反相高效液相色谱法测定大鼠肝组织中DNA的碱基含量

Determination of the Base Contents of Liver DNA of Rats by Reversed-Phase High Performance Liquid Chromatography

利用反相高效液相色谱法 ,采用SupelcosilLC 18色谱柱 (2 5 0mm× 4 6mmi d ,5 μm) ,以甲醇 0 0 5mol/LKH2 PO4缓冲液 (体积比为 2 0∶80 )为流动相 ,流速 0 8mL/min ,在 2 5 4nm波长处检测 ,对生活在高原 (海拔 2 3km)的习服大鼠肝组织中脱氧核糖核酸 (DNA)的碱基含量进行了检测 ,发现各碱基在DNA中所占的比例是相对稳定的 :腺嘌呤 (A) 2 8 8%、鸟嘌呤 (G) 2 3 3%、胞嘧啶 (C) 17 4 %、胸腺嘧啶 (T) 2 5 3% 。

The base contents of liver deoxyribonucleic acid (DNA) of rats living at an altitude of 2.3 km were determined by reversed phase high performance liquid chromatography. At first, 0 05 mol/L KH 2PO 4 (pH 4.0) was used to dissolve the DNA acid hydrolysis products with 8 bromoguanosine (Br 8G) as an internal standard. Then the DNA hydrolysis products with Br 8G were chromatographed on a Supelcosil LC 18 column with UV detection at 254 nm and eluted by the mobile phase of MeOH 0 05 mol/L KH 2PO 4 (pH 4.0) (20∶80, V/V ) at the flow rate of 0 8 mL/min. Under these conditions, several bases were separated effectively. From the results, the relatively constant proportions of the bases in DNA were found. The contents were 17.4% of cytosine (C), 28 8% of adenine (A), 23 3% of guanine (G) and 25 3% of thymine (T). RSDs of the deteremination of these bases were 1 7%, 1 5%, 1 3% and 2 1%, respectively. At the same time the methylation level of liver DNA of the rats determined by the internal standard method was 6 2%.