Capillary Electrophoresis of DNA in Hydroxyethylcellulose
Received date: 2012-11-19
Online published: 2012-12-25
Supported by
Project supported by the National Natural Science Foundation of China (No. 21205078), Research Fund for the Doctoral Program of Higher Education of China (No. 20123120110002), Teachers’ Innovation Ability Construction of University of Shanghai for Science and Technology (No. GDCX-Y-1205), the Innovation Program of Shanghai Municipal Education Commission (No. 13YZ073), Shanghai Committee of Science and Technology (No. 10540500700) and the Innovation Fund Project For Graduate Student of Shanghai (No. JWCXSL1202), and partly supported by the Leading Academic Discipline Project of Shanghai Municipal Government (S30502).
Using hydroxyethylcellulose (HEC) solution as polymer matrix, this work systematically studied the separation performance of 100 base pairs (bp) DNA ladder (100～1500 bp) by direct current electric field capillary electrophoresis (CE). In the present paper, we systematically investigated the influence of polymer concentration and molecular weight of HEC, electric field strength (E), the effective length (le) and the shape of the capillary, the temperature of the background electrolyte (BGE) on the separation performance of DNA. Furthermore, we compared the migration of DNA in polymer with the non-gel sieving model. Results show that: (1) When the concentration of HEC is above the entangled threshold c*, the mobility difference increases with the growth of molecular weight, whereas the mobility of DNA decreases with the rise of concentration of HEC. (2) The resolution of adjacent DNA fragments linearly increases with the effective length (le) of the capillary when le ranges from 4 to 12 cm. (3) The mobility of DNA increases with the growth of area ratio R (Slateral/Ssection), and thus the separation performance improves. (4) The increase of BGE temperature strengthens the diffusion effect of DNA, thus increases the mobility, and deteriorates the resolution. Based on the results above, we separated the φ×174-Hirc II digest by CE in an optimal electrophoretic condition. Experiment shows that rapid separation of φ×174-Hirc II digest was realized with high resolution. In our experiment, the fused-silica capillary is coated by acrylamide, and the background electrolyte (BGE) used for the sieving matrix contains 0.5×Tris-borate-EDTA (TBE) and 1×SYBR Green I. The DNA sample was injected for 2.0 s at an E of 100 V/cm. The self-build CE device involved is reliable and showed some remarkable achievements previously. Such a study is beneficial to the realization of rapid and effective separation of DNA, and allows deep insight into DNA migration in the polymer matrix under constant electric field.
Key words： capillary electrophoresis; hydroxyethylcellulose; DNA
Liu Chenchen , Li Zhenqing , Meng Fanming , Ni Yi , Dou Xiaoming , Yamaguchi Yoshinori . Capillary Electrophoresis of DNA in Hydroxyethylcellulose[J]. Acta Chimica Sinica, 2013 , 71(02) : 265 -270 . DOI: 10.6023/A12110935
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