Methods of Generating Nucleic Acid Fragments

• US 20110223638A1
• Filed: 03/02/2011
• Published: 09/15/2011
• Est. Priority Date: 03/10/2010
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Doudna, Jennifer A., Zhou, Kaihong, Wiedenheft, Blake
• Granted Patent: US 10,087,431 B2
• Time in Patent Office: Days
• US Class Current: 435/91.5
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

Provided herein are methods of using a Cas1 polypeptide to generate nucleic fragments from a DNA substrate. These methods may be performed in vitro or in vivo. Also provided are methods of screening for modulators of Cas1.

Claims

1. A method of generating nucleic acid fragments of substantially uniform length, the method comprising contacting a DNA substrate with a Cas1 polypeptide, wherein said contacting results in generation nucleic acid fragments of substantially uniform length.

2. The method of claim 1, wherein the contacting is performed in the presence of a divalent metal ion.

3. The method of claim 2, wherein the divalent metal ion is divalent magnesium ion.

4. The method of claim 2, wherein the divalent metal ion is divalent manganese ion.

5. The method of claim 1, wherein the DNA substrate is inside a cell.

6. The method of claim 1, wherein the DNA substrate is in vitro.

7. The method of claim 1, wherein the DNA substrate is double stranded linear DNA, single stranded DNA, circular DNA, or genomic DNA.

8. The method of claim 1, wherein said contacting is for about 0.1 hour to 24 hours.

9. The method of claim 1, wherein the nucleic acid fragments are about 30 bp-200 bp in length.

10. The method of claim 9, wherein the nucleic acid fragments are about 50 bp-100 bp in length.

11. The method of claim 1, further comprising sequencing said nucleic acid fragments.

12. The method of claim 1, wherein the contacting comprises contacting a surface comprising a biofilm, wherein the DNA is present in microorganisms present in the biofilm.

13. The method of claim 1, wherein the contacting comprises contacting an epithelial surface of an animal, wherein the DNA is present in the microorganisms present on the surface.

14. The method of claim 13, the epithelial surface is mucosal membrane.

15. The method of claim 13, the epithelial surface is skin.

16. The method of claim 1, further comprising cloning the nucleic acid fragments.

17. A kit comprising:

a) a purified Cas1 polypeptide; and

b) a buffer comprising magnesium or manganese.