Method for in vitro molecular evolution of protein function

• US 6,958,213 B2
• Filed: 12/12/2000
• Issued: 10/25/2005
• Est. Priority Date: 12/12/2000
• Status: Expired due to Fees

• Current Assignee: Alligator Bioscience AB
• Original Assignee: Alligator Bioscience AB
• Inventors: Carlsson, Roland, Furebring, Christina, Borrebaeck, Carl, Hager, Ann-Christin Malmborg
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): Chunduru, Suryaprabha
• Application Number:
  US09/734,801
• Publication Number:
  US 20020146780A1
• Time in Patent Office: 1,778 Days
• Field of Search: 435/6, 435/91.2, 435/69.1, 536/24.33, 536/24.32, 536/23.1, 536/22.1, 530/350
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR

Abstract

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 single stranded DNA constituting plus and minus strands of parent polynucleotide sequences;
  • (b) digesting the single stranded polynucleotide sequences with a nuclease other than DNase I to generate populations of single stranded 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;
  • (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 compared to the one or more protein motifs encoded by said parent polynucleotides.

Claims

1. 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 single stranded polynucleotide sequences constituting plus and minus strands of parent polynucleotide sequences;

b) digesting the single stranded polynucleotide sequences with an exonuclease to generate a first population of single stranded fragments comprising fragments generated from the plus strands and a second population of single stranded fragments comprising fragments generated from the minus strands, said second population being separate from said first population;

c) contacting said first population of single stranded fragments with said second population of single stranded fragments, whereby annealing occurs between said first population of single stranded fragments and said second population of single stranded fragments;

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 compared to the one or more protein motifs encoded by said parent polynucleotides.

2. The method as claimed in claim 1 wherein step (c) further comprises adding primer sequences that anneal to the 3′

and 5′

ends of at least one of the parent polynucleotides under annealing conditions.

3. A method as claimed in claim 1 wherein the exonuclease is BAL31.

4. A method as claimed in claim 1 wherein a parent polynucleotide sequence or sequences has been subjected to mutagenesis.

5. A method as claimed in claim 1 wherein the population of fragments generated in step b) are subjected to mutagenesis.

6. A method as claimed in claim 4 wherein mutagenesis is error prone PCR.

7. The method as claimed in claim 1, further comprising contacting the polynucleotide sequence of step d) with primer sequences that anneal to the 3′

and 5′

ends of at least one of the parent polynucleotides under annealing conditions and amplifying those hybrids that anneal to each other to generate a further polynucleotide sequence encoding one or more protein motifs having altered characteristics as compared to one or more protein motifs encoded by said parent polynucleotide.