Method for processing a library using ligation inhibition

• US 20030064404A1
• Filed: 09/24/2002
• Published: 04/03/2003
• Est. Priority Date: 09/25/2001
• Status: Active Grant

• 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.)
• Inventors: Kondo, Kazuhiro, Ohara, Osamu, Oishi, Michio
• Granted Patent: US 6,867,001 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Abstract

An objective of this invention is to provide a method which can specifically enrich a desired DNA with a long insert size from a DNA library and can provide a clone of the DNA directly. This invention provides a method of constructing a DNA library having increased proportion of a first double-stranded DNA therein by removing, from an original library containing the first double-stranded DNA to be increased in proportion, a second double-stranded DNA different from the first double-stranded DNA.

Claims

1. A method of constructing a circular DNA library having increased proportion of a first double-stranded DNA to be increased in proportion, by removing, from an original circular DNA library, a second double-stranded DNA that is not identical to the first double-stranded DNA, said method comprises the following steps of;

(1) linearizing a circular DNA in the original circular DNA library to obtain a linear DNA library;

(2) preparing a single-stranded DNA corresponding to the second double-stranded DNA;

(3) adding, to the linear DNA library, RecA protein and the second single-stranded DNA prepared in the step (2) to produce triplexed DNA through the binding of the second single-stranded DNA to the second double-stranded DNA via the RecA protein;

(4) subjecting the linear DNA containing the triplexed DNA prepared in the step (3) to self-ligation to selectively circularize a double-stranded DNA not having triplex structure;

(5) removing a linear DNA from the DNA subjected to treatment in the step (4) thereby constructing a DNA library having increased proportion of the first double-stranded DNA.

2. The method according to the claim 1, wherein the linear DNA is removed from the DNA subjected to treatment in the step (4) by transforming a host with the DNA obtained in the step (4) to select a host transformed with circular DNA using a drug.

3. A method of constructing a DNA library having increased proportion of a first double-stranded DNA to be increased in proportion, by removing, from an original circular DNA library, a second double-stranded DNA that is not identical to the first double-stranded DNA, said method comprises the following steps of;

(1) preparing a linear DNA library containing the first double-stranded DNA;

(2) preparing single-stranded DNAs corresponding to both ends of the second double-stranded DNA;

(3) adding, to the linear DNA, RecA protein and the second single-stranded DNA prepared in the step (2) to produce triplexed DNA through the binding of the second single-stranded DNA to the second double-stranded DNA via the RecA protein;

(4) subjecting the linear DNA containing the triplexed DNA prepared in the step (3) to ligation with a desired DNA to selectively circularize double-stranded DNA not having triplex structure;

(5) removing the linear DNAs from the DNA subjected to treatment in the step (4) thereby constructing a DNA library having increased proportion of the first double-stranded DNA.

4. The method according to the claim 3, wherein the linear DNA is removed from the DNA subjected to treatment in the step (4) by transforming a host with the DNA obtained in the step (4) to select a host transformed with circular DNA using a drug.