Analytical Sciences

Abstract − Analytical Sciences, 17(11), 1253 (2001).

Identifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution
Bi-Feng LIU, Qi-Guang XIE, and Ying-Tang LU 
Key Laboratory of MOE for Plant Developmental Biology, Wuhan University, Wuhan, 430072, P. R. China
It was demonstrated that a capillary electrophoresis (CE) method with a non-gel sieving solution has been developed to identify the orientation of DNA fragments in recombinant plasmids in molecular biology. The influences of the concentration of sieving polymer HEC, the applied electric field strength and sampling on CE separation were analyzed concerning the optimization of separation. YO-PRO-1 was used as a DNA intercalating reagent to facilitate fluorescence detection. Under the chosen conditions (buffer, 1 x TBE containing 1 uM YO-PRO-1 and 1.2% HEC; applied electric field strength, 200 V/cm; electrokinetic sampling: time, 5 s; voltage, -6 kV), three DNA markers (phi 174/HaeIII, pBR322/HaeIII and lambdaDNA/HindIII) were tested for further evaluating the relationship between the DNA size and the mobility. The established CE method conjugated with the enzymatic approach was successfully applied to identifying the DNA orientation of recombinant plasmid in transgene operations of a newly cloned gene from Arabidopsis Thaliana.