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 a s 460 kbp. These systems enable us to combine the rapid speed of high voltages and the separation power of pulsed field electrophoresis to a chieve high resolution and short run durations. We found that large DN A fragments can be separated at voltage gradients much higher than com monly 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 rel atively small DNA fragments (<50 kbp). We have also found an inverse r elationship 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 siz e range, DNA can be separated 8- to 25-fold faster and with better res olution than existing electrophoretic systems.