Synthesis of hybrid polynucleotide molecules using single-stranded polynucleotide molecules

• US 7,662,551 B2
• Filed: 11/18/2002
• Issued: 02/16/2010
• Est. Priority Date: 12/22/2000
• Status: Expired due to Fees

• Current Assignee: Alligator Bioscience AB
• Original Assignee: Alligator Bioscience AB
• Inventors: Kikuchi, Miho, Harayama, Shigeaki, Ohnishi, Kouhei
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/299,362
• Publication Number:
  US 20030138825A1
• Time in Patent Office: 2,647 Days
• Field of Search: 435/6, 435/91.1, 435/183, 435/320.1, 435/455, 435/462, 435/468, 435/91.2, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR

Abstract

A method to make libraries of hybrid polynucleotide molecules of two parental polynucleotide molecules utilizing single-stranded DNA was invented. Example of the method comprises several steps: (i) preparation of two single-stranded polynucleotide molecules comprising sequences containing one or more parts of homology and one or more parts of heterology, (ii) random or non-random fragmentation of said polynucleotides, (iii) hybridization of the fragmented molecules followed by de novo polynucleotide synthesis (i.e. polynucleotide chain elongation) on the hybridized molecules, (iv) separation of the chain elongation products (i.e. double-stranded polynucleotide molecules) into single-stranded polynucleotide molecules (denaturation) (v) hybridization of the resultant single-stranded polynucleotide molecules followed by de novo polynucleotide synthesis on the hybridized molecules, and (vi) repeating at least two further cycles of steps (iv) and (v).

Claims

1. A method for making libraries of hybrid polynucleotide molecules comprising the steps of:

(i) selecting and separately cloning a first single-stranded polynucleotide comprising a coding strand of a first family gene;

(ii) selecting and separately cloning a second single-stranded polynucleotide comprising a non-coding strand of a second family gene;

(iii) fragmenting the first and second single stranded polynucleotides thereby forming polynucleotide fragments;

(iv) incubating the polynucleotide fragments formed in step (iii) under conditions such that hybridization occurs to form heteroduplex molecules; and

(v) conducting nucleotide elongation on the heteroduplex molecules, wherein said first and second single-stranded polynucleotide molecules are used as starting molecules, thereby providing said library of hybrid polynucleotide molecules.

2. The method of claim 1, wherein the first family gene is different from the second family gene and wherein the first single-stranded polynucleotide comprises at least one homologous sequence and at least one sequence which is heterologous to the second single-stranded polynucleotide.

3. The method of claims 1 or 2 wherein mutations are introduced into hybrid polynucleotide molecules.

4. The method of claim 2, wherein said homologous sequence is at least 15 bases.

5. The method of claim 1, wherein said cloning of said first single stranded polynucleotides in step (i) and said cloning of said second single stranded polynucleotides in step (ii) are performed by cloning said first and second single stranded polynucleotides using a phagemid vector or a single-stranded DNA phage vector.

6. The method of claim 1, wherein said cloning of said first single stranded polynucleotides in step (i) and said cloning of said second single stranded polynucleotides in step (ii) are performed by asymmetric PCR.

8. The method of claims 1 or 7, wherein at least one of the two single-stranded polynucleotides is fragmented randomly.

9. The methods of claims 1 or 7, wherein both of the two single-stranded polynucleotides is fragmented randomly.

10. The method of claims 1 or 7, wherein mutations are introduced into at least one first and second single-stranded polynucleotides prior to production of heteroduplex molecules.

11. The method of claims 1 or 7, wherein mutations are introduced into both of the first and second single-stranded polynucleotides prior to production of the heteroduplex molecules.

7. A method for making libraries of hybrid polynucleotide molecules, which comprises:

(i) preparing and separately cloning two single stranded polynucleotide molecules comprising sequences which are complementary to each other,

(ii) fragmenting the two single-stranded polynucleotide molecules,

(iii) incubating the fragmented molecules under conditions such that hybridization of fragmented polynucleotide molecules occurs and de novo polynucleotide synthesis on the hybridized molecules occurs to produce elongated double stranded polynucleotide molecules,

(iv) denaturing the elongated double stranded polynucleotide molecules produced in step (iii) into single-stranded polynucleotide molecules,

(v) incubating the single-stranded polynucleotide molecules produced in step (iv) under conditions such that hybridization of single-stranded polynucleotide molecules occurs and de novo polynucleotide synthesis of hybridized molecules occurs, and

(vi) repeating at least two further cycles of (iv) and (v), thereby providing said library of hybrid polynucleotide molecules.

12. The method of claim 7, wherein said cloning of said two single stranded polynucleotide molecules in step (i) is performed by cloning said two single stranded polynucleotides using a phagemid vector or a single-stranded DNA phage vector.

13. The method of claim 7, wherein said cloning of said two single stranded polynucleotide molecules in step (i) is performed by asymmetric PCR.