Quantification of UV-induced cyclobutane pyrimidine dimers using an oligonucleotide chip assay.
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/m(2)). 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.
Kim
Min Jung
MJ
Research Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul 151-742, South Korea.
Lee
Su Chul
SC
Kang
Seong Ho
SH
Choo
Jaebum
J
Song
Joon Myong
JM
D001710:Biotin; D002138:Calibration; D053960:DNA Breaks / Q000528:radiation effects; D004249:DNA Damage / Q000235:genetics*; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism; D005456:Fluorescent Dyes; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods* / Q000592:standards; D009841:Oligonucleotides; D011740:Pyrimidine Dimers / Q000032:analysis* / Q000528:radiation effects; D014466:Ultraviolet Rays / Q000009:adverse effects; D014764:Viral Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; D009841:Oligonucleotides; D011740:Pyrimidine Dimers; D014764:Viral Proteins; D001710:Biotin; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)