Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate.

Abstract

A novel assay was developed for in vitro genetic recombination of DNA. Substrate and product DNA are cleaved with a restriction endonuclease [EcoRI], and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA was chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriction fragment, as does the DNA segment that was removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental [Escherichia coli] to recombinant DNA. This method was applied to an in vitro system that carries out integrative recombination of bacteriophage .lambda.. Of the different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and 3 kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. The in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found.