Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide).
We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.
Yoshioka
Hiroshi
H
Advanced Research Center for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 169-8555, Japan.
Mori
Yuichi
Y
Shimizu
Mitsuhiro
M
D004247:DNA / Q000302:isolation & purification*; D004563:Electrochemistry; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry*; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D011409:Propylene Glycols / Q000138:chemical synthesis / Q000737:chemistry*; D013696:Temperature; D014170:Transformation, Genetic; D014783:Viscosity
D011108:Polymers; D011409:Propylene Glycols; C012504:polypropylene glycol; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D011092:Polyethylene Glycols; D004247:DNA