METHODS FOR MANIPULATING BIOMOLECULES

• US 20120301926A1
• Filed: 05/29/2012
• Published: 11/29/2012
• Est. Priority Date: 05/27/2011
• Status: Abandoned Application

• 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
• Application Number:
  US13/482,542
• Publication Number:
  US 20120301926A1
• Time in Patent Office: Days
• US Class Current: 435/91.5
• 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:

introducing one or more nicks into a nucleic acid by subjecting a sample including a plurality of nucleic acids to nicking conditions; and

generating at least one double stranded break in at least one of the nucleic acids.

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

(a) introducing at least one nick into a double stranded nucleic acid; and

(b) forming a double stranded break in the nucleic acid by translating at least one nick.

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

6. The method of claim 2, wherein the translating includes polymerizing one or more unlabeled nucleotides onto the 3′

end of at least one nick.

7. The method of claim 2, wherein at least one of the nucleic acid fragments is not labeled.

8. The method of claim 2, wherein substantially all of the nucleic acid fragments are not labeled.

9. The method of claim 2, further comprising:

ligating at least one oligonucleotide adapter to at least one end of one or more nucleic acid fragments in the population of nucleic acid fragments.

10. The method of claim 2, wherein one strand of at least one end of a fragment of the population can be joined to one strand of a double-stranded oligonucleotide adaptor to generate a fragment-adaptor molecule having a break.

11. The method of claim 2, wherein both strands of at least one end of a fragment of the population can be joined to both strands of a double-stranded oligonucleotide adaptor.

12. The method of claim 2, further comprising modulating the nicking conditions so as to adjust the average size of the nucleic acid fragments.

13. A population of nucleic acid fragments generated by the method of claim 2.

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

(a) nicking a nucleic acid at least once on each strand; and

(b) nick translating the nicks thereby generating a double-stranded break to produce nucleic acid fragments.

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

to 3′

DNA polymerization/degradation reaction or a 5′

to 3′

DNA polymerization/strand displacement reaction.