Patent ID: 6451563
Filing Date: 2002-09-17
Classification: C12N

Abstract:
A method for obtaining a non-circular covalently closed double-stranded DNA molecule, the method comprising:(a) providing a first composition comprising a double-stranded DNA comprising a target DNA sequence; (b) excising the target DNA sequence from the DNA with an endonuclease to provide a second composition comprising the target DNA sequence; (c) providing a third composition comprising: (i) the target DNA sequence and (ii) one or more hairpin-shaped oligodesoxyribonucleotide(s); (d) ligating 3â€² and 5â€² strands of a hairpin-shaped oligodesoxyribonucleotide of (c) to respective ends of 3â€² and 5â€² strands of the target DNA sequence to provide a fourth composition comprising a mixture of: (i) covalently closed double-stranded DNA comprising the target sequence, and (ii) unligated contaminating nucleic acid material; (e) digesting components of the fourth composition in the presence of nucleases having affinity only for the contaminating nucleic acid material to provide a fifth composition comprising: (i) the covalently closed double-stranded DNA comprising the target sequence, and (ii) at least partially digested contaminating nucleic acid material, which is separable from the covalently closed double-stranded DNA comprising the target sequence by at least one physical or chemical characteristic comprising size; and (f) separating the covalently closed double-stranded DNA comprising the target sequence from one or more other components of the fifth composition to provide a sixth composition comprising the covalently closed double-stranded DNA, which sixth composition is substantially free of contaminating nucleic acid material, wherein any of steps (a) to (e) are temporally related in any manner that results in the formation of the fifth composition.