Dna extraction kit

8-OHdG formation was determined using an HPLC-ECD system according to the method of Asami et al. [27 (link)]. After each exposure to UV irradiation, calf thymus DNA was isolated from the reaction mixture using a DNA-extraction kit (Wako, Osaka, Japan) according to the manufacturer’s protocol, with minor modifications to prevent the formation of 8-OHdG during DNA isolation. Isolated DNA was then digested with nucleases to obtain 8-OHdG in the nucleoside form, after which the nucleosides were injected into a Purospher^® STAR RP-18e (5 μm, 4.0 × 250 nm, Merck Chemicals, Darmstat, Germany) connected to an HPLC system. The latter system consisted of a HITACHI (Tokyo, Japan) L-2130 pump and a UV 7000 detector (EYELA, Tokyo, Japan). Electrochemical detection was accomplished using an ECD (Coulochem^® III, Guard Cell 5020; ESA Inc., Dionex, Tokyo, Japan). The mobile phase consisted of 0.2 M Na₂PO₄ containing 6% methanol. The flow rate was 1.0 mL/min with the following applied conditions: E1: 150 mV, R: 1 μA, Filter: 10 s, output: 1.0 V, E2: 300 mV, R: 50 μA, Filter: 10 s, and output: 1.0 V. DNA-specific 8-OHdG was expressed in terms of the ratio of 8-OHdG to deoxyguanosine (2dG).

Genomic DNA was extracted from whole blood cells using a DNA Extraction Kit (WAKO Pure Chemical Ltd., Osaka, Japan), according to the manufacturer’s instructions. In order to amplify all exons and exon–intron boundaries in the Aα-, Bβ-, and γ-chain genes, 32 PCR primers were designed and DNA was amplified by PCR as described elsewhere [18 (link)]. PCR products were purified from agarose gels and directly sequenced using a BigDye^TM Terminator Cycle Sequencing Ready Reaction Kit (Thermo Fisher Scientific, Waltham, MA, USA) and 3500 Genetic Analyzer (Life Technologies, Carlsbad, CA, USA).