Methods and compositions for segregating target nucleic acid from mixed nucleic acid samples
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 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 do not bind to the cleaved target nucleic acid;
(iii) circularizing the cleaved target nucleic acid; wherein the circularized target nucleic acid is resistant to digestion with DNase or exonuclease; and
(iv) depleting the non-target nucleic acid.
2. The method of claim 1 , wherein the circularized target nucleic acid of (iii) is resistant to digestion with Plasmid Safe DNase, Lambda exonuclease, Exonuclease V (RecBCD) or Exonuclease III.
3. The method of claim 1 , wherein the endonuclease of (ii) is a restriction endonuclease that forms blunt ends on the non-target nucleic acid.
4. The method of claim 1 , 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.
5. The method of claim 1 , further comprising ligating a linker to the cleaved target nucleic acid, circularizing the cleaved target nucleic acid, and amplifying the target nucleic acid using a primer for the linker.
6. The method of claim 5 , wherein the amplifying is performed by rolling circle amplification.
7. The method of claim 5 , wherein the amplifying is performed by polymerase chain reaction.
8. The method of claim 5 , wherein the amplifying is performed using Phi29 DNA polymerase.
9. The method of claim 1 , wherein (i) and (ii) are performed simultaneously.
10. The method of claim 1 , wherein (iv) is performed before (ii).
11. The method of claim 1 , wherein the depleting is performed by gel isolation, size exclusion, or exonuclease digestion.
12. The method of claim 1 , wherein the mixed sample contains less than 10 pg of target nucleic acid.
13. The method of claim 1 , wherein the mixed sample further comprises humic acid, diesel soot, or an environmental or clinical contaminant.
14. The method of claim 1 , wherein the mixed sample contains nucleic acid from at least two different prokaryotic organisms.
15. The method of claim 1 , wherein the mixed sample contains nucleic acid from human and bacterial organisms.
16. The method of claim 1 , wherein the mixed s, p e contains nucleic acid from eukaryotic and prokaryotic organisms.
17. The method of claim 1 , wherein the mixed sample contains nucleic acid from an unknown organism.