彗星分析(comet assay)又稱單細胞電泳(SCGE)，目前廣泛的使用在分析藥物毒性和核酸修補機制，單細胞電泳所分析的是核酸斷裂程度，但是，短波長紫外線造成的核酸損傷是不會立即形成核酸斷裂，核酸的斷裂是修補過程的中間產物；也就是說單細胞電泳無法分析核酸鹼基型態改變的數量，而利用專一性的核酸切割酵素或是細胞核萃取物(nuclear extract)的處理切割，可以將型態改變的鹼基轉變為核酸斷裂而增加彗星分析對紫外線導致的去氧核醣核酸損傷檢測的敏感性，並增加彗星分析的應用性；本論文將研究探討彗星分析與核萃取物的應用。在本篇論文中，比較專一性核酸切割酵素Den V和細胞核粹取物對紫外線導致的去氧核醣核酸損傷的切割能力，發現核萃取物能夠較準確的評估紫外線(UVC)所造成DNA 損傷，接著以彗星分析來比較正常人類纖維母細胞 (MSU-1) 與來自XP病人分離出來的纖維母細胞 (XP22VI) 來證實彗星分析的正確性；最後利用彗星分析來比較腎臟癌細胞對短波長紫外線的修補能力和秋水仙胺抑制核酸切除修補機制的數據說明慧星分析在研究核酸修補機制上的應用性。

Comet assay, also called single cell gel electrophoresis (SCGE), is widely used to investigate genotoxin, and DNA repair system. UVC induced DNA adducts were cyclobutane pyrimidine dimmers (CPD) and 6-4 pyrimidine pyrimidone photoproducts (6-4PP). These adducts do not caused DNA breaks immediately, the DNA breaks were the intermediate of repair process that means conventional comet assay can’t estimate total amounts of UVC induced DNA adducts. To overcome this problem, people who demonstrated that damaged DNA was incubate with nuclear extract (NE) or T4 endonuclease V (Den V) to excised DNA adducts that would enhance the sensitivity of comet assay when UVC as damage source. In this study, I focused on the application of comet-NE assay. I found that comet-NE assay can detect more UV-induced DNA adducts than Den V. Besides, the accuracy of comet-NE assay also verified by distinguish different repair kinetic between XP22VI and MSU-1 cells. The well established comet-NE and comet-Den V assay were used to study repair kinetic of bladder cancer cells and colcemid effect on NER process.

Al-Baker, E. A., Oshin, M., Hutchison, C. J., and Kill, I. R. (2005). Analysis of UV-induced damage and repair in young and senescent human dermal fibroblasts using the comet assay. Mech Ageing Dev 126, 664-672.
Bau, D. T., Gurr, J. R., and Jan, K. Y. (2001). Nitric oxide is involved in arsenite inhibition of pyrimidine dimer excision. Carcinogenesis 22, 709-716.
Cadet, J., Sage, E., and Douki, T. (2005). Ultraviolet radiation-mediated damage to cellular DNA. Mutat Res 571, 3-17.
Collins, A. R., Dobson, V. L., Dusinska, M., Kennedy, G., and Stetina, R. (1997a). The comet assay: what can it really tell us? Mutat Res 375, 183-193.
Collins, A. R., Dusinska, M., Horvathova, E., Munro, E., Savio, M., and Stetina, R. (2001). Inter-individual differences in repair of DNA base oxidation, measured in vitro with the comet assay. Mutagenesis 16, 297-301.
Collins, A. R., Duthie, S. J., and Dobson, V. L. (1993). Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14, 1733-1735.
Collins, A. R., Mitchell, D. L., Zunino, A., de Wit, J., and Busch, D. (1997b). UV-sensitive rodent mutant cell lines of complementation groups 6 and 8 differ phenotypically from their human counterparts. Environ Mol Mutagen 29, 152-160.
de Boer, J., and Hoeijmakers, J. H. (2000). Nucleotide excision repair and human syndromes. Carcinogenesis 21, 453-460.
de Laat, W. L., Jaspers, N. G., and Hoeijmakers, J. H. (1999). Molecular mechanism of nucleotide excision repair. Genes Dev 13, 768-785.
Eveno, E., Bourre, F., Quilliet, X., Chevallier-Lagente, O., Roza, L., Eker, A. P., Kleijer, W. J., Nikaido, O., Stefanini, M., Hoeijmakers, J. H., and et al. (1995). Different removal of ultraviolet photoproducts in genetically related xeroderma pigmentosum and trichothiodystrophy diseases. Cancer Res 55, 4325-4332.
Friedberg, E. C. (2003). DNA damage and repair. Nature 421, 436-440.
Kumaravel, T. S., and Jha, A. N. (2006). Reliable Comet assay measurements for detecting DNA damage induced by ionising radiation and chemicals. Mutat Res 605, 7-16.
Li, H., Chang, T. W., Tsai, Y. C., Chu, S. F., Wu, Y. Y., Tzang, B. S., Liao, C. B., and Liu, Y. C. (2005). Colcemid inhibits the rejoining of the nucleotide excision repair of UVC-induced DNA damages in Chinese hamster ovary cells. Mutat Res 588, 118-128.
Li, P. Y., Chang, Y. C., Tzang, B. S., Chen, C. C., and Liu, Y. C. (2007). Antibiotic amoxicillin induces DNA lesions in mammalian cells possibly via the reactive oxygen species. Mutat Res 629, 133-139.
Mori, T., Nakane, M., Hattori, T., Matsunaga, T., Ihara, M., and Nikaido, O. (1991). Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6-4)photoproduct from the same mouse immunized with ultraviolet-irradiated DNA. Photochem Photobiol 54, 225-232.
Nishiwaki, Y., Kobayashi, N., Imoto, K., Iwamoto, T. A., Yamamoto, A., Katsumi, S., Shirai, T., Sugiura, S., Nakamura, Y., Sarasin, A., et al. (2004). Trichothiodystrophy fibroblasts are deficient in the repair of ultraviolet-induced cyclobutane pyrimidine dimers and (6-4)photoproducts. J Invest Dermatol 122, 526-532.
Pfeifer, G. P., You, Y. H., and Besaratinia, A. (2005). Mutations induced by ultraviolet light. Mutat Res 571, 19-31.
Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., and Linn, S. (2004). Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 73, 39-85.
Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175, 184-191.
Takahashi, Y., Moriwaki, S., Sugiyama, Y., Endo, Y., Yamazaki, K., Mori, T., Takigawa, M., and Inoue, S. (2005). Decreased gene expression responsible for post-ultraviolet DNA repair synthesis in aging: a possible mechanism of age-related reduction in DNA repair capacity. J Invest Dermatol 124, 435-442.
Tzeng, C. C., Liu, H. S., Li, C., Jin, Y. T., Chen, R. M., Yang, W. H., and Lin, J. S. (1996). Characterization of two urothelium cancer cell lines derived from a blackfoot disease endemic area in Taiwan. Anticancer Res 16, 1797-1804.
Wang, A. S., Ramanathan, B., Chien, Y. H., Goparaju, C. M., and Jan, K. Y. (2005). Comet assay with nuclear extract incubation. Anal Biochem 337, 70-75.