A method for removing unincorporated oligonucleotides from a reaction mixture, the method comprising:n(a) forming a mixture comprising:n(i) a DNA polymerase or nucleic acid ligase;(ii) a nuclease;(iii) an upstream oligonucleotide having a 3⬲ portion and a 5⬲ portion, wherein the 3⬲ portion comprises a 3⬲ recognition group and a 3⬲ terminal nucleotide; and(iv) a template nucleic acid;wherein (i) and (ii) are the same enzyme or seperate enzymes;(b) digesting the 3⬲ portion of the upstream oligonucleotide with the nuclease;(c) extending the digested upstream oligonucleotide with the polymerase or ligating the digested upstream oligonucleotide to a downstream oligonucleotide with the ligase, wherein the extending or ligating forms a polynucleotide product; and(d) contacting the mixture with a substrate comprising binding groups that bind the 3⬲ recognition group, to remove unincorporated upstream pligonucleotides from the reaction mixture.
›2.↳ 1The method of claim 1, wherein (i) is a DNA polymerase, and step (c) is extending the digested upstream oligonucleotide with the polymerase to form th…d2+23
The method of claim 1, wherein (i) is a DNA polymerase, and step (c) is extending the digested upstream oligonucleotide with the polymerase to form the polynucleotide product.
›3.↳ 2The method of claim 2, wherein the mixture further comprises a primer having a 3⬲ portion and a 5⬲ portion, wherein the 3⬲ portion comprises a 3⬲ reco…d3
The method of claim 2, wherein the mixture further comprises a primer having a 3⬲ portion and a 5⬲ portion, wherein the 3⬲ portion comprises a 3⬲ recognition group and a 3⬲ terminal nucleotide; and wherein both the upstream oligonucleotide and the primer comprise the same 3⬲ recognition group, wherein the template nucleic acid is double-stranded and the upstream oligonucleotide and primer hybridize to opposite strands of the template nucleic acid; wherein the method further comprises:ndigesting the 3⬲ portion of the primer with the nuclease;extending the digested primer with the polymerase to form a polynucleotide product; andcontacting the mixture with a substrate comprising binding groups that bind the 3⬲ recognition group, to remove unincorporated primers from the reaction mixture.
›4.↳ 2The method of claim 2, wherein the mixture further comprises a primer having a 3⬲ portion and a 5⬲ portion, wherein the 3⬲ portion comprises a 3⬲ reco…d3
The method of claim 2, wherein the mixture further comprises a primer having a 3⬲ portion and a 5⬲ portion, wherein the 3⬲ portion comprises a 3⬲ recognition group and a 3⬲ terminal nucleotide, and wherein the upstream oligonucleotide and the primer comprise different 3⬲ recognition groups, wherein the template nucleic acid is double-stranded and the upstream oligonucleotide and primer hybridize to opposite strands of the template nucleic acid; wherein the method further comprises:ndigesting the 3⬲ portion of the primer with the nuclease;extending the digested primer with the polymerase to form a polynucleotide product; andcontacting the mixture with a substrate comprising binding groups that bind the 3⬲ recognition group of the primer, to remove unincorporated primers from the reaction mixture.
›5.↳ 2The method of claim 2, wherein the mixture further comprises a primer that does not comprise a 3⬲ recognition group, wherein the template nucleic acid…d3
The method of claim 2, wherein the mixture further comprises a primer that does not comprise a 3⬲ recognition group, wherein the template nucleic acid is double-stranded and the upstream oligonucleotide and primer hybridize to opposite strands of the template nucleic acid.
›6.↳ 2The method of claim 2, wherein the polymerase is a DNA-directed DNA polymerase.d3
The method of claim 2, wherein the polymerase is a DNA-directed DNA polymerase.
›7.↳ 2The method of claim 2, wherein the polymerase is a reverse transcriptase.d3+3
The method of claim 2, wherein the polymerase is a reverse transcriptase.
›8.↳ 7The method of claim 7, wherein the mixture further comprises a DNA-directed DNA polymerase.d4+2
The method of claim 7, wherein the mixture further comprises a DNA-directed DNA polymerase.
›9.↳ 8The method of claim 8, wherein the reverse transcriptase and DNA-directed DNA polymerase are the same enzyme.d5+1
The method of claim 8, wherein the reverse transcriptase and DNA-directed DNA polymerase are the same enzyme.
›10.↳ 9The method of claim 9, wherein the enzyme is Anaerocellum thermophilum DNA polymerase, Bacillus pallidus DNA polymerase, Bacillus stearothermophilus D…d6
The method of claim 9, wherein the enzyme is Anaerocellum thermophilum DNA polymerase, Bacillus pallidus DNA polymerase, Bacillus stearothermophilus DNA polymerase, Carboxydothermus hydrogenoformans DNA polymerase, Thermoactinomyces vulgaris DNA polymerase, Thermoanaerobacter thermohydrosulfuricus DNA polymerase, Thermisipho africanus DNA polymerase, Thermotoga neapolitana DNA polymerase, Thermus aquaticus DNA polymerase, Thermus thermophilus DNA polymerase, or Thermus ZO5 DNA polymerase.
›11.↳ 2The method of claim 2, wherein the DNA polymerase and nuclease are the same enzyme.d3+2
The method of claim 2, wherein the DNA polymerase and nuclease are the same enzyme.
›12.↳ 11The method of claim 11, wherein the nuclease is a 3⬲-to-5⬲ exonuclease.d4+1
The method of claim 11, wherein the nuclease is a 3⬲-to-5⬲ exonuclease.
›13.↳ 12The method of claim 12, wherein the enzyme is Pyrococcus furiosus polymerase THERMALACE, DEEP VENT DNA polymerase (Pyrococcus sp. GB-D), VENT DNA poly…d5
The method of claim 12, wherein the enzyme is Pyrococcus furiosus polymerase THERMALACE, DEEP VENT DNA polymerase (Pyrococcus sp. GB-D), VENT DNA polymerase (Thermococcus litoralis), Bacillus stearothermophilus DNA polymerase, 9° Nm™ DNA polymerase (Thermococcus sp. strain 9° N-7), ACUPOL DNA polymerase, PROOFSTART DNA polymerase (Pyrococcus sp.), Pyrococcus woesei DNA polymerase, Thermococcus gorgonarius DNA polymerase, AMPLITHERM DNA polymerase, KOD DNA polymerase (Pyrococcus kodakarensis), Thermococcus fumicolans DNA polymerase, DYNAZYME EXT DNA polymerase (Thermus brockaianus), Thermosipho africanus DNA polymerase, Pyrodictium occultum DNA polymerase, Pyrococcus kodakarensis DNA polymerase, Thermotoga maritima DNA polymerase, Thermotoga neapolitana DNA polymerase, Bacillus pallidus DNA polymerase, Carboxydothermus hydrogenoformans DNA polymerase, Pyrococcus furiosus DNA polymerase, Pyrococcus sp. GB-D DNA polymerase, Thermococcus litoralis DNA polymerase, Thermococcus sp. strain 90° N-7 DNA polymerase, or Thermus brockaianus DNA polymerase.
›14.↳ 2The method of claim 2, wherein the DNA polymerase and nuclease are separate enzymes.d3+5
The method of claim 2, wherein the DNA polymerase and nuclease are separate enzymes.
›15.↳ 14The method of claim 14, wherein the polymerase is Thermus aquaticus DNA polymerase, Thermus thermophilus DNA polymerase, ZO5 DNA polymerase (Thermus s…d4
The method of claim 14, wherein the polymerase is Thermus aquaticus DNA polymerase, Thermus thermophilus DNA polymerase, ZO5 DNA polymerase (Thermus sp. ZO5), SPS17 DNA polymerase (Thermus sp. SPS17), Thermoactinomyces vulgaris DNA polymerase, Thermoanaerobacter thermohydrosulfuricus DNA polymerase, Anaerocellum thermophilum DNA polymerase, or FY7 DNA polymerase (Thermoanaerobacter thermohydrosulfuricus FY7).
›16.↳ 14The method of claim 14, wherein the nuclease is a mutant polymerase having 3⬲-to-5⬲ exonuclease activity that has lost its polymerase activity.d4+1
The method of claim 14, wherein the nuclease is a mutant polymerase having 3⬲-to-5⬲ exonuclease activity that has lost its polymerase activity.
›17.↳ 16The method of claim 16, wherein the nuclease is a mutant of Pyrococcusfuriosus polymerase THERMALACE, DEEP VENT DNA polymerase (Pyrococcus sp. GB-D), …d5
The method of claim 16, wherein the nuclease is a mutant of Pyrococcusfuriosus polymerase THERMALACE, DEEP VENT DNA polymerase (Pyrococcus sp. GB-D), VENT DNA polymerase (Thermococcus litoralis), Bacillus stearothermophilus DNA polymerase, 9° Nm™ DNA polymerase (Thermococcus sp. strain 90° N-7), ACUPOL DNA polymerase, PROOFSTART DNA polymerase (Pyrococcus sp.), Pyrococcus woesei DNA polymerase, Thermococcus gorgonarius DNA polymerase, AMPLITHERM DNA polymerase, KOD DNA Polymerase (Pyrococcus kodakarensis), Thermococcus fumicolans DNA Polymerase, DYNAZYME EXT DNA polymerase (Thermus brockaianus), Thermosipho africanus DNA polymerase, Pyrodictium occultum DNA polymerase, Pyrococcus kodakarensis DNA polymerase, Thermotoga maritima DNA polymerase, Thermotoga neapolitana DNA polymerase, Bacillus pallidus DNA polymerase, Carboxydothermus hydrogenoformans DNA polymerase, Pyrococcus furiosus DNA polymerase, Pyrococcus sp. GB-D DNA polymerase, Thermococcus litoralis DNA polymerase, Thermococcus sp. strain 90° N-7 DNA polymerase, or Thermus brockaianus DNA polymerase.
›18.↳ 14The method of claim 14, wherein the DNA polymerase is a mutant form of a wild-type DNA polymerase having 3⬲-to-5⬲ exonuclease activity, wherein the mu…d4+1
The method of claim 14, wherein the DNA polymerase is a mutant form of a wild-type DNA polymerase having 3⬲-to-5⬲ exonuclease activity, wherein the mutant form has lost its exonuclease activity.
›19.↳ 18The method of claim 18, wherein the polymerase is a mutant form of Pyrococcus furiosus polymerase THERMALACE, DEEP VENT DNA polymerase (Pyrococcus sp.…d5
The method of claim 18, wherein the polymerase is a mutant form of Pyrococcus furiosus polymerase THERMALACE, DEEP VENT DNA polymerase (Pyrococcus sp. GB-D), VENT DNA polymerase (Thermococcus litoralis), Bacillus stearothermophilus DNA polymerase, 9° Nm™ DNA polymerase (Thermococcus sp. strain 9° N-7), ACUPOL DNA polymerase, PROOFSTART DNA polymerase (Pyrococcus sp.), Pyrococcus woesei DNA polymerase, Thermococcus gorgonarius DNA polymerase, AMPLITHERM DNA polymerase, KOD DNA Polymerase (Pyrococcus kodakarensis), Thermococcus fumicolans DNA Polymerase, DYNAZYME EXT DNA polymerase (Thermus brockaianus), Thermosipho africanus DNA polymerase, Pyrodictium occultum DNA polymerase, Pyrococcus kodakarensis DNA polymerase, Thermotoga maritima DNA polymerase, Thermotoga neapolitana DNA polymerase, Bacillus pallidus DNA polymerase, Carboxydothermus hydrogenoformans DNA polymerase, Pyrococcus furiosus DNA polymerase, Pyrococcus sp. GB-D DNA polymerase, Thermococcus litoralis DNA polymerase, Thermococcus sp. strain 9° N-7 DNA polymerase, or Thermus brockaianus DNA polymerase.
›20.↳ 2The method of claim 2, wherein the mixture comprises two or more DNA polymerases having varying amounts of 3 ⬲-to-5⬲ exonuclease activity.d3
The method of claim 2, wherein the mixture comprises two or more DNA polymerases having varying amounts of 3 ⬲-to-5⬲ exonuclease activity.
›24.↳ 2multiple · 2, 23The method of claim 2 or 23, wherein the 3⬲ terminal nucleotide of the upstream oligonucleotide is modified with a blocking group that prevents extens…d3+3
Multiple dependency — claims 2, 23. Shown here under 2; it stands on every one of them.
The method of claim 2 or 23, wherein the 3⬲ terminal nucleotide of the upstream oligonucleotide is modified with a blocking group that prevents extension or ligation of the undigested upstream oligonucleotide.
›25.↳ 24The method of claim 24, wherein the blocking group is a 3⬲-deoxynucleotide.d4
The method of claim 24, wherein the blocking group is a 3⬲-deoxynucleotide.
›26.↳ 24The method of claim 24, wherein the blocking group is 3⬲-phosphoglycoaldehyde, 3⬲-phosphate, 3⬲-mercapto, or 3⬲-amrno.d4
The method of claim 24, wherein the blocking group is 3⬲-phosphoglycoaldehyde, 3⬲-phosphate, 3⬲-mercapto, or 3⬲-amrno.
›27.↳ 24The method of claim 24, wherein the blocking group comprises the 3⬲ recognition group.d4
The method of claim 24, wherein the blocking group comprises the 3⬲ recognition group.
›28.↳ 2multiple · 2, 23The method of claim 2 or 23, wherein the upstream oligonucleotide cannot be extended or ligated unless the 3⬲ recognition group is removed.d3
Multiple dependency — claims 2, 23. Shown here under 2; it stands on every one of them.
The method of claim 2 or 23, wherein the upstream oligonucleotide cannot be extended or ligated unless the 3⬲ recognition group is removed.
›21.↳ 1The method of claim 1, wherein the nuclease is inactive until an activation step is applied.d2+1
The method of claim 1, wherein the nuclease is inactive until an activation step is applied.
›22.↳ 21The method of claim 21, wherein the nuclease is PROOFSTART DNA polymerase.d3
The method of claim 21, wherein the nuclease is PROOFSTART DNA polymerase.
›23.↳ 1The method of claim 1, wherein (i) is a nucleic acid ligase, and step (c) is ligating the digested upstream oligonucleotide to a downstream oligonucle…d2
The method of claim 1, wherein (i) is a nucleic acid ligase, and step (c) is ligating the digested upstream oligonucleotide to a downstream oligonucleotide with the ligase to form the polynucleotide product.
›29.↳ 1The method of claim 1, wherein the nuclease is a 3⬲-to-5⬲ exonuclease.d2
The method of claim 1, wherein the nuclease is a 3⬲-to-5⬲ exonuclease.
›30.↳ 1The method of claim 1, wherein the 3⬲ terminal nucleotide comprises all or part of the 3⬲ recognition group.d2
The method of claim 1, wherein the 3⬲ terminal nucleotide comprises all or part of the 3⬲ recognition group.
›31.↳ 1The method of claim 1, wherein an internal nucleotide of the upstream oligonucleotide comprises all or part of the 3⬲ recognition group.d2
The method of claim 1, wherein an internal nucleotide of the upstream oligonucleotide comprises all or part of the 3⬲ recognition group.
›32.↳ 1The method of claim 1, wherein the 3⬲ portion of the upstream oligonucleotide is non- complementary with the template.d2
The method of claim 1, wherein the 3⬲ portion of the upstream oligonucleotide is non- complementary with the template.
›33.↳ 1The method of claim 1, wherein all the nucleosides within the 3⬲ portion of the upstream oligonucleotide are linked by linkages that are resistant to …d2+2
The method of claim 1, wherein all the nucleosides within the 3⬲ portion of the upstream oligonucleotide are linked by linkages that are resistant to hydrolysis by the nuclease.
›34.↳ 33The method of claim 33, wherein the linkages are methyl phosphonate linkages.d3
The method of claim 33, wherein the linkages are methyl phosphonate linkages.
›35.↳ 33The method of claim 33, wherein the linkages are phosphorothionate linkages.d3
The method of claim 33, wherein the linkages are phosphorothionate linkages.
›36.↳ 1The method of claim 1, wherein all the nucleosides within the 3⬲ portion of the upstream oligonucleotide are linked by phosphodiesterase linkages, and…d2+1
The method of claim 1, wherein all the nucleosides within the 3⬲ portion of the upstream oligonucleotide are linked by phosphodiesterase linkages, and the 5⬲ portion of the upstream oligonucleotide comprises a linkage that is resistant to hydrolysis.
›37.↳ 36The method of claim 36, wherein the linkage resistant to hydrolysis is a methyl phosphonate linkage or a phosphorothionate linkage.d3
The method of claim 36, wherein the linkage resistant to hydrolysis is a methyl phosphonate linkage or a phosphorothionate linkage.
›38.↳ 1The method of claim 1, wherein the 3⬲ portion of the upstream oligonucleotide consists of L nucleotides.d2
The method of claim 1, wherein the 3⬲ portion of the upstream oligonucleotide consists of L nucleotides.
›39.↳ 1The method of claim 1, wherein the template nucleic acid is DNA.d2
The method of claim 1, wherein the template nucleic acid is DNA.
›40.↳ 1The method of claim 1, wherein the template nucleic acid is RNA.d2
The method of claim 1, wherein the template nucleic acid is RNA.
›41.↳ 1The method of claim 1, wherein the substrate is a size-exclusion-chromatography resin.d2
The method of claim 1, wherein the substrate is a size-exclusion-chromatography resin.
›42.↳ 1The method of claim 1, wherein the recognition group is a group recognized by an antibody, and the binding group is the antibody.d2+3
The method of claim 1, wherein the recognition group is a group recognized by an antibody, and the binding group is the antibody.
›43.↳ 42The method of claim 42, wherein the recognition group is digoxygenin.d3
The method of claim 42, wherein the recognition group is digoxygenin.
›44.↳ 42The method of claim 42, wherein the recognition group is fluorescein.d3
The method of claim 42, wherein the recognition group is fluorescein.
›45.↳ 42The method of claim 42, wherein the recognition group is biotin.d3
The method of claim 42, wherein the recognition group is biotin.
›46.↳ 1The method of claim 1, wherein the recognition group is biotin and the binding group is avidin or streptavidin.d2
The method of claim 1, wherein the recognition group is biotin and the binding group is avidin or streptavidin.
›47.↳ 1The method of claim 1, wherein the recognition group comprises phenylboronic acid and the binding group comprises salicylhydroxamic acid.d2
The method of claim 1, wherein the recognition group comprises phenylboronic acid and the binding group comprises salicylhydroxamic acid.
›48.↳ 1The method of claim 1, wherein the recognition group comprises salicylhydroxamic acid and the binding group comprises phenylboronic acid.d2
The method of claim 1, wherein the recognition group comprises salicylhydroxamic acid and the binding group comprises phenylboronic acid.
›49.↳ 1The method of claim 1, wherein the recognition group is polyhistidine and the binding group is nickel cation.d2
The method of claim 1, wherein the recognition group is polyhistidine and the binding group is nickel cation.
›50.↳ 1The method of claim 1, wherein the recognition group is a nucleotide sequence of the upstream oligonucleotide and the binding group is a complementary…d2
The method of claim 1, wherein the recognition group is a nucleotide sequence of the upstream oligonucleotide and the binding group is a complementary nucleotide sequence.
›51.↳ 1The method of claim 1, wherein the upstream oligonucleotide comprises a modified nucleotide 5⬲ to the 3⬲ recognition group, and wherein the nuclease c…d2+7
The method of claim 1, wherein the upstream oligonucleotide comprises a modified nucleotide 5⬲ to the 3⬲ recognition group, and wherein the nuclease cleaves the upstream oligonucleotide at the modified nucleotide.
›52.↳ 51The method of claim 51, wherein the nuclease cleaves the upstream oligonucleotide at the modified nucleotide when the modified nucleotide is present i…d3+6
The method of claim 51, wherein the nuclease cleaves the upstream oligonucleotide at the modified nucleotide when the modified nucleotide is present in a duplex preferentially over when it is not in a duplex.
›53.↳ 52The method of claim 52, wherein the modified nucleotide is a ribonucleotide and the nuclease is an RINAse H.d4+1
The method of claim 52, wherein the modified nucleotide is a ribonucleotide and the nuclease is an RINAse H.
›54.↳ 53The method of claim 53, wherein the RNAse H is Thermus thermophilus DNA polymerase, Thermus thermophilus RINAse H, human RNAse H, or E. ccli RNAse H.d5
The method of claim 53, wherein the RNAse H is Thermus thermophilus DNA polymerase, Thermus thermophilus RINAse H, human RNAse H, or E. ccli RNAse H.
›55.↳ 52The method of claim 52, wherein the modified nucleotide comprises 8-oxo-7,8-dihydro-2⬲-deoxyguanosine; 7-methylguanine; 2,6-diamino-4-hydroxy-5-N-meth…d4
The method of claim 52, wherein the modified nucleotide comprises 8-oxo-7,8-dihydro-2⬲-deoxyguanosine; 7-methylguanine; 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine; 4,6-diamino-5-formamidopyrimidine; 5-hydroxy-2 ⬲-deoxycytidine; 5-hydroxy-2⬲-deoxyuridine; or N7-methylguanine; and the nuclease is formamido-pyrimidine-DNA glycosylase; and the mixture further comprises a 3⬲ phosphatase.
›56.↳ 52The method of claim 52, wherein the modified nucleotide comprises 7,8-dihydro-8-oxoguanine; formamidopyrimidine; 2,6-diamino-4-hydroxy-5-formamidopyri…d4
The method of claim 52, wherein the modified nucleotide comprises 7,8-dihydro-8-oxoguanine; formamidopyrimidine; 2,6-diamino-4-hydroxy-5-formamidopyrimidine; or 8-oxoguanine; and the nuclease is 8-oxoguanine DNA glycosylase; and the mixture further comprises an AP endonuclease.
›57.↳ 52The method of claim 52, wherein the modified nucleotide comprises 5,6-dihydrothymine; 6-hydroxy-5,6-dihydrothymine; cis-thymine glycol; trans-thymine …d4
The method of claim 52, wherein the modified nucleotide comprises 5,6-dihydrothymine; 6-hydroxy-5,6-dihydrothymine; cis-thymine glycol; trans-thymine glycol; 5-hydroxy-5-methylhydantoin; methyltartonyl urea; urea; 5-hydroxycytosine; 5-hydroxyuracil; uracil glycol; dihydrouracil; 6-hydroxyuracil; glycol; ureidoisobutyric acid; 5-hydroxy-6-hydrothymine; 5,6-dihydrouracil; 5-hydroxy-6-hydrouracil; 5-hydroxy-2⬲-deoxycytidine; 5-hydroxy-2⬲-deoxyuridine; and the nuclease is endonuclease III or thymine glycol-DNA glycosylase; and the mixture further comprises an AP endonuclease.
›58.↳ 52The method of claim 52, wherein the modified nucleotide is an AP nucleotide and the nuclease is an AP endonuclease.d4
The method of claim 52, wherein the modified nucleotide is an AP nucleotide and the nuclease is an AP endonuclease.
›59.↳ 1The method of claim 1, wherein the mixture further comprises a 3⬲ phosphatase.d2+1
The method of claim 1, wherein the mixture further comprises a 3⬲ phosphatase.
›60.↳ 59The method of claim 59, wherein the 3⬲ phosphatase is exonuclease III, exonuclease IV, or yeast AP endonuclease.d3
The method of claim 59, wherein the 3⬲ phosphatase is exonuclease III, exonuclease IV, or yeast AP endonuclease.
›61.↳ 1The method of claim 1, wherein the 5⬲ portion of the upstream oligonucleotide comprises a 5⬲ recognition group that is different from the 3⬲ recogniti…d2+1
The method of claim 1, wherein the 5⬲ portion of the upstream oligonucleotide comprises a 5⬲ recognition group that is different from the 3⬲ recognition group.
›62.↳ 61The method of claim 61, further comprising step (e): contacting the mixture with a substrate comprising binding groups that bind the 5⬲ recognition gr…d3
The method of claim 61, further comprising step (e): contacting the mixture with a substrate comprising binding groups that bind the 5⬲ recognition group.
