Partial homologous recombination of DNA chain

• US 7,220,548 B2
• Filed: 03/10/2004
• Issued: 05/22/2007
• Est. Priority Date: 03/13/2003
• Status: Expired due to Fees

• Current Assignee: Aisin Cosmos R&D Co., Ltd. (Aisin Corp.), Kazusa DNA Research Institute Foundation.
• Original Assignee: Aisin Cosmos R&D Co., Ltd. (Aisin Corp.), Kazusa DNA Research Institute Foundation.
• Inventors: Ohara, Osamu, Oishi, Michio, Kondo, Kazuhiro
• Primary Examiner(s): Shibuya; Mark L.
• Assistant Examiner(s): Lundgren; Jeffrey S.
• Application Number:
  US10/798,750
• Publication Number:
  US 20040180374A1
• Time in Patent Office: 1,168 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1082 Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2537/119 Triple helix formation

Abstract

The present invention provides a method of constructing a circular DNA library having an increased content of a desired first dsDNA by removing a second dsDNA using RecA protein to introduce a target single strand nucleic acid by homologous recombination at the 3′ terminal portion of the second dsDNA, whereby the target DNA has a 3′ terminal portion that differs from the 3′ terminal portion of the second dsDNA to prevent circularization, thereby creating a triple stranded DNA portion at the 3′ terminal end of the second dsDNA, adding Exonuclease I to digest the displaced first strand of the second dsDNA, ligating the DNA fragments to circularize the desired first dsDNA, removing the linear second dsDNA, thereby constructing the circularized DNA library having an increased content of the desired first dsDNA.

Claims

1. A method of constructing a DNA library having an increased content of a first dsDNA by removing a second dsDNA, which is different from the first dsDNA, from a DNA library containing the first dsDNA whose content is to be increased and the second dsDNA, comprising:

(1) adding a third ss nucleic acid, which contains a homologous sequence to the DNA library, said third ss nucleic acid containing a sequence that is homologous to a 3′

terminal portion of a first strand of the second dsDNA, the homologous sequence being located at a position other than the 3′

terminal portion of the third ss nucleic acid, said third ss nucleic acid having a 3′

terminal sequence that is different from that of the second dsDNA;

(2) adding a RecA protein to the DNA library, thereby catalyzing homologous recombination between the 3′

terminal portion of the first strand of the second dsDNA and the third ss nucleic acid to form a triple stranded portion at the 3′

terminal portion of the second dsDNA consisting of the first strand of the second dsDNA, the third ss nucleic acid, and a second strand of the second dsDNA;

(3) adding Exonuclease I to the DNA library containing a homologous recombinant (triple stranded portion) to digest the first strand of the second dsDNA of the triple stranded portion;

(4) ligating a DNA fragment to circularize the first dsDNA; and

(5) removing linear DNA not reacted in the ligation treatment of (4), thereby constructing the DNA library having an increased content of the first dsDNA.

2. The method according to claim 1 wherein the DNA library is a circular DNA library, further comprising a treatment for cleaving circular dsDNA prior to step (1).

3. The method according to claim 1, wherein the ligation is self-ligation.

4. A method of constructing a DNA library having an increased content of a first dsDNA, said first dsDNA comprised of a first nucleic acid strand and a second nucleic acid strand, by condensing the first dsDNA from a DNA library containing the first dsDNA whose content is to be increased, comprising:

(1) mixing a third ss nucleic acid which contains a homologous sequence to a 3′

terminal portion of a first nucleic acid strand of the first dsDNA and contains a sequence capable of providing a restriction site at the 3′

terminal portion thereof, and a fourth ss nucleic acid which contains a sequence capable of hybridizing to the 3′

terminal portion of the third ss nucleic acid and forming the restriction site at the hybridized portion with the third ss nucleic acid and a label, and hybridizing the 3′

terminal portion of the third ss nucleic acid and the fourth ss nucleic acid to form a fifth nucleic acid to forming a restriction site at the double stranded portion of the fifth nucleic acid;

(2) adding a RecA protein and the fifth nucleic acid obtained in step (1) to the DNA library and leading to homologous recombination between a part of the first dsDNA and a portion of the third ss nucleic acid of the fifth nucleic acid to form a triple stranded portion formed of a first nucleic acid strand of the first dsDNA, the portion of the third ss nucleic acid, and a second nucleic acid strand of the first dsDNA, the 3′

terminal of the fourth nucleic acid of the fifth nucleic acid flanked by the 5′

terminal of the second nucleic acid strand of the first dsDNA;

(3) adding Exonuclease I to the DNA library obtained in step (2) to digest the first nucleic acid strand of the first dsDNA of the triple stranded portion;

(4) recovering a complex containing the fourth ss nucleic acid from the DNA library via the label;

(5) cleaving the restriction site of the complex recovered in step (4) by an appropriate restriction enzyme;

(6) ligating a DNA fragment cleaved in step (5) to circularize the first dsDNA; and

(7) removing a linear DNA not reacted in step (6), thereby constructing the DNA library having an increased content of the first dsDNA.

5. The method according to claim 4 wherein the DNA library is a circular DNA library, further comprising a treatment for cleaving circular dsDNA prior to step (1).

6. The method according to claim 4, wherein the ligation is self-ligation.