Method for making linear, covalently closed DNA constructs

US6451563B1 · kind B1 · utility

Assignee

• Mologen Forschungs Entwicklungs und Vertriebs GmbH · DE

Inventors

• Burghardt Wittig · Berlin, DE
• Claas Junghans · Berlin, DE
• Matthias Schroff · Berlin, DE

Key dates

• Filing date: Feb 14, 2000
• Grant date: Sep 17, 2002
• Expiry date: Feb 14, 2020

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/66
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A process to obtain linear double-stranded covalently closed DNA “dumbbell” constructs from plasmids by restriction digest, subsequent ligation with hairpin oligodesoxyribonucleotides, optionally in the presence of restriction enzyme, and a final digestion with endo- and exonucleolytic enzymes that degrade all contaminating polymeric DNA molecules but the desired construct. The invention also provides a process to obtain said dumbbell constructs employing endonuclease class II enzymes. Furthermore, the invention provides a process to obtain linear, covalently closed DNA molecules, such as plasmids, free from contamination by genomic DNA, by submitting the DNA preparation to a facultative endonucleolytic degradation step and an obligatory exonucleolytic degradation step.