Separation of large DNA molecules with high voltage pulsed field gel electrophoresis.
We have developed two high voltage (3 kV and 10 kV) high speed pulsed field gel electrophoresis systems for the separation of DNA as large as 460 kbp. These systems enable us to combine the rapid speed of high voltages and the separation power of pulsed field electrophoresis to achieve high resolution and short run durations. We found that large DNA fragments can be separated at voltage gradients much higher than commonly used. Yeast chromosomes as large as 460 kbp can be separated in 4 h at 20 V/cm and 1-50 kbp DNA can be rapidly separated in 30 min at 55 V/cm. This is 25 times faster in mobility for the separation of relatively small DNA fragments (< 50 kbp). We have also found an inverse relationship between the voltage applied and the size separation limit at that particular voltage gradient (55 V/cm limits the separation to 50 kbp while 20 V/cm can separate up to 460 kbp). Depending on the size range, DNA can be separated 8- to 25-fold faster and with better resolution than existing electrophoretic systems.
Department of Pharmacology, University of North Carolina at Chapel Hill 27599-7365.
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D000260:Adenoviruses, Human; D001482:Base Composition; D015825:Chromosomes, Fungal; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D004271:DNA, Fungal / Q000302:isolation & purification; D004279:DNA, Viral / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000295:instrumentation / Q000379:methods*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000295:instrumentation / Q000379:methods*; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae; D012685:Sepharose
D004271:DNA, Fungal; D004279:DNA, Viral; D004247:DNA; D012685:Sepharose