Method for processing a library using ligation inhibition

• US 6,867,001 B2
• Filed: 09/24/2002
• Issued: 03/15/2005
• Est. Priority Date: 09/25/2001
• 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: Kondo, Kazuhiro, Ohara, Osamu, Oishi, Michio
• Primary Examiner(s): Sitton, Jehanne
• Assistant Examiner(s): BAUSCH, SARAE L
• Application Number:
  US10/253,319
• Publication Number:
  US 20030064404A1
• Time in Patent Office: 903 Days
• Field of Search: 435/6, 435/91.1, 435/5, 536/23.1
• US Class Current: 506/17
• 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 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 comprising the following steps:

(1) linearizing circular DNA in an original circular DNA library comprising a first double-stranded DNA and a second double-stranded DNA to form a linear DNA library;

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

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

(4) subjecting the mixture formed in step (3) to self-ligation to selectively circularize double-stranded DNA that dose not contain a triplex; and

(5) removing noncircular DNA from the ligated mixture formed in step (4) to produce a circular DNA library having increased proportion of the first double-stranded DNA in comparison to the original DNA library.

2. The method of claim 1, wherein in step (5) the noncircular DNA is removed from the circularized DNA formed in step (4) by transforming a host with the circularized DNA in step (4) using drug selection.

3. A method of constructing a circular DNA library having increased proportion of a first double-stranded DNA 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 comprising the following steps:

(1) linearizing circular DNA in an original circular DNA library comprising a first double-stranded DNA and a second double-stranded DNA to form a linear DNA library;

(2) preparing a single-stranded DNA mixture comprising a first single-stranded DNA corresponding to the 5′

end of the second double-stranded DNA and a second single-stranded DNA corresponding to the 3′

end of the second double-stranded DNA;

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

(4) subjecting the second mixture formed in step (3) to ligation with a desired DNA under conditions to selectively circularize double-stranded DNA that does not contain a triplex; and

(5) removing noncircular DNA from the ligated second mixture formed in step (4) to produce a circular DNA library having increased proportion of the first double-stranded DNA in comparison to the original DNA library.

4. The method of claim 3, wherein in step (5) the noncircular DNA is removed from the circularized DNA formed in step (4) by transforming a host with the circularized DNA in step (4) using drug selection.