Isolation of target nucleic acids
The invention provides methods of isolating a target nucleic acid in a sample. A primer is hybridized to the target. A polymerase and modified nucleotide resistant to nuclease degradation are used to extend the primer to create a modified polynucleotide. The sample is exposed to a nuclease, thereby isolating the modified polynucleotide. Optionally, the target nucleic acid may be further protected by binding a protein in a sequence specific manner to one end of the target nucleic acid to create a protected target nucleic acid resistant to nuclease degradation. Thus, after exposing the sample to a nuclease, the modified polynucleotide and protected target nucleic acid are isolated.
1. A method for isolating a target nucleic acid, the method comprising:
hybridizing at least one primer to a target nucleic acid in a sample comprising non-target nucleic acids;
extending the primer using a polymerase and modified nucleotides that are resistant to nuclease degradation to create a modified polynucleotide that protects the target nucleic acid from nuclease degradation; and
exposing the sample to an exonuclease to digest the non-target nucleic acids, thereby isolating the target nucleic acid.
2. The method of claim 1 , wherein the modified nucleotides comprise modified nucleotide triphosphates.
3. The method of claim 2 , wherein the modified nucleotide triphosphates comprise alpha-phosphorothioate nucleotide triphosphates, morpholino triphosphates, peptide nucleic acids, peptide nucleic acid analogs, or sugar modified nucleotide triphosphates.
4. The method of claim 3 , wherein the modified nucleotide triphosphates are selected from the group consisting of 2′-Deoxycytidine-5′-O-(1-Thiotriphosphate), 2′-O-methyl modified nucleotide triphosphate, 2′-fluoro modified nucleotide, 2′-O-Methyladenosine-5′-Triphosphate, 2′-O-Methylcytidine-5′-Triphosphate, 2′-O-Methylguanosine-5′-Triphosphate, 2′-0-Methyluridine-5′-Triphosphate, 2′-O-Methylinosine-5′-Triphosphate, 2′-O-Methyl-2-aminoadenosine-5′-Triphosphate, 2′-O-Methylpseudouridine-5′-Triphosphate, 2′-O-Methyl-5-methyluridine-5′-Triphosphate, 2′-O-Methyl-N6-Methyladenosine-5′-Triphosphate, 2′-Fluoro-2′-deoxyadenosine-5′-Triphosphate, 2′-Fluoro-2′-deoxycytidine-5′-Triphosphate, 2′-Fluoro-2′-deoxyguanosine-5′-Triphosphate, 2′-Fluoro-2′-deoxyuridine-5′-Triphosphate, and 2′-Fluoro-thymidine-5′-Triphosphate.
5. The method of claim 1 , wherein natural nucleotides are used in combination with modified nucleotides.
6. The method of claim 1 , wherein two primers are used for hybridization.
7. The method of claim 6 , further comprising amplification of the modified polynucleotide.
8. The method of claim 2 , further comprising dephosphorylating the target nucleic acid using a phosphatase.
9. The method of claim 1 , further comprising detecting the target nucleic acid.
10. The method of claim 1 , wherein the target comprises circulating tumor DNA.
11. The method of claim 10 , wherein the sample is a blood sample, serum sample, or plasma sample.
12. A method for isolating a target nucleic acid, the method comprising:
hybridizing at least one primer to a first end of a target nucleic acid in a sample comprising non-target nucleic acids;
extending the primer using a polymerase and modified nucleotides that are resistant to nuclease degradation to create a modified polynucleotide that protects the target nucleic acid from nuclease digestion;
binding at least one protein to a second end of the target nucleic acid in a sequence-specific manner to create protected target nucleic acid resistant to nuclease degradation; and
exposing the sample to an exonuclease to digest the non-target nucleic acids, thereby isolating the protected target nucleic acid.
13. The method of claim 12 , wherein the modified nucleotides comprise modified nucleotide triphosphates.
14. The method of claim 12 , wherein natural nucleotides are used in combination with modified nucleotides.
15. The method of claim 13 , wherein the modified nucleotide triphosphates comprise alpha-phosphorothioate nucleotide triphosphates, morpholino triphosphates, peptide nucleic acids, peptide nucleic acid analogs, or sugar modified nucleotide triphosphates.
16. The method of claim 15 , wherein the modified nucleotide triphosphates are selected from the group consisting of 2′-Deoxycytidine-5′-O-(1-Thiotriphosphate), 2′-O-methyl modified nucleotide triphosphate, 2′-fluoro modified nucleotide, 2′-O-Methyladenosine-5′-Triphosphate, 2′-O-Methylcytidine-5′-Triphosphate, 2′-O-Methylguanosine-5′-Triphosphate, 2′-0-Methyluridine-5′-Triphosphate, 2′-O-Methylinosine-5′-Triphosphate, 2′-O-Methyl-2-aminoadenosine-5′-Triphosphate, 2′-O-Methylpseudouridine-5′-Triphosphate, 2′-O-Methyl-5-methyluridine-5′-Triphosphate, 2′-O-Methyl-N6-Methyladenosine-5′-Triphosphate, 2′-Fluoro-2′-deoxyadenosine-5′-Triphosphate, 2′-Fluoro-2′-deoxycytidine-5′-Triphosphate, 2′-Fluoro-2′-deoxyguanosine-5′-Triphosphate, 2′-Fluoro-2′-deoxyuridine-5′-Triphosphate, and 2′-Fluoro-thymidine-5′-Triphosphate.
17. The method of claim 13 , wherein the at least one protein comprises a Cas endonuclease complexed with a guide RNA that targets the Cas endonuclease to a region of the target nucleic acid.
18. The method of claim 17 , wherein the Cas endonuclease is catalytically inactive.
19. The method of claim 13 , further comprising detecting the target nucleic acid.
20. The method of claim 19 , wherein the detecting step comprises using hybridization, spectrophotometry, sequencing, electrophoresis, amplification, fluorescence detection, chromatography, DNA staining, or microscopy.
21. The method of claim 13 , wherein the sample is a blood sample, serum sample, plasma sample, urine sample, saliva sample, semen sample, feces sample, phlegm sample, or liquid biopsy.