METHODS FOR MANIPULATING BIOMOLECULES

• US 20140343265A1
• Filed: 06/25/2014
• Published: 11/20/2014
• Est. Priority Date: 05/27/2011
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: CHEN, Zhoutao, DUAN, Xiaoping, PARK, Kyusung
• Granted Patent: US 11,542,535 B2
• Time in Patent Office: Days
• US Class Current: 536/23.1
• 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 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12P 19/40 having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

In some embodiments, the present teachings provide compositions, systems, methods and kits for generating a population of nucleic acid fragments. In some embodiments, nucleic acids can be fragmented enzymatically. For example, methods for generating a population of nucleic acid fragments can include a nucleic acid nicking reaction. In one embodiment, the methods can include a nick translation reaction. A nicking reaction can introduce nicks at random positions on either strand of a double-stranded nucleic acid. A nick translation reaction can move the position of nicks to a new position so that the new positions of two of the nicks are aligned to create a double-stranded break. In some embodiments, methods for generating a population of nucleic acid fragments can include joining at least one end of a fragmented nucleic acid to one or more oligonucleotide adaptors.

Claims

1. A method for generating a population of nucleic acid fragments comprising:

(a) nicking a double stranded nucleic acid molecule at least once on each strand, wherein the nucleic acid molecule does not include uracil; and

(b) fragmenting the nucleic acid molecule by nick translating at least one nick in each strand and generating at least one double-stranded break in the nucleic acid molecule, thereby producing two or more nucleic acid fragments.

2. The method of claim 1, wherein the nicking comprises enzymatic nicking.

3. The method of claim 1, wherein the nick translating comprises a 5′

to 3′

DNA polymerization/degradation reaction or a 5′

to 3′

DNA polymerization/strand displacement reaction.

4. The method of claim 1, wherein the nick translating includes polymerizing one or more unlabeled nucleotides onto the 3′

end of at least one nick.

5. The method of claim 1, wherein at least one of the nucleic acid fragments is not labeled.

6. The method of claim 1, wherein substantially all of the nucleic acid fragments are not labeled.

7. The method of claim 1, further including joining both strands of at least one end of a nucleic acid fragment to both strands of a double-stranded oligonucleotide adaptor.

8. The method of claim 1, further comprising adjusting the average size of the nucleic acid fragments by modulating the reaction conditions for the nicking conditions.

9. A population of nucleic acid fragments generated by the method of claim 1.

10. The method of claim 1, further including a single-stranded nucleic acid binding protein.

11. A method for nucleic acid fragmentation, comprising:

(a) providing a solution including a plurality of double stranded nucleic acid molecules that do not include uracil; and

(b) subjecting at least two different nucleic acid molecules of the plurality to the methods of claim 1.

12. The method of claim 11, further including fragmenting that at least two different nucleic acid molecules of the plurality in the same reaction mixture.

13. The method of claim 11, further including a single-stranded nucleic acid binding protein.