50] Conversion of circular DNA to linear strands for mapping

Abstract

Publisher Summary This chapter describes the conversion of circular DNA to linear strands for mapping. Circular DNA molecules are a suitable substrate for restriction endonucleases. When properly treated, covalently closed circular DNAs can be converted by a multihitting restriction endonuclease to full-length linear molecules. A restriction endonuclease digestion of DNA can be inhibited by ethidium bromide (EtdBr). The optimal concentration of ethidium bromide required to limit the cleavage of covalently closed circular DNA by a multihitting restriction endonuclease can only be determined by titration. In order to obtain optimal yields it is necessary to determine the temperature at which the restriction endonuclease is most active. After determining the optimal temperature the yield of form III can be maximized by titrating the EtdBr while maintaining all other variables constant. At temperatures with greater enzymatic activity, more EtdBr is required to produce similar effects to those achieved at other temperatures. The technique presented allows for gel calibration and restriction endonuclease site mapping of circular DNAs without the introduction of external standards.