Method to eliminate linear DNA from mixture containing nicked-circular supercoiled and linear plasmid DNA

摘要

Preparations of circular plasmid DNA in either supercoiled or nicked-circular form often are contaminated with undesired linear DNA fragments arising from shearing/degradation of chromosomal DNA or linearization of plasmid DNA itself. We report a simple enzymatic method, using a combination of λ exonuclease and RecJf, for the selective removal of linear DNA from such mixtures. λ exonuclease digests one strand of linear duplex DNA in the 5’ to 3’ direction, whereas RecJf, a single-strand-specific exonuclease, digests the remaining complementary single strand into mononucleotides. This combination of exonucleases can remove linear DNA from a mixture of linear and supercoiled DNA, leaving the supercoiled form intact. Furthermore, the inability of λ exonuclease to initiate digestion at nicks or gaps enables the removal of undesired linear DNA when nicked-circular DNA has been enzymatically prepared from supercoiled DNA. This method can be useful in the preparation of homogeneous circular plasmid DNA required for therapeutic applications and biophysical studies.