Open AccessIdentifying the Orientation of DNA Fragment in Recombinant Plasmid by Capillary Electrophoresis with a Non-Gel Sieving Solution
Author(s) -
BiFeng Liu,
Qiguang Xie,
YingTang Lu
Publication year - 2001
Publication title -
analytical sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.392
H-Index - 73
eISSN - 1348-2246
pISSN - 0910-6340
DOI - 10.2116/analsci.17.1253
Subject(s) - haeiii , chemistry , capillary electrophoresis , plasmid , recombinant dna , dna , pbr322 , chromatography , electrophoresis , gel electrophoresis , microbiology and biotechnology , restriction fragment , restriction enzyme , base pair , biochemistry , gene , restriction fragment length polymorphism , polymerase chain reaction , biology
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 microM 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 lambda DNA/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.