Process for obtaining recombined nucleotide sequences in vitro, libraries of sequences and sequences thus obtained

• US 7,718,786 B2
• Filed: 06/03/2005
• Issued: 05/18/2010
• Est. Priority Date: 08/12/1998
• Status: Expired due to Fees

• Current Assignee: Proteus Corporation
• Original Assignee: Proteus Corporation
• Inventors: Lefevre, Fabrice, Dupret, Daniel, Masson, Jean-Michel
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US11/143,455
• Publication Number:
  US 20050221378A1
• Time in Patent Office: 1,810 Days
• Field of Search: 435/320.1, 435/6, 536/23.1, 536/22.1
• US Class Current: 536/23.1
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR

Abstract

Ligation-mediated method of recombining polynucleotides in vitro. Polynucleotides from a library are fragmented and the fragments are hybridized to an assembly template. The hybridized fragments are iteratively re-hybridized and ligated until the ends of the hybridized fragments are adjacent to the ends of other hybridized fragments on the assembly template. A final ligation produces recombined polynucleotides.

Claims

1. A recombined polynucleotide library produced from an initial library of polynucleotide fragments comprising the steps of:

(a) hybridizing the fragments to an assembly template;

(b) ligating together the hybridized fragments that have adjacent ends with a ligase;

(c) denaturing the ligated fragments from the assembly template; and

(d) repeating at least the hybridizing, ligating and denaturing steps multiple times;

wherein said recombined polynucleotide library;

1) is obtained without the use of polymerase in steps (a) to (d),

2) comprises only the fragments present in the initial library of polynucleotide fragments and sequences having one or several properties of the sequences of the initial library, and

3) comprises randomly recombined polynucleotides comprising said ligated fragments.

2. A recombined polynucleotide library produced from an initial library of polynucleotide fragments comprising the steps of:

(a) hybridizing, to an assembly template, fragments of polynucleotides derived from the initial library of polynucleotide fragments comprised of at least two different polynucleotides;

(b) ligating together the hybridized fragments that have adjacent ends with a ligase;

(c) denaturing the ligated fragments from the assembly template; and

(d) repeating at least the hybridizing, ligating and denaturing steps multiple times, thereby forming random recombinant polynucleotides comprised of ligated fragments;

wherein said recombined polynucleotide library;

1) is obtained without the use of polymerase in steps (a) to (d),

2) comprises only the fragments present in the initial library of polynucleotide fragments and sequences having one or several properties of the sequences of the initial library, and

3) comprises randomly recombined polynucleotides comprising said ligated fragments.

3. A recombined polynucleotide library derived from an initial library of polynucleotide sequences comprising ligated fragments of said initial library of polynucleotide sequences, said fragments being aligned in accordance with a complementary assembly template, wherein the recombined polynucleotide library comprises only the fragments present in the initial library of polynucleotide sequences, said recombined polynucleotide library includes at least one recombined sequence having one or several properties of the sequences of the initial library, and wherein said recombined polynucleotide library comprises randomly recombined polynucleotides comprising said ligated fragments.