Methods and compositions 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 composition 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 EDTA at a concentration of at least 10 mM.
2. The composition of claim 1 , further comprising:
(iii) a mixed sample comprising a target nucleic and a non-target nucleic acid.
3. The composition of claim 2 , 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.
4. The composition of claim 2 , wherein the mixed sample contains less than 10 pg of target nucleic acid.
5. The composition of claim 2 , wherein the mixed sample further comprises humic acid, diesel soot, or an environmental or clinical contaminant.
6. The composition 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.
7. The composition of claim 1 , wherein the buffer contains a Ca2+ concentration of at least 50 mM.
8. The composition of claim 1 , wherein the buffer contains a Mg2+ concentration of less than 10 mM.
9. A composition 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 non-processive endonuclease is a non-processive restriction enzyme.
10. The composition of claim 9 , further comprising:
(iii) a mixed sample comprising a target nucleic and a non-target nucleic acid.
11. The composition of claim 10 , 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.
12. The composition of claim 10 , wherein the mixed sample contains less than 10 pg of target nucleic acid.
13. The composition of claim 10 , wherein the mixed sample further comprises humic acid, diesel soot, or an environmental or clinical contaminant.
14. The composition of claim 9 , 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.
15. The composition of claim 9 , wherein the buffer contains a Ca2+ concentration of at least 50 mM.
16. The composition of claim 9 , wherein the buffer contains a Mg2+ concentration of less than 10 mM.
17. A composition 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 non-processive endonuclease is DpnI.
18. The composition of claim 17 , further comprising:
(iii) a mixed sample comprising a target nucleic and a non-target nucleic acid.
19. The composition of claim 18 , 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.
20. The composition of claim 18 , wherein the mixed sample contains less than 10 pg of target nucleic acid.
21. The composition of claim 18 , wherein the mixed sample further comprises humic acid, diesel soot, or an environmental or clinical contaminant.
22. The composition of claim 17 , 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.
23. The composition of claim 17 , wherein the buffer contains a Ca2+ concentration of at least 50 mM.
24. The composition of claim 17 , wherein the buffer contains a Mg2+ concentration of less than 10 mM.