Kim Y, Yeung E S
Ames Laboratory-US Department of Energy, Iowa State University 50011, USA.
J Chromatogr A. 1997 Sep 26;781(1-2):315-25. doi: 10.1016/s0021-9673(97)00472-x.
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.
我们已经证明，使用填充聚环氧乙烷的毛细管电泳可以分离测序梯中长达1000个碱基对（bp）的DNA碱基（966 bp处原始数据的分辨率 = 0.5）。在不同实验条件下测试了这种筛分基质的分离性能。发现电场强度在链爬行的起始以及分离效率中起着关键作用。为了实现良好的分离，需要优化凝胶组成和浓度，但总凝胶浓度应在2.5%至3.0%之间。我们观察到毛细管长度会影响理论塔板数和DNA的最大可读长度。对于高达500 bp的测序，可以使用相对低粘度的溶液，这极大地方便了运行之间筛分基质的更换。
