Use of site-specific nicking endonucleases to create single-stranded regions and applications thereof

• US 6,660,475 B2
• Filed: 12/15/2000
• Issued: 12/09/2003
• Est. Priority Date: 12/15/2000
• Status: Active Grant

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Jack, William E., Schildkraut, Ira, Menin, Julie Forney
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US09/738,444
• Publication Number:
  US 20030022317A1
• Time in Patent Office: 1,089 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to the use of site-specific nucleic acid nicking enzymes to create single-stranded regions in duplex nucleic acids. Such single-stranded regions can take the form of gaps interior to the duplex, or terminal single-stranded regions. Single-stranded termini can be crafted to allow linkage of various elements via base-pairing with elements containing a complementary single-stranded region. This joining is useful, for example, in an ordered, oriented assembly of DNA modules to create cloning or expression vectors. This joining is also useful in attaching detection probes and purifying DNA molecules containing the single-stranded region. Gaps are useful in similar applications, including attaching detection or purification probes.

Claims

1. A method for creating target single strand regions in a plurality of double stranded DNA molecules for use in joining the DNA molecules, comprising:

(a) nicking at least two sites bordering a target region within the DNA molecules with at least one site-specific nicking endonuclease;

(b) subjecting the nicked DNA molecules from step (a) to conditions that selectively denature the target region to create the target single stranded region; and

(c) joining the DNA molecules from step (b) by means of the target single strand regions.

2. A method of claim 1 wherein the at least two sites bordering the target region are located on a single strand of the double stranded DNA so that the target single stranded region comprises a gap in the double stranded DNA.

3. A method for creating a target single strand region at a terminus of a linear double stranded DNA molecule for use in joining the DNA molecule to a second DNA molecule by means of the single strand region, or for detecting, purifying or selectively mutagenizing the DNA molecule, comprising

(a) nicking at least one site bordering the target region at the terminus of the linear double stranded DNA with at least one site-specific nicking endonuclease;

(b) subjecting the nicked DNA molecules from step (a) to conditions that selectively denature the target region to create the target single stranded region; and

(c) joining the DNA molecule to a second DNA molecule by means of the single strand region, or detecting, purifying or selectively mutagenizing the DNA molecule by means of the single strand region.

4. The method of claim 3 wherein the DNA terminus is pre-existing.

5. The method of claim 3 wherein the DNA terminus is formed by site-specific endonuclease cleavage.

6. A method for creating target single strand regions in a double stranded DNA molecule for use in detecting, purifying or selectively mutagenizing the DNA molecule, the method comprising:

(a) nicking at least two sites bordering a target region in the DNA molecule with at least one site-specific nicking endonuclease; and

(b) subjecting the nicked DNA molecules from step (a) to conditions that selectively denature the target region for creating the target single stranded region; and

(c) detecting, purifying or selectively mutagenizing the DNA molecule by means of the target single strand region.

7. A method of claim 6, wherein the two sites bordering the target region are both located on a single strand of the double stranded DNA so that the target single stranded region comprises a gap in the double stranded DNA.