Kits for segregating target nucleic acid from mixed nucleic acid samples
View Patent ↗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.
1. A kit for segregating a target nucleic acid from a mixed sample containing a target nucleic acid and a non-target nucleic acid, comprising:
(i) a non-processive endonuclease that binds to the target nucleic acid, but does not cleave the target nucleic acid; and
(ii) a buffer having conditions suitable for the non-processive endonuclease to bind the target nucleic acid, but not cleave the target nucleic acid; wherein the buffer contains a Ca2+ concentration of at least 50 mM.
2. The kit of claim 1 , wherein the buffer contains a Ca2+ concentration that is at least 500times greater than the Mg2+ concentration of the buffer.
3. The kit of claim 1 , wherein the buffer contains a Mg2+ concentration of less than 10 mM.
4. The kit of claim 3 , wherein the buffer contains EDTA at a concentration of at least 10 mM.
5. The kit of claim 3 , wherein the buffer contains EDTA at a concentration of about 100 mM.
6. The kit of claim 1 , wherein the buffer contains EDTA at a concentration of at least 10 mM.
7. The kit of claim 1 , wherein the buffer contains EDTA at a concentration of about 100 mM.
8. The kit of claim 1 , wherein the non-processive endonuclease binds N4-methylcytosine or N6-methyladenine.
9. The kit of claim 1 , wherein the non-processive endonuclease is inhibited by C5-methylcytosine.
10. The kit of claim 1 , wherein the non-processive endonuclease is a non-processive restriction enzyme.
11. The kit of claim 1 , wherein the non-processive endonuclease is DpnI.
12. The kit of claim 1 , wherein the non-proccssive endonuclease comprises a detectable label.
13. The kit of claim 1 , wherein the non-processive endonuclease is biotinylated.
14. A kit for segregating a target nucleic acid from a mixed sample containing a target nucleic acid and a non-target nucleic acid, comprising:
(i) a non-processive endonuclease that binds to the target nucleic acid, but does not cleave the target nucleic acid; and
(ii) a buffer having conditions suitable for the non-processive endonuclease to bind the target nucleic acid, but not cleave the target nucleic acid; wherein the buffer contains a Mg2+ concentration of less than 10 mM.
15. The kit of claim 14 , wherein the buffer contains EDTA at a concentration of at least 10 mM.
16. The kit of claim 14 , wherein the non-processive endonuclease binds N4-methylcytosine or N6-methyladenine.
17. The kit of claim 14 , wherein the non-processive endonuclease is inhibited by C5-methylcytosine.
18. The kit of claim 14 , wherein the non-processive endonuclease is a non-processive restriction enzyme.
19. The kit of claim 14 , wherein the non-processive endonuclease is DpnI.
20. The kit of claim 14 , wherein the non-processive endonuclease comprises a detectable label.
21. The kit of claim 14 , wherein the non-processive endonuclease is biotinylated.
22. A kit for segregating a target nucleic acid from a mixed sample containing a target nucleic acid and a non-target nucleic acid, comprising:
(i) a non-processive endonuclease that binds to the target nucleic acid, but does not cleave the target nucleic acid; wherein the non-processive endonuclease comprises a detectable label; and
(ii) a buffer having conditions suitable for the non-processive endonuclease to bind the target nucleic acid, but not cleave the target nucleic acid.
23. The kit of claim 22 , wherein the non-processive endonuclease is biotinylated.
24. The kit of claim 22 , wherein the non-processive endonuclease binds N4-methylcytosine or N6-methyladenine.
25. The kit of claim 22 , wherein the non-processive endonuclease is inhibited by C5-methylcytosine.
26. The kit of claim 22 , wherein the non-processive endonuclease is a non-processive restriction enzyme.
27. The kit of claim 22 , wherein the non-processive endonuclease is DpnI.
28. The kit of claim 22 , further comprising:
(iii) a solid support material that binds the non-processive endonuclease.
29. The kit of claim 28 , further comprising:
(iv) a binder specific for biotinylation.
30. The kit of claim 29 , further comprising:
(v) a methylated nucleic acid positive control.