Improved phenol emulsion DNA reassociation technique (PERT) using thermal cycling.

Phenol emulsion reassociation technique (PERT) greatly enhances the rate of DNA hybridization in solution over typical hybridization conditions (1) and has successfully been used to accelerate competitive DNA reassociation for performing subtraction based genomic cloning (2-5). PERT is accomplished through the establishment and maintenance of a phenol emulsion, typically by mechanical vortexing or shaking. While using this technique, we encountered emulsion stability problems and found stability was directly dependent upon DNA concentration. At low DNA concentrations and small volumes the emulsions became difficult to maintain even with vigorous agitation. Since it is at low DNA concentrations, when hybridization times using standard conditions are prohibitively long that PERT is most useful, we sought alternative mechanisms for maintaining a phenol emulsion. We describe a method to maintain a phenol emulsion in the presence of low DNA concentrations which does not require mechanical agitation. Rather, an emulsion is established and maintained by thermal cycling the PERT hybridization reaction.