Quantification of UV-induced cyclobutane pyrimidine dimers using an oligonucleotide chip assay

• Published in: Analytical and bioanalytical chemistry Vol. 397; no. 6; pp. 2271 - 2277
• Main Authors: Kim, Min Jung, Lee, Su Chul, Kang, Seong Ho, Choo, Jaebum, Song, Joon Myong
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 01.07.2010; Springer-Verlag; Springer
• Subjects: Analytical Chemistry; Assaying; Biochemistry; Biotin; Calibration; Carbocyclic compounds; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chips; Cyclobutane; Cyclobutane pyrimidine dimer; Deoxyribonuclease (Pyrimidine Dimer) - metabolism; Detectors; Dimers; DNA; DNA Breaks - radiation effects; DNA Damage - genetics; Enzymes; Exact sciences and technology; Fluorescence; Fluorescent Dyes; Food Science; Laboratory Medicine; Lesion-specific enzyme-induced break assay; Mathematical analysis; Monitoring/Environmental Analysis; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotide Array Sequence Analysis - standards; Oligonucleotide chip; Oligonucleotides; Pyrimidine Dimers - analysis; Pyrimidine Dimers - radiation effects; Pyrimidines; Short Communication; Spectrometric and optical methods; T4 endonuclease V; Ultraviolet Rays - adverse effects; Viral Proteins - metabolism; Oligonucleotide chip; Lesion-specific enzyme-induced break assay; Cyclobutane pyrimidine dimer; T4 endonuclease V; Chemical analysis; Gel electrophoresis; Enzyme; Use; Laser induced fluorescence; DNA chip; Immobilization; Fluorescence spectrometry; Pyrimidine; Cyclobutane pyrimidine dimmer; Fluorophore; DNA; Standard curve; Irradiation; Dimer; Oligonucleotide; Yield; Cyclobutane; Lesion; Reliability; Quantitative analysis
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-010-3793-6

A lesion-specific enzyme-induced DNA strand break assay was developed for an oligonucleotide chip for the determination of UVB-induced cyclobutane pyrimidine dimers (CPDs). A 20-mer of fluorophore-labeled and biotinylated oligonucleotide was immobilized on the chip. CPDs in DNA on the chip were formed by UVB irradiation (312 nm). T4 endonuclease V (T4N5) was used to excise the CPD site as T4N5 sensitively and specifically detects CPDs. The fluorophore-labeled DNA fragments were detected by a laser-induced fluorescence (LIF) detection system. The number of CPDs induced by UVB was determined based on a mathematical equation obtained from a predetermined calibration curve. The yield of UVB-induced CPDs was 1.73 CPDs per megabase per (kJ/m²). The reliability of this value was proved by its similarity to reference values obtained from gel electrophoresis. The developed assay has strong potential to quantify most kinds of UV-induced DNA lesions.