Fast DNA separations using poly(ethylene oxide) in non-denaturing medium with temperature programming.
We demonstrated fast DNA separations in low viscosity entangled solutions with a temperature gradient in a non-denaturing separation medium. The separations were carried out in a solution of commercially available poly(ethylene oxide) (PEO) [1 x Tris(hydroxymethyl)aminomethane borate buffer, without urea] with a temperature gradient of 2 degrees C/min. The performance was compared with that of a solution of PEO with urea at ambient temperature. We found that the former condition gives sufficient resolution for accurate base calling and that in general, it gave better separation for fragments larger than 450 base pairs (bp). Most importantly, the separation speed approaches 30 bp/min. In addition, we describe a simple yet reliable gel preparation protocol for such separations.
Fung
E N
EN
Ames Laboratory, U.S. Department of Energy and Department of Chemistry, Iowa State University 50011, USA.
Pang
H M
HM
Yeung
E S
ES
D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D011092:Polyethylene Glycols; D012996:Solutions; D013696:Temperature; D014508:Urea; D014783:Viscosity
D012996:Solutions; D011092:Polyethylene Glycols; D014508:Urea; D004247:DNA