样品稀释对毛细管电泳分离检测DNA片段的影响

Influence of Sample Dilution on Separation and Detection of DNA Fragments by Capillary Electrophoresis

通过理论推导和实验验证表明；适当稀释DNA样品溶液，采用流体力学进样或电动进样都不会较大地减低峰高，而DNA片段毛细管电泳的分离效率和分离度还能有所提高。采用稀释样品的方法可提高DNA样品的使用效率。采用羟乙基纤维素无胶筛分介质分离了DNA片段。用激光诱导荧光（氩离子激光器，488nm）电荷耦合器件检测。用低浓度的筛分介质（0．4％）分离了分子质量较大的ADNA－HindⅢ全部8个片段（12bp～23130bP）。用高浓度的筛分介质（1．6％）分离分子质量较小的pBR322－HaeⅢ22个片段（18bp～587bp）。将pBR322－HaeⅢDNA样品溶液稀释10倍时，123bp和124bp两个片段得到了分离。

Capillary electrophoresis has become an important and useful method to separate and determine DNA fragments. In molecular biochemistry, the volume of DNA sample is very small (μL level)and DNA sample is liable to be contaminated and degraded. According to theoretical inference and experiments, we propose that dilution of DNA sample solution can increase separation efficiency and resolution without evidently reducing height of peaks. By this method, the usage efficiency of DNA sample can be improved. It is also demonstrated the separation and detection of DNA fragments by capillary electrophoresis with hydroxyethyl cellulose non-gel sieving matrix and with laser-induced fluorescence charge-coupled device as detector.By using lower concentration non-gel matrix(0. 4% ), all 8 larger size fragments of λ DNA/Hind Ⅲ (125 bp-23 130 bp) can be completely separated. Twenty smaller size fragments of pBR322-Hae Ⅲ DNA(18 bp-587 bp)can be separated by higher concentration (1. 6% ) non-gel matrix. As ratio of sample dilution is 10, two adjacent fragment (123 bp and 124 bp) of pBR322-Hae Ⅲ DNA can be separated.