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Multiplexed, UVC ‐Induced, Sequence‐Dependent DNA Damage Detection
Author(s) -
Nair Sindhu G.,
Loppnow Glen R.
Publication year - 2013
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/php.12066
Subject(s) - oligonucleotide , dna damage , molecular beacon , dna , pyrimidine dimer , chemistry , guanine , thymine , cytosine , cuvette , microbiology and biotechnology , nucleotide , biochemistry , biology , gene , physics , quantum mechanics
The exposure of DNA to ultraviolet (UV) radiation causes sequence‐dependent damage. Thus, there is a need for an analytical technique that can detect damage in large numbers of DNA sequences simultaneously. In this study, we have designed an assay for UVC‐induced DNA damage in multiple oligonucleotides simultaneously by using a 96‐well plate and a novel automated sample mover. The UVC‐induced DNA damage is measured using smart probes, analogs of molecular beacons in which guanosine nucleotides act as the fluorescence quencher. Our results show that the oligonucleotide damage constants obtained with this method are reproducible and similar to those obtained in cuvettes. The calibration curve for poly‐ dT shows good linearity ( R 2 = 0.96), with limits of detection (LOD) and quantification (LOQ) equal to 55 and 183 n m , respectively. The results show that the damage kinetics upon irradiation is sensitive to the different types of photoproducts formed in the different sequences used; i.e . poly‐A oligonucleotides containing guanine are damaged at a faster rate than poly‐A oligonucleotides containing either thymine or cytosine. Thus, detecting DNA damage in a 96‐well plate and quantifying the damage with smart probes are a simple, fast and inexpensive mix‐and‐read technique for multiplexed, sequence‐specific DNA damage detection.