分光光度法测定脱氧核糖核酸——基于其与亚甲蓝的反应

Spectrophotometric Determination of Deoxyribonucleic Acid Based on Its Reaction with Methylene Blue

试验发现在pH 6.5的B-R缓冲溶液中在加脱氧核糖核酸(DNA)后亚甲蓝溶液在其吸收峰666 nm波长处的吸收明显减弱(即色泽减褪),而且吸光度的减弱(ΔA)程度与DNA的质量浓度在10.0 mg.L-1以内保持线性关系,其相关系数为0.9998,此反应对DNA的检出限为0.10 mg.L-1,分别以蛋白酶、胰蛋白酶及溶菌酶作为基体定量加入DNA配制成3种合成样品,按所提出方法进行分析,测得回收率结果在98.4%~101.0%之间,分析结果的RSD(n=11)值在1.1%~3.1%之间。对反应条件(包括亚甲蓝的浓度、反应的温度、酸度及时间等因素)作了试验和选定,还对反应机理作了简要的讨论。

It was found that in a B-R buffer solution of pH 6.5,the absorption of methylene blue at its absorption maxima of 666 nm was remarkably decreased upon the addition of DNA,and linear relationship was obtained between the magnitude of decrease in absorbance(ΔA) and concentration of DNA in the range within 10.0 mg·L-1,with correlation coefficient of 0.999 8 and detection limit of 0.10 mg·L-1.The proposed method was applied to the determination of DNA in 3 simulated samples based on proteinase,trypsin and bacte...