Methods and compositions for segregating target nucleic acid from mixed nucleic acid samples

Patent metadata

Field	Value
Publication number	US-8927218-B2
Application number	US-201213533489-A
Country	US
Kind code	B2
Filing date	Jun 26, 2012
Priority date	Jun 27, 2011
Publication date	Jan 6, 2015
Grant date	Jan 6, 2015

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.

First claim

What is claimed is:

1. A method for segregating a target nucleic acid from a mixed sample containing the target nucleic acid and a non-target nucleic acid, comprising:
(i) contacting the mixed sample with a non-processive endonuclease;
wherein the non-processive endonuclease binds the target nucleic acid, but does not cleave the target nucleic;
wherein a complex of the non-processive endonuclease and the target nucleic acid is formed; and
(ii) segregating a first fraction of the sample containing the complex from a second fraction of the sample containing the non-target nucleic acid;
wherein the mixed sample contains nucleic acid from at least two different prokaryotic organisms, from human and bacterial organisms, from eukaryotic and prokaryotic organisms, from at least two different eukaryotic organisms, or from an unknown organism.

2. The method of claim 1 , wherein the mixed sample contains humic acid, diesel soot, or an environmental or clinical contaminant.

3. The method of claim 1 , wherein the contacting is in a buffer having conditions suitable for the non-processive endonuclease to bind the target nucleic acid, but not cleave the target nucleic acid.

4. The method of claim 3 , wherein the buffer contains a Mg 2+ concentration suitable for the non-processive endonuclease to bind the target nucleic acid, but not cleave the target nucleic acid.

5. The method of claim 3 , wherein the buffer contains a Ca 2+ concentration of at least 50 mM.

6. The method of claim 3 , wherein the buffer contains a Ca 2+ concentration that is at least 500 times greater than the Mg 2+ concentration of the buffer.

7. The method of claim 1 , wherein the non-processive endonuclease binds a methylated nucleotide.

8. The method of claim 7 , wherein the non-processive endonuclease binds N4-methylcytosine or N6-methyladenine.

9. The method of claim 1 , wherein the non-processive endonuclease is inhibited by C5-methylcytosine.

10. The method of claim 1 , wherein the non-processive endonuclease is present at molar ratios equal to or greater than the target DNA.

11. The method of claim 1 , wherein the non-processive endonuclease comprises a detectable label.

12. The method of claim 1 , wherein the non-processive endonuclease has less than 10% of the catalytic activity of a control endonuclease that binds the target nucleic acid and cleaves the target nucleic acid.

13. The method of claim 1 , wherein the non-processive endonuclease is a recombinase, resolvase, transposase, integrase, repair enzyme, or phosphothioation protein.

14. The method of claim 1 , wherein the segregated nucleic acid is at least 90% of a target genome.

15. The method of claim 1 , wherein the method requires less than 80 minutes to complete.

16. The method of claim 1 , wherein at least 90% of the target nucleic acid in the mixed sample is segregated into the first fraction.

17. The method of claim 1 , wherein the mixed sample contains at least 100.000 times the amount of non-target nucleic acid compared to the amount of target nucleic acid.

18. The method of claim 1 , wherein the mixed sample contains less than 10 pg of target nucleic acid.

19. The method of claim 1 , further comprising:
(iii) contacting the first fraction of (ii) with a non-processive endonuclease;
wherein the non-processive endonuclease binds the target nucleic acid, but does not cleave the target nucleic acid;
wherein a complex of the non-processive endonuclease and the target nucleic acid is formed; and
(vi) segregating a fraction containing the complex of (iii) from a fraction of the sample containing the non-target nucleic acid.

20. The method of claim 1 , wherein the non-processive endonuclease is bound to a solid substrate.

21. A method for segregating a target nucleic acid from a mixed sample containing the target nucleic acid and a non-target nucleic acid, comprising:
(i) contacting the mixed sample with a non-processive restriction enzyme:
wherein the non-processive restriction enzyme binds the target nucleic acid, but does not cleave the target nucleic acid:
wherein a complex of the non-processive restriction enzyme and the target nucleic acid is formed; and
(ii) segregating a first fraction of the sample containing the complex from a second fraction of the sample containing the non-target nucleic acid.

22. The method of claim 21 , wherein the non-processive restriction enzyme is DpnI.

23. The method of claim 21 , wherein the mixed sample contains humic acid, diesel soot, or an environmental or clinical contaminant.

24. The method of claim 21 , wherein the mixed sample contains nucleic acid from at least two different prokaryotic organisms, from human and bacterial organisms, from eukaryotic and prokaryotic organisms, from at least two different eukaryotic organisms, or from an unknown organism.

25. The method of claim 21 , wherein the contacting is in a buffer having conditions suitable for the non-processive restriction enzyme to bind the target nucleic acid, but not cleave the target nucleic acid.

26. The method of claim 21 , wherein the method requires less than 80 minutes to complete.

27. The method of claim 21 , wherein at least 90% of the target nucleic acid in the mixed sample is segregated into the first fraction.

28. The method of claim 21 , wherein the mixed sample contains less than 10 pg of target nucleic acid.

29. The method of claim 21 , wherein the non-processive restriction enzyme is bound to a solid substrate.

Assignees

• Forsyth Roger Allyn
• Flir Systems

Inventors

• Forsyth Roger Allyn

Classifications

• C12Q1/34

  involving hydrolase · CPC title

• C12N15/1003

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title