Quantitation of ultraviolet-induced single-strand breaks using oligonucleotide chip.
A simple, accurate and robust methodology was established for the direct quantification of ultraviolet (UV)-induced single-strand break (SSB) using oligonucleotide chip. Oligonucleotide chips were fabricated by covalently anchoring the fluorescent-labeled ssDNAs onto silicon dioxide chip surfaces. Assuming that the possibility of more than one UV-induced SSB to be generated in a small oligonucleotide is extremely low, SSB formation was investigated quantifying the endpoint probe density by fluorescence measurement upon UV irradiation. The SSB yields obtained based on the highly sensitive laser-induced fluorometric determination of fluorophore-labeled oligonucleotides were found to coincide well with that predicted from a theoretical extrapolation of the results obtained for plasmid DNAs using conventional agarose gel electrophoresis. The developed method has the potential to serve as a high throughput, sample-thrifty, and time saving tool to realize more realistic, and direct quantification of radiation and chemical-induced strand breaks. It will be especially useful for determining the frequency of SSBs or lesions convertible to SSBs by specific cleaving reagents or enzymes.
Pal
Sukdeb
S
Research Institute of Pharmaceutical Sciences and College of Pharmacy, Seoul National University, Seoul 151-742, South Korea.
Kim
Min Jung
MJ
Choo
Jaebum
J
Kang
Seong Ho
SH
Lee
Kyeong-Hee
KH
Song
Joon Myong
JM
D002138:Calibration; D004249:DNA Damage / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000235:genetics / Q000528:radiation effects*; D046228:Microarray Analysis / Q000379:methods*; D015394:Molecular Structure; D009841:Oligonucleotides / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D009841:Oligonucleotides