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;
extending the primer using a polymerase and modified nucleotides that are resistant to nuclease degradation to create a modified polynucleotide;
exposing the sample to a nuclease; and
isolating the modified polynucleotides.
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′-O-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 , wherein the nuclease comprises an exonuclease.
10 . The method of claim 1 , further comprising detecting the target nucleic acid.
11 . The method of claim 10 , wherein the detecting step comprises using hybridization, spectrophotometry, sequencing, electrophoresis, amplification, fluorescence detection, chromatography, DNA staining, or microscopy.
12 . The method of claim 1 , wherein the sample is a blood sample, serum sample, plasma sample, urine sample, saliva sample, semen sample, feces sample, phlegm sample, or liquid biopsy.
13 . 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;
extending the primer using a polymerase and modified nucleotides that are resistant to nuclease degradation to create a modified polynucleotide;
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;
exposing the sample to a nuclease; and
isolating the modified polynucleotides and protected target nucleic acid.
14 . The method of claim 13 , wherein the modified nucleotides comprise modified nucleotide triphosphates.
15 . The method of claim 13 , wherein natural nucleotides are used in combination with modified nucleotides.
16 . The method of claim 14 , wherein the modified nucleotide triphosphates comprise alpha-phosphorothioate nucleotide triphosphates, morpholino triphosphates, peptide nucleic acids, peptide nucleic acid analogs, or sugar modified nucleotide triphosphates.
17 . The method of claim 16 , 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′-O-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.
18 . The method of claim 14 , 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.
19 . The method of claim 18 , wherein the Cas endonuclease is catalytically inactive.
20 . The method of claim 14 , wherein the nuclease comprises an exonuclease.
21 . The method of claim 14 , further comprising detecting the target nucleic acid.
22 . The method of claim 21 , wherein the detecting step comprises using hybridization, spectrophotometry, sequencing, electrophoresis, amplification, fluorescence detection, chromatography, DNA staining, or microscopy.
23 . The method of claim 14 , wherein the sample is a blood sample, serum sample, plasma sample, urine sample, saliva sample, semen sample, feces sample, phlegm sample, or liquid biopsy.