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建立了高效液相色谱/紫外法测定刺参组织全基因组DNA甲基化水平的方法,并运用此方法对喹噁啉药物处理过的刺参样品DNA甲基化水平进行分析。色谱条件为:色谱柱为ZORBAX SB-Aq(4.6 mm×250mm;5μm);柱温为30℃;检测波长为280 nm;进样量20μL;流动相为甲醇-7 mmol/L乙酸铵(7∶93),流速为1.0 mL/min。5-甲基脱氧胞苷和脱氧胞苷的质量浓度在0.05~1.0 mg/L范围内时,线性关系良好,相关系数(r)均为0.999 9。5-甲基脱氧胞苷和脱氧胞苷的检出限均为0.05 mg/L,在加标浓度为0.05,0.25,1.0 mg/L时,回收率为90.4%~100.6%,批内、批间相对标准偏差均小于4%。采用CTAB法提取刺参组织DNA,酶解后进行HPLC测定,结果表明:喹噁啉药物处理过的组织样品DNA甲基化水平低于对照组,该类药物通过改变DNA甲基化水平而影响基因的正常表达,可能是其产生遗传毒性的一种机制,建立的方法可以很好地用于全基因组DNA总甲基化水平的检测。
A method for the determination of methylation level of whole genome DNA in sea cucumbers by high performance liquid chromatography / ultraviolet spectrophotometry was established. The DNA methylation levels of the samples treated with quinoxaline were also analyzed. The chromatographic conditions were: the column was ZORBAX SB-Aq (4.6 mm × 250 mm; 5 μm); the column temperature was 30 ℃; the detection wavelength was 280 nm; the injection volume was 20 μL; the mobile phase was methanol-7 mmol / : 93) at a flow rate of 1.0 mL / min. The linearity of 5-methyl deoxycytidine and deoxycytidine was good in the range of 0.05-1.0 mg / L, the correlation coefficients (r) were 0.999 9.5-methyl deoxycytidine and deoxycytidine The detection limits were both 0.05 mg / L. The recoveries ranged from 90.4% to 100.6% at the spiked concentrations of 0.05, 0.25 and 1.0 mg / L. The relative standard deviations (RSDs) were less than 4%. CTAB method was used to extract the DNA of S. alata. After enzyme digestion, the results of HPLC assay showed that DNA methylation level of quinoxaline-treated tissue samples was lower than that of the control group, and these drugs affected the DNA methylation level The normal expression of the gene may be a mechanism of its genotoxicity, and the established method can be well used for the detection of the total methylation level of the whole genome DNA.