Process of clean cloning

US8883420B2 · kind B2 · utility

Assignee

• Icon Genetics GmbH · DE

Inventors

• Sylvestre Marillonnet · Bodenwerder, DE
• Stefan Werner · Halle (Saale), DE
• Carola Engler · Bodenwerder, DE
• Romy Kandzia · Bodenwerder, DE
• Frank Thieme · Halle (Saale), DE
• Ernst Weber · Halle (Saale), DE

Key dates

• Filing date: Oct 8, 2009
• Grant date: Nov 11, 2014
• Expiry date: Jun 15, 2030

Classification

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

Abstract

A process of inserting a nucleic acid sequence of interest into an acceptor nucleic acid is provided. The process comprises amplifying by PCR a DNA comprising in the following order a sequence segment U, a nucleic acid sequence segment of known nucleotide sequence K2, and a nucleic acid sequence segment of known sequence K3. The process further comprises treating the linear double-stranded DNA molecules from the PCR amplification with an exonuclease to obtain a single-stranded overhang at the first end of the DNA and a single-stranded overhang comprising nucleic acid segments K2 and K3 at the second end of the DNA. The process additionally comprises annealing the product of the exonuclease treatment to a linearized double-stranded acceptor nucleic acid which has been designed to complement the single-stranded overhangs of the product of the exonuclease treatment.