Methods and Compositions for Segregating Target Nucleic Acid From Mixed Nucleic Acid Samples

• US 20180112206A1
• Filed: 10/12/2017
• Published: 04/26/2018
• Est. Priority Date: 06/27/2011
• Status: Active Grant

• Current Assignee: Flir Detection, Inc. (Teledyne Technologies Incorporated)
• Original Assignee: Flir Detection, Inc. (Teledyne Technologies Incorporated)
• Inventors: Forsyth, Roger Allyn
• Granted Patent: US 10,190,113 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 38/46 Hydrolases (3)
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: C12Q 1/06 Quantitative determination
• CPC Class Codes: C12Q 1/34 involving hydrolase
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

The invention provides methods, compositions and kits for segregating a target nucleic acid from a mixed nucleic acid sample. The methods, compositions and kits comprise a non-processive endonuclease (e.g., a restriction enzyme) or an antibody that binds the target nucleic acid (e.g., has methylation specificity). The mixed nucleic acid sample can comprise prokaryotic and eukaryotic nucleic acid and/or nucleic acid from more than one prokaryotic or eukaryotic organisms.

Claims

1-48. -48. (canceled)

49. A method for enriching a target nucleic acid in a mixed sample containing the target nucleic acid and a non-target nucleic acid, comprising:

(i) digesting the mixed sample with a methylation-sensitive or methylation-dependent endonuclease that cleaves the target nucleic acid and not the non-target nucleic acid;

(ii) digesting the sample with an endonuclease;

wherein the endonuclease cleaves the non-target nucleic acid and not the target nucleic acid, resulting in non-target nucleic acid ends that are incompatible with the cleaved target nucleic acid;

(iii) ligating a linker to the cleaved target nucleic acid, or circularizing the cleaved target nucleic acid; and

(iv) depleting the non-target nucleic acid.

50. The method of claim 49, wherein the circularized target nucleic acid of (iii) is resistant to digestion with DNase or exonuclease.

51. The method of claim 50, wherein the circularized target nucleic acid of (iii) is resistant to digestion with Plasmid Safe DNase, Lambda exonuclease, Exonuclease V (RecBCD) or Exonuclease III.

52. The method of claim 49, wherein the linker is resistant to the depletion of (iv).

53. The method of claim 49, wherein the linker comprises a modification selected from inverted dT, phosphorothioation, stem loop, methylation, hydroxymethylation, adenylation, glucosylation and 3′

OH end overhangs of 4 nucleotides or longer.

54. The method of claim 49, wherein the endonuclease of (ii) is a restriction endonuclease that forms blunt ends on the non-target nucleic acid.

55. The method of claim 49, wherein a restriction endonuclease recognition sequence of the non-target nucleic acid is located within a restriction endonuclease recognition sequence of the target nucleic acid.

56. The method of claim 49, further comprising amplifying the target nucleic acid using a primer for the linker.

57. The method of claim 56, wherein the amplifying is performed by rolling circle amplification.

58. The method of claim 56, wherein the amplifying is performed by polymerase chain reaction.

59. The method of claim 56, wherein the amplifying is performed using Phi29 DNA polymerase.

60. The method of claim 49, wherein (i) and (ii) are performed simultaneously.

61. The method of claim 49, wherein (iv) is performed before (ii).

62. The method of claim 49, wherein the depleting is performed by gel isolation, size exclusion, or exonuclease digestion.

63-80. -80. (canceled)