Quantitation of ultraviolet-induced single-strand breaks using oligonucleotide chip
Introduction
The principal methods for quantitating SSBs in DNA are (a) alkaline sucrose gradient sedimentation; (b) gel electrophoresis; and (c) alkaline elution. Alkaline sucrose gradient sedimentation for DNA analysis requires 104–106 radioactively labeled mammalian cell nuclei and the detection limit is ∼1–2 sites per 108 Da (3.3–6.6 × 10−3 sites kb−1 that corresponds to 0.5–5 fmol of damage in 50–500 ng of DNA) [1]. A fluorimetric method of DNA detection can also be used for analysis of nonradioactive DNA by alkaline sucrose sedimentation, but requires approximately 500 ng of cellular DNA [1]. Agarose gel electrophoresis allows for quantitative determination of radiation- and chemical-induced lesions in DNA that result in SSBs or alkali-labile sites or lesions that can be converted chemically or enzymatically to SSBs or alkali-labile sites [1], [2]. The method is advantageous for quantitation of lesions in DNA that cannot be labeled easily with radioactivity as the DNA in gel is detected by staining with a fluorophore [3], [4]. The limiting sensitivity for static-field electrophoresis is about one SSB per million bases [1], whereas unidirectional pulsed-field gradient gel electrophoresis [2] and orthogonal alternating-field gel electrophoresis [5], [6] have been used to overcome reptation-limited dispersion of longer DNA molecules in neutral agarose gels. Low levels of DNA damage can be detected by alkaline elution (1 per 2–3 × 109 Da or about 1 × 10−4 kb−1), but it also requires 105–2 × 106 radioactive or nonradioactive mammalian cells [7], [8] corresponding to 500–2000 ng of DNA.
In this paper we present our contribution, outlined in Fig. 1a and b, to explore the possibility of determining UVC-induced SSBs using short single-stranded oligonucleotides immobilized onto silicon wafer-based chips. The procedure for measuring SSBs using chip-based assay is evolved from the idea that in case of oligonucleotides with a few hundreds of base pairs, the possibility of UV-induced multi-hits, resulting in SSBs, per molecule is negligible. Considering the fact that the average number of UVC-induced SSBs even in plasmid DNAs is in the range of 10−5–10−6 kbp−1 J−1 m−2[9], it can be assumed that the possibility of more than one single-strand breaks in an oligonucleotide probe as short as 20 mer is practically nil. Therefore, if fluorophore-labeled oligonucleotides, immobilized on chip, are exposed to UV radiation while keeping the chip in an appropriate buffer medium, the amount of oligonucleotides, back calculated from the fluorescence measurement of the supernatant, theoretically represents the total number of single-strand breaks.
We showed that SSBs determination by our analysis method gave accurate measures of SSB yields, i.e., comparable to the predicted values. On silicon dioxide chip surface we anchored fluorescent-ss oligonucleotides, verifying the surface coverage upon digestion with alkaline phosphatase (ALP), a hydrolase enzyme. The average surface coverage value was subsequently used to calculate the number of expected breaks based on a theoretical extrapolation of previous works [9], [10] determining the average number of UV-induced SSBs for plasmid DNAs using agarose gel electrophoresis. Unlike gel-based methods, the chip-based assay allows for high-throughput and rapid determination of UV-induced SSB using small single-stranded oligonucleotides.
In addition, LIF detection using a fused silica capillary as sample holder [11] enabled highly sensitive quantitative fluorescence measurement of fluorescent probes over a wide dynamic range and thereby permitted the use of a small sample volume and very low concentration of probes.
Section snippets
Chemicals
Toluene, (3-aminopropyl)triethoxysilane (APTES), ethylenediamine tetraacetic acid, disodium salt (EDTA), Tris–HCl aqueous solution (1 M), potassium dihydrogenphosphate (KH2PO4), dipotassium hydrogenphosphate (K2HPO4), sodium chloride (NaCl), magnesium chloride (MgCl2), and alkaline phosphatase from Escherichia coli were obtained from Sigma (St. Louis, MO, USA). Glutaraldehyde was purchased from Electron Microscopy Sciences (Fort Washington, PA, USA). Silicon wafers (100) with a 1-μm-thick oxide
Fluorescence measurement calibration
Since the fluorescence quantum yield of fluorescein is dependent on the substrate to which it is bound [12], the corresponding fluorescence calibration plot was determined for fluorescein-labeled oligonucleotide used in this study. Fluorescence intensity (λex = 488 nm, λem = 520 nm) was measured four times for each individual sample at concentrations in the range 10−9–10−7 M, prepared from 10−4 M stock solution of oligonucleotide, in the digestion buffer (Tris–HCl), after digestion of the
Conclusion
The chip-based method offered a rapid, simple approach to quantify the UVC-induced SSBs determination. The closeness of experimentally obtained and predicted values of UV-induced SSBs indirectly validated the chip-based assays and ALP-digestion assay. Our results provide a basis for measurement of SSB induced by UV radiation using oligonucleotide chip. However, it is imperative that the flexibility of the method and its usefulness for evaluating the radiation or chemical-induced DNA strand
Acknowledgements
This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Ministry of Science & Technology (MOST), Government of Korea under the contract numbers R01-2007-000-20238-0 (2007) and M20702000005-08N0200-00510.
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