USE OF SECONDARY PULSED-FIELD GEL-ELECTROPHORESIS IN SEPARATION OF LARGE DNA

It has been reported that secondary pulsed field gel (SPFG) electropho resis can dramatically increase the speed of separation of large DNA m olecules without a decrease in resolution (Zhang, T. Y., Smith, C. L., and Canter, C. R. (1991) Nucleic Acids Res. 19, 1291-1296). However, our attempts to duplicate previous SPFG conditions were unsuccessful. We therefore sought to more precisely define the effects of secondary pulsing on the separation of large DNA and to determine the value of t he technique in separating molecules up to 1100 kb. Here we report on two of the key SPFG parameters, namely the frequency and duration of t he secondary pulse on the migration and resolution of DNA in SPFG. We found that the size range of separation is determined by the sum of th e duration of the primary and secondary pulses. Under optimal conditio ns, a 25-70% increase in velocity can be achieved without loss in reso lution. (C) 1995 Academic Press, Inc.