Separation of DNA sequencing fragments up to 1000 bases by using poly(ethylene oxide)-filled capillary electrophoresis

被引:86
作者
Kim, Y
Yeung, ES
机构
[1] IOWA STATE UNIV,US DOE,AMES LAB,AMES,IA 50011
[2] IOWA STATE UNIV,DEPT CHEM,AMES,IA 50011
关键词
poly(ethylene oxide) gels; capillary columns; DNA;
D O I
10.1016/S0021-9673(97)00472-X
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We have demonstrated that DNA bases up to 1000 base pairs (bp) in a sequencing ladder can be separated using poly(ethylene oxide)-filled capillary electrophoresis (resolution of raw data = 0.5 at 966 bp). Separation performance of this sieving matrix has been tested under different experimental conditions. It was found that the electric field strength played a critical role in the onset of reptation and thus the separation efficiency. Optimized gel composition and concentration is required for good separation, but the total gel concentration should lie between 2.5 and 3.0%. We observed that the capillary length influences the number of theoretical plates and the maximum readable length of DNA. For sequencing up to 500 bp, relatively nonviscous solutions can be used, greatly facilitating the replacement of the sieving matrix in between runs. (C) 1997 Elsevier Science B.V.
引用
收藏
页码:315 / 325
页数:11
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