Separation of DNA sequencing fragments up to 1000 bases by using poly(ethylene oxide)-filled capillary electrophoresis.
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.
Kim
Y
Y
Ames Laboratory-US Department of Energy, Iowa State University 50011, USA.
Yeung
E S
ES
D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D053938:DNA Fragmentation; D019075:Electrophoresis, Capillary / Q000379:methods*; D009994:Osmolar Concentration; D011092:Polyethylene Glycols / Q000737:chemistry*; D017422:Sequence Analysis, DNA / Q000379:methods*
D011092:Polyethylene Glycols; D004247:DNA