Method for in vitro molecular evolution of protein function using varied exonuclease digestion in two polynucleotide populations

US7262012B2 · kind B2 · utility

Assignee

• ALLIGATOR BIOSCIENCE AB · SE

Inventors

• Christina Furebring · Lund, SE
• Roland Carlsson · Lund, SE
• Carl Arne Krister Borrebaeck · Lund, SE
• Ann-Christin Malmborg Hager · Helsingborg, SE

Key dates

• Filing date: May 16, 2003
• Grant date: Aug 28, 2007
• Expiry date: Aug 12, 2023

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12N15/1058
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

A method for in vitro molecular evolution of protein function The invention provides a method for generating a polynucleotide sequence or population of sequences from parent single-stranded polynucleotide sequences encoding one or more protein motifs, comprising the steps of (a) providing a first population of single-stranded polynucleotide molecules and a second population of single-stranded polynucleotide molecules, the first and second populations together constituting plus and minus strands of parent polynucleotide sequences, (b) carrying out a reaction for digesting the first and second populations of single-stranded polynucleotide molecules with an exonuclease to generate corresponding populations of single-stranded polynucleotide fragments, (c) contacting said fragments generated from the plus strands with fragments generated from the minus strands and optionally, adding primer sequences that anneal to the 3′ and 5′ends of at least one of the parent polynucleotides under annealing conditions, and (d) amplifying the fragments that anneal to each other to generate at least one polynucleotide sequence encoding one or more protein motifs having altered characteristics as compare…