Universal restriction endonuclease

• US 4,935,357 A
• Filed: 02/05/1986
• Issued: 06/19/1990
• Est. Priority Date: 02/05/1986
• Status: Expired due to Term

• Current Assignee: New England Biolabs Incorporated
• Original Assignee: New England Biolabs Incorporated
• Inventors: Szybalski, Waclaw
• Primary Examiner(s): Wiseman, Thomas G.
• Assistant Examiner(s): Patterson, Jr., Charles L.
• Application Number:
  US06/826,220
• Time in Patent Office: 1,595 Days
• Field of Search: 435/91, 435/199, 435/6, 435/7, 935/19
• US Class Current: 435/91.53
• 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 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Abstract

A universal restriction endonuclease for cleaving a target DNA at a predetermined site. The universal restriction endonuclease utilizes a tailored oligodeoxynucleotide adapter in conjunction with a restriction enzyme which cuts DNA at a known distance from its recognition site. The oligodeoxynucleotide adapter mimics the relationship which exists in such enzymes between its recognition site and cut site.

Claims

1. A universal restriction endonuclease composition which cleaves a target DNA at a predetermined site comprising:

(a) an oligonucleotide adapter which comprises a double-stranded nucleotide sequence corresponding to the recognition site of the restriction endonuclease and a single-stranded nucleotide sequence complementary to the predetermined site of the target DNA; and

(b) a restriction endonuclease;

wherein the oligodeoxynucleotide adapter is designed to mimic the relationship which exists between the recognition site and cleavage site of the restriction endonuclease.

2. The universal restriction endonuclease composition of claim 1, wherein the oligodeoxynucleotide adapter and restriction endonuclease are covalently bound.

3. The universal restriction endonuclease composition of claim 1, wherein the restriction endonuclease is a Class II enzyme selected from the group consisting of BbvI, BbvII, BinI, FokI, HgaI, HphI, MboII, MnlI, SfaNI, TaqII, and Tth111II.

4. The universal restriction endonuclease composition of claim 3, wherein the restriction endonuclease is FokI.

5. The universal restriction endonuclease composition of claim 1, wherein the restriction endonuclease is a Class III enzyme selected from the group consisting of EcoP1, EcoP15, HineI, and HinfIII.

6. A method for cleaving a target DNA at a predetermined site comprising contacting the target DNA in an appropriate buffer solution with a universal restriction endonuclease composition in an amount and for a time sufficient to cause the desired cleavage to occur, said universal restriction endonuclease composition comprising:

(a) an oligonucleotide adapter which comprises a double-stranded nucleotide sequence corresponding to the recognition site of the restriction endonuclease and a single-stranded nucleotide sequence complementary to the predetermined site of the target DNA; and

(b) a restriction endonuclease;

wherein the oligodeoxynucleotide adapter is designed to mimic the relationship which exists between the recognition site and cleavage site of the restriction endonuclease.

7. The method of claim 6, wherein the oligonucleotide adapter and restriction endonuclease are covalently bound.

8. The method of claim 6, wherein the contacting of the universal restriction endonuclease composition with the target DNA comprises:

(a) complexing the oligodeoxynucleotide adapter to the predetermined site of the target DNA; and

(b) adding the appropriate restriction endonuclease to the DNA-adapter complex.

9. The method of claim 8, wherein the target DNA is single stranded.

10. The method of claim 9, comprising the further step of extending the 3'"'"'-OH terminus of the oligodeoxynucleotide adapter along the single-stranded target DNA to form a double-stranded DNA containing the predetermined cut site prior to addition of the appropriate restriction endonuclease.

11. The method of claim 6, wherein the oligodeoxynucleotide adapter is complexed with the restriction endonuclease and contacting comprises complexing the complex with the target DNA.

12. The method of claim 11 wherein the target DNA is single-stranded.