Quantitative assay of photoinduced DNA strand breaks by real-time PCR

被引：5

作者：

Wiczk, Justyna ^{[1
]}

Westphal, Kinga ^{[1
]}

Rak, Janusz ^{[1
]}

机构：

[1] Univ Gdansk, Fac Chem, Wita Stwosza 63, PL-80308 Gdansk, Poland

来源：

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS | 2016年 / 128卷

关键词：

Real-time PCR; Mass spectrometry; Photosensitizing nucleobases; Single strand breaks; Photodamage; MASS-SPECTROMETRY; UV-IRRADIATION; DUPLEX DNA; DAMAGE; RADIATION; SEQUENCE; OLIGONUCLEOTIDES; PHOTOREACTION; MECHANISMS; GENERATION;

D O I：

10.1016/j.jpba.2016.06.023

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320 nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies. (C) 2016 Elsevier B.V. All rights reserved.

引用

页码：480 / 484

页数：5

共 30 条

[1] Photodynamic Therapy of Cancer: An Update [J].

Agostinis, Patrizia ;

Berg, Kristian ;

Cengel, Keith A. ;

Foster, Thomas H. ;

Girotti, Albert W. ;

Gollnick, Sandra O. ;

Hahn, Stephen M. ;

Hamblin, Michael R. ;

Juzeniene, Asta ;

Kessel, David ;

Korbelik, Mladen ;

Moan, Johan ;

Mroz, Pawel ;

Nowis, Dominika ;

Piette, Jacques ;

Wilson, Brian C. ;

Golab, Jakub .

CA-A CANCER JOURNAL FOR CLINICIANS, 2011, 61 (04) :250-281

[2] Cytotoxic and genotoxic effects of silver nanoparticles in testicular cells [J].

Asare, Nana ;

Instanes, Christine ;

Sandberg, Wiggo J. ;

Refsnes, Magne ;

Schwarze, Per ;

Kruszewski, Marcin ;

Brunborg, Gunnar .

TOXICOLOGY, 2012, 291 (1-3) :65-72

[3]

Ausubel F.M., 2002, SHORT PROTOCOLS MOL

[4] Interstrand cross-link induction by UV radiation in bromodeoxyuridine-substituted DNA: Dependence on DNA conformation [J].

Cecchini, S ;

Masson, C ;

La Madeleine, C ;

Huels, MA ;

Sanche, L ;

Wagner, JR ;

Hunting, DJ .

BIOCHEMISTRY, 2005, 44 (51) :16957-16966

[5] Investigation of the origin of the sequence selectivity for the 5-halo-2′-deoxyuridine sensitization of DNA to damage by UV-irradiation [J].

Chen, TQ ;

Cook, GP ;

Koppisch, AT ;

Greenberg, MM .

JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2000, 122 (16) :3861-3866

[6] Electron-Induced Elimination of the Bromide Anion from Brominated Nucleobases. A Computational Study [J].

Chomicz, Lidia ;

Rak, Janusz ;

Storoniak, Piotr .

JOURNAL OF PHYSICAL CHEMISTRY B, 2012, 116 (19) :5612-5619

[7] Analysis of apoptosis by cytometry using TUNEL assay [J].

Darzynkiewicz, Zbigniew ;

Galkowski, Dariusz ;

Zhao, Hong .

METHODS, 2008, 44 (03) :250-254

[8]

Dorak Tevfik M., 2009, PCR REAL TIME

[9] Base damage and single-strand break repair: Mechanisms and functional significance of short- and long-patch repair subpathways [J].

Fortini, Paola ;

Dogliotti, Eugenia .

DNA REPAIR, 2007, 6 (04) :398-409

[10] EVIDENCE FOR DNA DOUBLE-STRAND BREAKS AS THE CRITICAL LESIONS IN YEAST-CELLS IRRADIATED WITH SPARSELY OR DENSELY IONIZING-RADIATION UNDER OXIC OR ANOXIC CONDITIONS [J].

FRANKENBERG, D ;

FRANKENBERGSCHWAGER, M ;

BLOCHER, D ;

HARBICH, R .

RADIATION RESEARCH, 1981, 88 (03) :524-532

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