SYNTHESIS OF HYBRID POLYNUCLEOTIDE MOLECULES USING SINGLE-STRANDED POLYNUCLEOTIDE MOLECULES

• US 20080146461A1
• Filed: 02/25/2008
• Published: 06/19/2008
• Est. Priority Date: 12/22/2000
• Status: Active Grant

• Current Assignee: Alligator Bioscience AB
• Original Assignee: Alligator Bioscience AB
• Inventors: Kikuchi, Miho, Harayama, Shigeaki, Ohnishi, Kouhei
• Granted Patent: US 8,071,289 B2
• Time in Patent Office: Days
• US Class Current: 506/23
• 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 in which double-stranded polynucleotide molecules are not used as starting materials.

2. The method of claim 1, wherein two types of single-stranded polynucleotide molecules are used as starting materials and wherein the first-type molecule comprises stretches of sequences containing one or more parts of homology and one or more parts of heterology to the complementary sequence of the second-type molecule.

3. The method of claim 2, wherein the single-stranded polynucleotide molecules are fragmented and used as templates for de novo polynucleotide synthesis to create hybrid polynucleotide molecules.

4. The method of claim 2, wherein mutations are introduced into hybrid polynucleotide molecules prior, during or after the production of the hybrid polynucleotide molecules.

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

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

(ii) randomly or non-randomly 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,

(iv) denaturing the resultant elongated double-stranded polynucleotide molecules into single-stranded polynucleotide molecules,

(v) incubating the resultant single-stranded polynucleotide molecules under conditions such that hybridization of single-stranded polynucleotide molecules occurs and de novo polynucleotide synthesis on the hybridized molecules occurs, and

(vi) repeating at least two further cycles of steps (iv) and (v).