Lehmann A;Yamashita S;Ogi T
着色性干皮病是一种常染色体隐性遗传病。受影响的患者表现出极端的阳光敏感性和皮肤癌易感性。XP在大多数情况下与核苷酸切除修复缺陷（NER）有关，这是负责从DNA中去除光损伤的过程。通过核苷酸掺入修复补丁来测量NER活性，称为“非程序DNA合成（UDS）”，是XP诊断和NER研究最常用的测定方法之一。我们用5-乙炔基-2 '-脱氧尿苷（EdU）代替胸苷，建立了一种快速、准确的测定UDS的方法。掺入修复补丁的EdU可以直接与荧光叠氮化物衍生物缀合，从而避免了对放射性标记的胸苷或掺入的溴脱氧尿苷（BrdU）的变性和抗体检测的需要。我们证明，EdU掺入试验是兼容的常规技术，如免疫荧光染色和标记的细胞与微乳胶珠。重要的是，我们可以在制备检测盖玻片的半天内完成整个UDS检测;这种技术可能被证明是XP诊断的有用方法。
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.
登录
查看更多内容
影响因子：
3.8
作者：
Kleijer, Wim J.;Laugel, Vincent;Lehmann, Alan R.
通讯作者：
Lehmann, Alan R.
影响因子：
2.6
作者：
ARLETT, CF;GREEN, MHL;MAYNE, LV
通讯作者：
MAYNE, LV
影响因子：
6.5
作者：
Moriwaki, SI;Stefanini, M;Kraemer, KH
通讯作者：
Kraemer, KH
DOI：
10.1016/s0027-5107(02)00219-1
发表时间：
2002-11-30
影响因子：
2.3
作者：
Qiao, YW;Spitz, MR;Wei, QY
通讯作者：
Wei, QY
影响因子：
3
作者：
Kleijer, Wim J.;van der Sterre, Marianne L. T.;Jaspers, Nicolaas G. J.
通讯作者：
Jaspers, Nicolaas G. J.