Oligonucleotides and Analogs Labeled with Energy Transfer Dyes

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers facilitate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure RZC(O)RRwhere Ris a Calkyl attached to the donor dye, C(O) is a carbonyl group, Zis either NH, sulfur or oxygen, Ris a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and Rincludes a functional group which attaches the linker to the acceptor dye. 123.-. (canceled)24. An energy transfer dye , comprising:a xanthene donor dye configured to absorb excitation energy at a first wavelength and transferring energy in response;an acceptor dye configured to absorb the energy transferred by the donor dye, and fluorescing at a second wavelength in response; anda non-nucleosidic linker linking the donor dye to the acceptor dye, wherein the non-nucleosidic linker is a.) configured to efficiently transfer energy from the donor dye to the acceptor dye, b.) is attached to the 4′ position of the xanthene donor dye, and c.) comprises a functional group selected from the group consisting of an alkene, diene, alkyne, a five membered ring and a six membered ring having at least one unsaturated bond or fused ring structure.25. The energy transfer dye of claim 24 , wherein the acceptor dye is a xanthene or cyanine dye.26. The energy transfer dye of claim 25 , wherein when the acceptor dye is a xanthene dye claim 25 , then the non-nucleosidic linker is attached to the xanthene acceptor dye at a 4′ claim 25 , 5 or 6 ring position.27. The energy transfer dye of claim 25 , wherein the acceptor dye is selected from the group consisting of 4 claim 25 ,7 dichlorofluorescein dyes claim 25 , asymmetric ...

Propargyl Substituted Nucleoside Compounds and Methods

Disclosed, among other things, are compounds having the structure 1107-. (canceled)109. The compound of claim 108 , wherein Wis H.110. The compound of claim 108 , wherein Wis H claim 108 , azido claim 108 , amino claim 108 , fluoro claim 108 , chloro claim 108 , or methoxy.111. The compound of claim 108 , wherein Wis —OH.112. The compound of claim 108 , wherein Wis —OH.113. The compound of claim 108 , wherein Wis monophosphate claim 108 , diphosphate claim 108 , or triphosphate.114. The compound of claim 108 , wherein Wis triphosphate.115. The compound of claim 108 , wherein LABEL is selected from the group consisting of a fluorescent dye claim 108 , an energy transfer dye claim 108 , a quencher claim 108 , and biotin.116. The compound of claim 108 , wherein LABEL is a fluorescent dye comprising a rhodamine dye claim 108 , a fluorescein dye claim 108 , a rhodol dye claim 108 , a cyanine dye claim 108 , a phthalocyanine dye or a squaraine dye.117. The compound of claim 108 , wherein the fluorescent dye is a fluorescein dye claim 108 , a rhodamine dye or an energy transfer dye.118. The compound of claim 108 , wherein LABEL is an energy transfer dye comprising a donor dye covalently attached to an acceptor dye claim 108 , wherein either the donor dye or the acceptor dye is covalently attached to the linker claim 108 , and the acceptor dye is capable of absorbing light emitted by the donor dye.119. The compound of claim 118 , wherein the donor dye comprises a fluorescein dye or a rhodamine dye.120. The compound of claim 118 , wherein the acceptor dye comprises one of a rhodamine dye claim 118 , a fluorescein dye claim 118 , a cyanine dye or a non-fluorescent quencher.126. The compound of claim 125 , wherein LABEL is selected from the group consisting of a fluorescent dye claim 125 , an energy transfer dye claim 125 , a quencher claim 125 , and biotin.127. The compound of claim 125 , wherein LABEL is an energy transfer dye comprising a donor dye covalently attached to an ...

MUTANT DNA POLYMERASES

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides. 1. An isolated mutant DNA polymerase capable of selectively transiently-binding a labeled nucleotide and selectively incorporating a terminator nucleotide.2. The isolated mutant DNA polymerase of claim 1 , comprising a mutant RB69 DNA polymerase.3. The isolated mutant DNA polymerase of claim 1 , comprising an amino acid sequence according to any one of SEQ ID NOS:2-8.4. The isolated mutant DNA polymerase of further comprises a reporter moiety.5. The isolated mutant DNA polymerase of claim 4 , wherein the reporter moiety is an energy transfer donor moiety.6. The isolated mutant DNA polymerase of claim 5 , wherein the energy transfer donor moiety is a fluorescent dye or a nanoparticle.7. A system comprising a mutant DNA polymerase of bound to a DNA template and a primer claim 1 , and a nucleotide transiently-bound to the mutant DNA polymerase.8. An isolated mutant DNA polymerase comprising an amino acid sequence that is at least 80% claim 1 , 85% claim 1 , 90% claim 1 , 95% claim 1 , 97% or 99% identical to any amino acid sequence selected from the group consisting of: SEQ ID NO: 1 claim 1 , SEQ ID NO: 2 claim 1 , SEQ ID NO:3 claim 1 , SEQ ID NO:4 claim 1 , SEQ ID NO:5 claim 1 , SEQ ID NO:6 claim 1 , SEQ ID NO:7 and SEQ ID NO:8. This application is a continuation of U.S. Nonprovisional application Ser. No. 12/790,768, filed on May 28, 2010 which claims the filing date benefit of U.S. Provisional Application No. 61/184,774, filed on Jun. 5, 2009; 61/242,762, filed on Sep. 15, 2009; 61/263,320, filed on Nov. 20, 2009; and 61/295,533, filed on Jan. 15, 2010. The contents of each of the foregoing ...

Polymer particles, nucleic acid polymer particles and methods of making and using the same

The disclosure relates to methods of making polymer particles, said methods including the steps of: making an aqueous gel reaction mixture; forming an emulsion having dispersed aqueous phase micelles of gel reaction mixture in a continuous phase; adding an initiator oil comprising at least one polymerization initiator to the continuous phase; and performing a polymerization reaction in the micelles. Further, the initiator oil is present in a volume % relative to a volume of the aqueous gel reaction mixture of between about 1 vol % to about 20 vol %. The disclosure also relates to methods of making nucleic acid polymer particles having the same method steps and wherein the aqueous gel reaction mixture includes a nucleic acid fragment, such as a primer.

MODIFIED NUCLEOTIDES AND USES THEREOF

In some embodiments, the disclosure relates generally to methods, as well as related, systems, compositions, kits and apparatuses, for nucleic acid analysis that involve the use of modified nucleotides, including terminator nucleotides and/or tagged nucleotides, in a template-dependent nucleotide incorporation reaction. In some embodiments, the nucleic acid analysis can be conducted at a single reaction site, or at a plurality of reaction sites in an array of reaction sites. Optionally, the array contains a plurality of reaction sites having about 1-100 million, or about 100-250 million, or about 200-500 million, or about 500-900 million, or more reaction sites. Optionally, each reaction site is in contact with, operatively coupled, or capacitively coupled to one or more sensors that are ion-sensitive FETs (isFETs) or chemically-sensitive FETs (chemFETs) sensors. Optionally, the reaction sites are in fluid communication with each other. 1. A method for detecting a plurality of nucleotide incorporations , comprising:a) providing a surface having 100-700 million reaction sites, wherein each reaction site is attached or operatively linked to at least one sensor, and the reaction sites contain (i) a polymerase and (ii) a nucleic acid template and (iii) an extendible end;b) contacting the reaction sites with a first solution containing one or more terminator nucleotides;c) incorporating at least one type of a terminator nucleotide at the extendible ends and generating non-extendible ends having terminator moieties, and generating nucleotide incorporation products within the reaction sites; andd) detecting the nucleotide incorporation products within the reaction sites using the sensors.2. The method of claim 1 , further comprising removing claim 1 , cleaving or converting the terminator moieties from the non-extendible ends to generate extendible ends by contacting the terminator moieties on the non-extendible ends with at least one cleaving agent.3. The method of claim ...

NUCLEOTIDE TRANSIENT BINDING FOR SEQUENCING METHODS

Provided herein are compositions and systems for use in polymerase-dependent, nucleotide transient-binding methods. The methods are useful for deducing the sequence of a template nucleic acid molecule and single nucleotide polymorphism (SNP) analyses. The methods rely on the fact that the polymerase transient-binding time for a complementary nucleotide is longer compared to that of a non-complementary nucleotide. The labeled nucleotides transiently-binds the polymerase in a template-dependent manner, but does not incorporate. The methods are conducted under any reaction condition that permits transient binding of a complementary or non-complementary nucleotide to a polymerase, and inhibits nucleotide incorporation. 1. A method for identifying a nucleotide bound to a polymerase , comprising:a) contacting at least one type of a labeled nucleotide to an immobilized complex having a first polymerase bound to a template nucleic acid molecule that is bound to a polymerization initiation site, under suitable conditions to transiently-bind the at least one type of labeled nucleotide to the polymerase in a nucleic acid template-dependent manner and to inhibit nucleotide polymerization by the polymerase;b) detecting the transiently-bound labeled nucleotide; andc) identifying the labeled nucleotide transiently-bound to the polymerase.2. The method of claim 1 , wherein the suitable conditions in step (a) comprise: (i) reducing the levels or omission of a metal cation that permits nucleotide incorporation and/or addition of a cation that inhibits nucleotide incorporation; (ii) the polymerase selectively binds the nucleotide in a template-dependent manner and exhibits reduced nucleotide incorporation activity; (iii) the at least one type of labeled nucleotide is a labeled non-incorporatable nucleotide; and/or (iv) the polymerization initiation site is a non-extendible polymerization initiation site.3. The method of claim 1 , wherein the suitable conditions in step (a) comprise: ( ...

MUTANT RB69 DNA POLYMERASE

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides. 1. An isolated mutant DNA polymerase capable of selectively transiently-binding a labeled nucleotide and selectively incorporating a terminator nucleotide.2. The isolated mutant DNA polymerase of claim 1 , comprising a mutant RB69 DNA polymerase.3. The isolated mutant DNA polymerase of claim 1 , comprising an amino acid sequence according to any one of SEQ ID NOS:2-8.4. The isolated mutant DNA polymerase of further comprises a reporter moiety.5. The isolated mutant DNA polymerase of claim 4 , wherein the reporter moiety is an energy transfer donor moiety.6. The isolated mutant DNA polymerase of claim 5 , wherein the energy transfer donor moiety is a fluorescent dye or a nanoparticle.7. A system comprising a mutant DNA polymerase of bound to a DNA template and a primer claim 1 , and a nucleotide transiently-bound to the mutant DNA polymerase.8. A nucleic acid molecule encoding the mutant DNA polymerase of .9. The nucleic acid molecule of which is DNA or RNA.10. A vector comprising the nucleic acid molecule of .11. The vector of further comprising a promoter sequence joined with the nucleic acid molecule encoding the mutant DNA polymerase.12. The vector of claim 11 , wherein the promoter is constitutive or inducible.13. A host cell carrying the vector of .14. The host cell of which is a phage claim 13 , a prokaryote cell or a eukaryote cell.15. A method for producing a mutant DNA polymerase polypeptide claim 14 , comprising culturing the host cell of under conditions suitable for the host cell to produce the mutant DNA polymerase polypeptide.16. The mutant DNA polymerase produced by the method ...

Polymer particles, nucleic acid polymer particles and methods of making and using the same

The disclosure relates to methods of making polymer particles, said methods including the steps of: making an aqueous gel reaction mixture; forming an emulsion having dispersed aqueous phase micelles of gel reaction mixture in a continuous phase; adding an initiator oil comprising at least one polymerization initiator to the continuous phase; and performing a polymerization reaction in the micelles. Further, the initiator oil is present in a volume % relative to a volume of the aqueous gel reaction mixture of between about 1 vol % to about 20 vol %. The disclosure also relates to methods of making nucleic acid polymer particles having the same method steps and wherein the aqueous gel reaction mixture includes a nucleic acid fragment, such as a primer.

MUTANT RB69 DNA POLYMERASE

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides. 1. An isolated mutant DNA polymerase comprising an amino acid sequence according to any one of SEQ ID NOS: 3-8.2. The isolated mutant DNA polymerase of further comprising a reporter moiety.3. The isolated mutant DNA polymerase of claim 2 , wherein the reporter moiety is an energy transfer donor moiety.4. The isolated mutant DNA polymerase of claim 3 , wherein the energy transfer donor moiety is a fluorescent dye or a nanoparticle.5. A system comprising a mutant DNA polymerase of bound to a DNA template and a primer claim 1 , and a nucleotide transiently-bound to the mutant DNA polymerase.6. A nucleic acid molecule comprising a nucleotide sequence encoding the mutant DNA polymerase of .7. A vector comprising the nucleic acid molecule of .8. The vector of claim 7 , further comprising a promoter sequence which is operably joined to the nucleic acid sequence encoding the mutant DNA polymerase.9. The vector of claim 8 , wherein the promoter is constitutive or inducible.10. A host cell carrying the vector of .11. A host cell carrying the vector of .12. A method for producing a mutant DNA polymerase polypeptide claim 11 , comprising culturing the host cell of under conditions suitable for the host cell to produce the mutant DNA polymerase polypeptide.13. The mutant DNA polymerase produced by the method of . This application is a divisional of U.S. application Ser. No. 15/200,670, filed Jul. 1, 2016, now allowed, which is a divisional of U.S. application Ser. No. 14/191,997, filed on Feb. 27, 2014, now U.S. Pat. No. 9,399,767, which is a continuation of U.S. application Ser. No. 13/600,416, filed on ...

NUCLEOTIDE TRANSIENT BINDING FOR SEQUENCING METHODS

Provided herein are compositions and systems for use in polymerase-dependent, nucleotide transient-binding methods. The methods are useful for deducing the sequence of a template nucleic acid molecule and single nucleotide polymorphism (SNP) analyses. The methods rely on the fact that the polymerase transient-binding time for a complementary nucleotide is longer compared to that of a non-complementary nucleotide. The labeled nucleotides transiently-binds the polymerase in a template-dependent manner, but does not incorporate. The methods are conducted under any reaction condition that permits transient binding of a complementary or non-complementary nucleotide to a polymerase, and inhibits nucleotide incorporation. 120-. (canceled)21. A method for identifying a nucleotide bound to a polymerase , comprising: wherein the first polymerase is included in a first immobilized complex comprising (i) the first polymerase, (ii) a first template nucleic acid molecule, and (iii) a first polymerization initiation site,', 'wherein the first immobilized complex is linked to a first site on a surface and the surface includes a plurality of immobilized complexes linked to other sites on the same surface', 'wherein the first polymerase lacks exonuclease activity, and', 'wherein the first type of nucleotide includes a detectable moiety;', 'detecting the first type of transiently-bound nucleotide; and', 'identifying the first type of transiently-bound nucleotide., 'transiently binding in a template-dependent manner, and without polymerizing, a first type of nucleotide to a first polymerase in the presence of a cation that inhibits nucleotide incorporation by the first polymerase,'}22. The method of claim 21 , further comprising: removing the first type of nucleotide that is transiently bound to the first polymerase.23. The method of claim 22 , further comprising: contacting the first immobilized complex with a second type of nucleotide under suitable conditions for the first ...

NUCLEOTIDE TRANSIENT BINDING FOR SEQUENCING METHODS

Provided herein are compositions and systems for use in polymerase-dependent, nucleotide transient-binding methods. The methods are useful for deducing the sequence of a template nucleic acid molecule and single nucleotide polymorphism (SNP) analyses. The methods rely on the fact that the polymerase transient-binding time for a complementary nucleotide is longer compared to that of a non-complementary nucleotide. The labeled nucleotides transiently-binds the polymerase in a template-dependent manner, but does not incorporate. The methods are conducted under any reaction condition that permits transient binding of a complementary or non-complementary nucleotide to a polymerase, and inhibits nucleotide incorporation. 1. A method for identifying a nucleotide bound to a polymerase , comprising:a) contacting at least one type of a labeled nucleotide to an immobilized complex having a first polymerase bound to a template nucleic acid molecule that is bound to a polymerization initiation site, under suitable conditions to transiently-bind the at least one type of labeled nucleotide to the polymerase in a nucleic acid template-dependent manner and to inhibit nucleotide polymerization by the polymerase;b) detecting the transiently-bound labeled nucleotide; andc) identifying the labeled nucleotide transiently-bound to the polymerase.2. A system comprising:a) a polymerase which lacks exonuclease activity complexed with a template nucleic acid molecule which is bound to a polymerization initiation site; a nucleotide including a detectable moiety which is transiently bound to the active site of the polymerase in a template-dependent manner; and a cation that inhibits nucleotide incorporation by the polymerase, present at a concentration that inhibits nucleotide incorporation by the polymeraseb) a detection system comprising excitation illumination elements, optical transmission elements and detectors.3. The system of claim 2 , wherein the transiently-bound nucleotide is ...

MUTANT RB69 DNA POLYMERASE

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides. 1. An isolated nucleic acid comprising a nucleotide sequence encoding a mutant DNA polymerase having the amino acid sequence of SEQ ID NO:2.2. A vector comprising the nucleic acid sequence of .3. The vector of claim 2 , further comprises a promoter sequence which is operably joined to the nucleic acid sequence encoding the mutant DNA polymerase.4. The vector of claim 3 , wherein the promoter is constitutive or inducible.5. A host cell carrying the vector of .6. A host cell carrying the vector of .7. A method for producing a mutant DNA polymerase polypeptide claim 6 , comprising culturing the host cell of under conditions suitable for the host cell to produce the mutant DNA polymerase polypeptide.8. The mutant DNA polymerase produced by the method of . This application is a divisional of U.S. Nonprovisional application Ser. No. 14/191,997, filed on Feb. 27, 2014, which is a continuation of U.S. Nonprovisional application Ser. No. 13/600,416, filed on Aug. 31, 2012, now issued U.S. Pat. No. 8,703,461, which is a continuation of U.S. Nonprovisional application Ser. No. 12/790,768, filed on May 28, 2010, now abandoned, which claims the filing date benefit of U.S. Provisional Application No. 61/263,320, filed on Nov. 20, 2009; and the subject application is a continuation of U.S. Non-provisional application Ser. No. 14/991,230, filed Jan. 8, 2016, which is a continuation of U.S. Non-provisional application Ser. No. 14/108,166, filed on Dec. 16, 2013, now issued U.S. Pat. No. 9,255,258, which is a continuation of U.S. Non-provisional application Ser. No. 12/790,760, filed on May 28, 2010, now issued ...

Thiolated nucleotide analogues for nucleic acid synthesis

The present disclosure provide systems, compositions, methods, reagents, kits and products for extending a nucleic acid that includes incorporating a nucleotide residue at a terminus of a nucleic acid using a polymerase enzyme and at least one nucleotide, wherein the at least one nucleotide includes a thiophosphate moiety, and wherein the at least one nucleotide is resistant to hydrolysis by phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of a phosphatase. In some embodiments, the nucleotide incorporation can be conducted in the presence of at least on chelation moiety that is configured to bind an orthophosphate moiety.

Polymer particles, nucleic acid polymer particles and methods of making and using the same

The disclosure relates to methods of making polymer particles, said methods including the steps of: making an aqueous gel reaction mixture; forming an emulsion having dispersed aqueous phase micelles of gel reaction mixture in a continuous phase; adding an initiator oil comprising at least one polymerization initiator to the continuous phase; and performing a polymerization reaction in the micelles. Further, the initiator oil is present in a volume % relative to a volume of the aqueous gel reaction mixture of between about 1 vol % to about 20 vol %. The disclosure also relates to methods of making nucleic acid polymer particles having the same method steps and wherein the aqueous gel reaction mixture includes a nucleic acid fragment, such as a primer.

Nucleotide transient binding for sequencing methods

Provided herein are compositions and systems for use in polymerase-dependent, nucleotide transient-binding methods. The methods are useful for deducing the sequence of a template nucleic acid molecule and single nucleotide polymorphism (SNP) analyses. The methods rely on the fact that the polymerase transient-binding time for a complementary nucleotide is longer compared to that of a non-complementary nucleotide. The labeled nucleotides transiently-binds the polymerase in a template-dependent manner, but does not incorporate. The methods are conducted under any reaction condition that permits transient binding of a complementary or non-complementary nucleotide to a polymerase, and inhibits nucleotide incorporation.

Fluorescent nucleobase conjugates having anionic linkers

Provided are nucleotide-dye conjugates and related compounds in which a dye is linked to a nucleobase directly or indirectly by an anionic linker. The anionic character of the linker is provided by one or more anionic moieties which are present in the linker, such as phosphate, phosphonate, sulfonate, and carboxylate groups. When the dye is a provided as a donor/acceptor dye pair, the anionic linker can be located between the donor and the acceptor, or between the nucleobase and either the donor or acceptor, or both. In one embodiment, conjugates of the invention provide enhanced electrophoretic mobility characteristics to sequencing fragments, e.g., for dideoxy sequencing using labeled terminators.

Universal nucleotides for nucleic acid analysis

The invention includes methods and kits for making and analyzing primer extension products incorporating one or more universal bases, including methods and kits for nucleic acid sequencing and microsatellite analysis.

4, 7-Dichlororhodamine dyes

A class of 4,7-dichlororhodamine compounds useful as fluorescent dyes are disclosed having the structure ##STR1## wherein R 1 -R 6 are hydrogen, fluorine, chlorine, lower alkyl lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, lining group, or combinations thereof or, when taken together, R 1 and R 6 is benzo, or, when taken together, R 4 and R 5 is benzo; Y 1 -Y 4 are hydrogen or lower alkyl or, when taken together, Y 1 and R 2 is propano and Y 2 and R 1 is propano, or, when taken together, Y 3 and R 3 is propano and Y 4 and R 4 is propano; and X 1 -X 3 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl carboxylate, sulfonic acid, --CH 2 OH, and linking group. In another aspect, the invention includes reagents labeled with the 4,7-dichlororhodamine dye compounds, including deoxynucleotides, dideoxynucleotides, and polynucleotides. In an additional aspect, the invention includes methods utilizing such dye compounds and reagents including dideoxy polynucleotide sequencing and fragment analysis methods.

Propargylethoxyamino nucleotides

Propargylethoxyamino nucleosides are disclosed having the structure ##STR1## wherein R 1 and R 2 are --H, lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W 1 is --H or --OH; W 2 is --OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'-position; and W 3 is --PO 4 , --P 2 O 7 , --P 3 O 10 , phosphate analog, or --OH. Additionaly, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

Substituted propargylethoxyamido nucleosides, oligonucleotides and methods for using same

Substituted propargylethoxyamido nucleosides are disclosed having the structure ##STR1## wherein X is selected from the group consisting of amino alkanoic acid, alkylamino benzoic acid, α-amino acid, and 4-amino-2-butynoic acid. R 1 and R 2 taken separately are selected from the group consisting of --H, lower alkyl, protecting group, and label; R 3 is selected from the group consisting of --H and lower alkyl. B is a 7-deazapurine, purine, or pyrimidine nucleoside base. When B is purine or 7-deazapurine, the sugar moiety is attached at the N 9 -position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N 1 -position of the pyrimidine. When B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine. W 1 is selected from the group consisting of --H and --OH. W 2 is --OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'-position. W 3 is selected from the group consisting of --PO 4 , --P 2 O 7 , --P 3 O 10 , phosphate analog, and --OH. Additionaly, a primer extension method is provided employing the above X-substituted propargylethoxyamido nucleosides, and polynucleotides including the above X-substituted propargylethoxyamido nucleosides is provided.

Fluorescent nucleobase conjugates having anionic linkers

Provided are nucleotide-dye conjugates and related compounds in which a dye is linked to a nucleobase directly or indirectly by an anionic linker. The anionic character of the linker is provided by one or more anionic moieties which are present in the linker, such as phosphate, phosphonate, sulfonate, and carboxylate groups. When the dye is a provided as a donor/acceptor dye pair, the anionic linker can be located between the donor and the acceptor, or between the nucleobase and either the donor or acceptor, or both. In one embodiment, conjugates of the invention provide enhanced electrophoretic mobility characteristics to sequencing fragments, e.g., for dideoxy sequencing using labeled terminators.

Polynucleotide sequence detection assays

Polynucleotide sequence detection assays and analysis

Methods and software for associating mobility probes with target macromolecules are discussed. By encoding the identities of macromolecules of interest with a universal set of tag portions complementary to a universal set of mobility probes, reactions varying in their input starting material may be identified using the same universal set of mobility probes. This allows the universal collection of mobility probes to be used in a target macromolecule-independent manner. Software is used to decode the associations between the mobility probes and a given target macromolecular identity.

Energy transfer dyes with enchanced fluorescence

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R21Z1C(O)R22R28 where R21 is a C1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z1 is either NH, sulfur or oxygen, R22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R28 includes a functional group which attaches the linker to the acceptor dye.

Energy transfer dyes with enhanced fluorescence

Energy transfer fluorescent dyes, reagents incorporating the dyes, kits and methods for using the dyes and reagents are provided. The energy transfer fluorescent dyes include a donor dye which absorbs light at a first wavelength and emits excitation energy in response, the donor dye including a xanthene ring structure having a 4' ring position, an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, and a linker attaching the donor dye to the acceptor dye, the linker having a 4' end which includes a R 1 XC(O)R 2 group where R 1 is a C 1-5 alkyl attached to the 4' ring position of the donor dye, X selected from the group consisting of NH, sulfur and oxygen, C(O) is a carbonyl group, and R 2 includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon. Alternatively, the energy transfer fluorescent dyes include a donor dye which absorbs light at a first wavelength and emits excitation energy in response, the donor dye including a xanthene ring structure, an acceptor dye which is either a xanthene, cyanine, phthalocyanine or squaraine dye which is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, the acceptor having an emission maximum that is greater than about 600 nm or at least about 100 nm greater than the absorbance maximum of the donor dye, and a linker attaching the donor dye to the acceptor dye.

Method for detecting oligonucleotides using energy transfer dyes with long stoke shift

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 26 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Oligonucleotides and analogs labeled with energy transfer dyes

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Oligonucleotides and analogs labeled with energy transfer dyes

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers facilitate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Kits useful for sequencing nucleic acids

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers facilitate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Methods of labeling nucleic acids with energy transfer dyes

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Oligonucleotides and analogs labeled with energy transfer dyes

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers facilitate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Methods of analyzing polynucleotides employing energy transfer dyes

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers facilitate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Methods of labeling polynucleotides with energy transfer dyes

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Regents labeled with energy transfer dyes

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Energy transfer dyes with enhanced fluorescence

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Energy transfer dyes with enhanced fluorescence

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Methods of analyzing polynucleotides employing energy transfer dyes

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Methods and compositions for synthesis of labelled oligonucleotides and analogs on solid-supports

Methods and compositions to label oligonucleotides and analogs directly on a solid-support having the structure where S is a solid-support, A is a cleavable linker, X is a moiety with three or more attachment sites, L is a label, Y is a nucleophile, i.e. O, NH, NR or S, and P 1 is an acid cleavable protecting group are provided. The labelled solid-support is reacted in a cyclical fashion to synthesize a labelled oligonucleotide on a solid-support in the 5′ to 3′ direction, having the structure: Labelled oligonucleotides are also synthesized by reacting: (i) a label reagent bearing functionality consisting of carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid, (ii) an oligonucleotide on solid support with nucleophilic functionality, and (iii) a coupling reagent, whereby an ester, amide, thioester, sulfonamide, sulfonate, phosphonate, phosphoramidate, phosphorothioate, or phosphate bond is formed. The labelling reaction may be conducted at label sites including the 5′ terminus, the 3′ terminus, a nucleobase, an internucleotide linkage, a sugar, amino, sulfide, hydroxyl, and carboxyl.

Energy transfer dyes with enhanced fluorescence

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Labelled oligonucleotides synthesized on solid-supports

Methods and compositions to label oligonucleotides and analogs directly on a solid-support having the structure where S is a solid-support, A is a cleavable linker, X is a moiety with three or more attachment sites, L is a label, Y is a nucleophile, i.e. O, NH, NR or S, and P 1 is an acid cleavable protecting group are provided. The labelled solid-support is reacted in a cyclical fashion to synthesize a labelled oligonucleotide on a solid-support in the 5′ to 3′ direction, having the structure: Labelled oligonucleotides are also synthesized by reacting: (i) a label reagent bearing functionality consisting of carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid, (ii) an oligonucleotide on solid support with nucleophilic functionality, and (iii) a coupling reagent, whereby an ester, amide, thioester, sulfonamide, sulfonate, phosphonate, phosphoramidate, phosphorothioate, or phosphate bond is formed. The labelling reaction may be conducted at label sites including the 5′ terminus, the 3′ terminus, a nucleobase, an internucleotide linkage, a sugar, amino, sulfide, hydroxyl, and carboxyl.

4,7-dichlororhodamine dyes

A class of 4,7-dichlororhodamine compounds useful as fluorescent dyes are disclosed having structure formula (I) wherein R1-R6 are hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group, or combinations thereof, or, when taken together, R1 and R6 is benzo, or, when taken together, R4 and R5 is benzo; Y1-Y4 are hydrogen or lower alkyl, or, when taken together, Y1 and R2 is propano and Y2 and R1 is propano, or, when taken together, Y3 and R3 is propano and Y4 and R4 is propano; and X1-X3 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonic acid, -CH2OH, and a linking group. In another aspect, the invention includes reagents labeled with the 4,7-dichlororhodamine dye compounds, including deoxynucleotides, dideoxynucleotides, and polynucleotides. In an additional aspect, the invention includes methods utilizing such dye compounds and reagents including dideoxy polynucleotide sequencing and fragment analysis methods.

4,7-dichlororhodamine dyes useful as molecular probes

A set of 4,7-dichlororhodamine compounds useful as fluorescent dyes are disclosed having the structures (I) and (VI) wherein R1-R6 are hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group, or, when taken together, R1 and R6 is benzo, or, when taken together, R4 and R5 is benzo; R7-R10, R12-R16 and R18 may be hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group; R11 and R17 may be hydrogen, lower alkyl, lower alkene, lower alkyne, phenyl, aryl, linking group; Y1-Y4 are hydrogen, lower alkyl, or cycloalkyl, or, when taken together, Y1 and R2, Y2 and R1, Y3 and R3, and/or Y4 and R4 is propano, ethano, or substituted forms thereof; and X1-X3 taken separately are hydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonate, hydroxymethyl, and linking group, or any combinations thereof. In another aspect, the invention includes reagents labeled with the 4,7-dichlororhodamine dye compounds, including deoxynucleotides, dideoxynucleotides, and polynucleotides. In an additional aspect, the invention includes methods utilizing such dye compounds and reagents including dideoxy polynucleotide sequencing and fragment analysis methods.

Polynucleotide sequence detection assays

The present teachings relate to methods, compositions, and kits for detecting one or more target polynucleotide sequences in a sample. In some embodiments of the present teachings, oligonucleotides are hybridized to complementary target polynucleotides and are ligated together to form a ligation product. In some embodiments of the present teachings, the ligation product can be amplified, and the identity and quantity of the target polynucleotides determined based on sequence introduced in the ligation reaction. Some embodiments of the present teachings allow for the detection of target polynucleotide sequences by hybridization of a mobility probe to sequence information introduced in the ligation reaction. Some embodiments of the present teachings provide for highly multiplexed detection, identification, and quantification of a plurality of target polynucleotides using a variety of analytical procedures.

Substituted propargylethoxyamido nucleosides

Substituted propargylethoxyamido nucleosides are disclosed having structure (I) wherein X is selected from the group consisting of amino alkanoic acid, alkylamino benzoic acid, α-amino acid, and 4-amino-2-butynoic acid. R1 and R2 taken separately are selected from the group consisting of -H, lower alkyl, protecting group, and label; R3 is selected from the group consisting of -H and lower alkyl. B is a 7-deazapurine, purine, or pyrimidine nucleoside base. When B is purine or 7-deazapurine, the sugar moiety is attached at the N9-position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N1-position of the pyrimidine. When B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine. W¿1? is selected from the group consisting of -H and -OH. W2 is -OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'-position. W3 is selected from the group consisting of -PO4, -P2O7, -P3O10, phosphate analog, and -OH. Additionally, a primer extension method is provided employing the above X-substituted propargylethoxyamido nucleosides, and polynucleotides including the above X-substituted propargylethoxyamido nucleosides is provided.

Nucleic Acid Terminators Incorporating a Cationic Moiety and Methods for Their Use

Disclosed are methods and kits applicable to sequencing methods, such as Sanger dideoxy sequencing methods. The methods and kits disclosed utilize a cationically charged nucleic acid terminator in combination with a discriminatory polymerase.

Sulfonated diarylrhodamine dyes as fluorescent labels

Sulfonated diarylrhodamine compounds are useful as fluorescent labels of nucleosides, nucleotides, polynucleotides, and polypeptides. The compounds find particular application in the area of fluorescent nucleic acid analysis, e.g., automated DNA sequencing and fragment analysis, detection of probe hybridization in hybridization arrays, detection of nucleic acid amplification products, and the like.

Energy transfer dyes with enhanced fluorescence

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers facilitate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R21Z1C(O)R22R28 where R21 is a C1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z, is either NH, sulfur or oxygen, R22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R28 includes a functional group which attaches the linker to the acceptor dye.

4,7-dichlororhodamine dyes labeled polynucleotides

A set of 4,7-dichlororhodamine compounds useful as fluorescent dyes are disclosed having the structures wherein R 1 -R 6 are hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group, or, when taken together, R 1 and R 6 is benzo, or, when taken together, R 4 and R 5 is benzo; R 7 -R 10 , R 12 -R 16 and R 18 may be hydrogen, fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, amido, nitrile, lower alkoxy, linking group; R 11 and R 17 may be hydrogen, lower alkyl, lower alkene, lower alkyne, phenyl, aryl, linking group; Y 1 -Y 4 are hydrogen, lower alkyl, or cycloalkyl, or, when taken together, Y 1 and R 2 , Y 2 and R 1 Y 3 and R 3 , and/or Y 4 and R 4 is propano, ethano, or substituted forms thereof, and X 1 -X 3 taken separately are hydrogen, chlorine, fluorine, lower alkyl, carboxylate, sulfonate, hydroxymethyl, and linking group, or any combinations thereof. In another aspect, the invention includes reagents labeled with the 4,7-dichlororhodamine dye compounds, including deoxynucleotides, dideoxynucleotides, and polynucleotides. In an additional aspect, the invention includes methods utilizing such dye compounds and reagents including dideoxy polynucleotide sequencing and fragment analysis methods.

Fluorescent nucleobase conjugates having anionic linkers

Provided are nucleotide-dye conjugates and related compounds in which a dye is linked to a nucleobase directly or indirectly by an anionic linker. The anionic character of the linker is provided by one or more anionic moieties which are present in the linker, such as phosphate, phosphonate, sulfonate, and carboxylate groups. When the dye is a provided as a donor/acceptor dye pair, the anionic linker can be located between the donor and the acceptor, or between the nucleobase and either the donor or acceptor, or both. In one embodiment, conjugates of the invention provide enhanced electrophoretic mobility characteristics to sequencing fragments, e.g., for dideoxy sequenci ng using labeled terminators.

Methods and compositions for synthesis of labelled oligonucleotides and analogs on solid-supports

Mobility-modifying cyanine dyes

The present invention provides a novel class of fluorescent cyanine dye compounds that are modified at one of the heterocyclic ring nitrogen atoms with a mobility-modifying moiety that permits the electrophoretic mobilities of polynucleotides labeled with the mobility-modifying cyanine dyes to be adjusted or tuned in a predictable fashion while retaining enzymatic activity. The ability to predictably tune the relative electrophoretic mobilities of the dyes permits the creation of sets of mobility-matched fluorescent dyes of a variety of structures for a variety of applications, including fluorescence-based 4-color nucleic acid sequencing reactions.

Energy transfer dyes with improved fluorescence

Metal chelation post incorporation detection methods

The present disclosure provides methods relating to detecting, identifying and measuring nucleic acids that includes binding metal ions to nucleic acid polymer backbones having negative charges formed as a result of nucleotide incorporation, and detecting or measuring hydrogen ions that are generated as a result of forming metal chelate complexes by addition of chelating agents.

Hydrophilic polymeric particles and methods for making same

A method of forming a particle includes, in a disperse phase within an aqueous suspension, polymerizing a plurality of mer units of a hydrophilic monomer having a hydrophobic protection group, thereby forming a polymeric particle including a plurality of the hydrophobic protection groups. The method further includes converting the polymeric particle to a hydrophilic particle.

Sulfonated diarylrhodamine dyes

Substituted propargylethoxyamido nucleosides

Substituted propargylethoxyamido nucleosides are disclosed having structure (I) wherein X is selected from the group consisting of amino alkanoic acid, alkylamino benzoic acid, α-amino acid, and 4-amino-2-butynoic acid. R1 and R2 taken separately are selected from the group consisting of -H, lower alkyl, protecting group, and label; R3 is selected from the group consisting of -H and lower alkyl. B is a 7-deazapurine, purine, or pyrimidine nucleoside base. When B is purine or 7-deazapurine, the sugar moiety is attached at the N9-position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N1-position of the pyrimidine. When B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine. W¿1? is selected from the group consisting of -H and -OH. W2 is -OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'-position. W3 is selected from the group consisting of -PO4, -P2O7, -P3O10, phosphate analog, and -OH. Additionally, a primer extension method is provided employing the above X-substituted propargylethoxyamido nucleosides, and polynucleotides including the above X-substituted propargylethoxyamido nucleosides is provided.

Mobility Modifying Cyanine Dyes

Substituted propargylethoxyamido nucleosides

【課題】ポリメラーゼ酵素の基質として有用なヌクレオチド化合物、プライマー伸長反応でこのようなヌクレオチド化合物を使用する方法、およびこのようなヌクレオチド化合物を含有するポリヌクレオチドの提供。 【解決手段】下記の置換プロパルギルエトキシアミドヌクレオシドの開示：Ｘは、アミノアルカン酸、アルキルアミノ安息香酸、α−アミノ酸、および４−アミノ−２−ブチン酸からなる群から選択される。Ｒ １ およびＲ ２ は、別個に、−Ｈ、低級アルキル、保護基、および標識からなる群から選択される；Ｒ ３ は、−Ｈおよび低級アルキルからなる群から選択される。Ｂは、７−デアザプリン、プリン、またはピリミジンヌクレオシド塩基である。さらに、プライマー伸長方法が提供され、これは、上記Ｘ置換プロパルギルエトキシアミドヌクレオシドを使用し、そして、上記Ｘ置換プロパルギルエトキシアミドヌクレオシドを含有するポリヌクレオチドが提供される。 【選択図】なし

Mutant dna polymerases

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides.

Synthesis of labelled oligonucleotides on solid-supports

Methods and compositions to label oligonucleotides and analogs directly on a solid-support having the structure (I), where S is a solid-support, A is a cleavable linker, X is a moiety with three or more attachment sites, L is a label, Y is a nucleophile, i.e. O, NH, NR or S, and P1 is an acid cleavable protecting group are provided. The labelled solid-support is reacted in a cyclical fashion to synthesize a labelled oligonucleotide on a solid-support in the 5' to 3' direction, having the structure (II). Labelled oligonucleotides are also synthesized by reacting: (i) a label reagent bearing functionality consisting of carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid, (ii) an oligonucleotide on solid support with nucleophilic functionality, and (iii) a coupling reagent, whereby an ester, amide, thioester, sulfonamide, sulfonate, phosphonate, phosphoramidate, phosphorothioate, or phosphate bond is formed. The labelling reaction may be conducted at label sites including the 5' terminus, the 3' terminus, a nucleobase, an internucleotide linkage, a sugar, amino, sulfide, hydroxyl, and carboxyl.

Fluorescent nucleobase conjugates having anionic linkers

Propargyl substituted nucleoside compounds and methods

Disclosed, among other things, are compounds having the structure wherein X comprises a bond or a linker, LABEL comprises at least one detectable label, W 1 taken alone is —H or —OH, W 2 is —OH or a non-extendable moiety, W 3 when taken alone is —H or when taken together with W 1 is —CH 2 —O—, and W 4 is OH, monophosphate, diphosphate, or triphosphate. Also disclosed are labeled polynucleotide compounds and methods of use thereof.

Substituted propargylethoxyamido nucleosides

Substituted propargylethoxyamido nucleosides are disclosed having structure (I) wherein X is selected from the group consisting of amino alkanoic acid, alkylamino benzoic acid, .alpha.-amino acid, and 4-amino-2-butynoic acid. R1 and R2 taken separately are selected from the group consisting of -H, lower alkyl, protecting group, and label; R3 is selected from the group consisting of -H and lower alkyl. B is a 7-deazapurine, purine, or pyrimidine nucleoside base. When B is purine or 7-deazapurine, the sugar moiety is attached at the N9-position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N1-position of the pyrimidine. When B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine. W1 is selected from the group consisting of -H and -OH. W2 is -OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3'-position. W3 is selected from the group consisting of -PO4, -P2O7, -P3O10, phosphate analog, and -OH. Additionally, a primer extension method is provided employing the above X-substituted propargylethoxyamido nucleosides, and polynucleotides including the above X-substituted propargylethoxyamido nucleosides is provided.

Universal nucleotides for nucleic acid analysis

The invention includes methods and kits for making and analyzing primer extension products incorporating one or more universal bases, including methods and kits for nucleic acid sequencing and microsatellite analysis.

Energy transfer dyes with enhanced fluorescence

Novel linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye are provided. These linkers faciliate the efficient transfer of energy between a donor and acceptor dye in an energy transfer dye. One of these linkers for linking a donor dye to an acceptor dye in an energy transfer fluorescent dye has the general structure R 21 Z 1 C(O)R 22 R 28 where R 21 is a C 1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z 1 is either NH, sulfur or oxygen, R 22 is a substituent which includes an alkene, diene, alkyne, a five and six membered ring having at least one unsaturated bond or a fused ring structure which is attached to the carbonyl carbon, and R 28 includes a functional group which attaches the linker to the acceptor dye.

Synthesis of labelled oligonucleotides on solid-supports

Methods and compositions to label oligonucleotides and analogs directly on a solid-support having the structure where S is a solid-support, A is a cleavable linker, X is a moiety with three or more attachment sites, L is a label, Y is a nucleophile, i.e. O, NH, NR or S, and P<SUB>1 </SUB>is an acid cleavable protecting group are provided. The labelled solid-support is reacted in a cyclical fashion to synthesize a labelled oligonucleotide on a solid-support in the 5' to 3' direction, having the structure: Labelled oligonucleotides are also synthesized by reacting: (i) a label reagent bearing functionality consisting of carboxylic acid, sulfonic acid, phosphonic acid, or phosphoric acid, (ii) an oligonucleotide on solid support with nucleophilic functionality, and (iii) a coupling reagent, whereby an ester, amide, thioester, sulfonamide, sulfonate, phosphonate, phosphoramidate, phosphorothioate, or phosphate bond is formed. The labelling reaction may be conducted at label sites including the 5' terminus, the 3' terminus, a nucleobase, an internucleotide linkage, a sugar, amino, sulfide, hydroxyl, and carboxyl.

Propargylethoxyamino nucleotide primer extensions

Propargylethoxyamino nucleosides are disclosed having the structure wherein R 1 and R 2 are —H, lower alkyl, or label; B is a 7-deazapurine, purine, or pyrimidine nucleoside base; W 1 is —H or —OH; W 2 is —OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3′-position; and W 3 is —PO 4 , —P 2 O 7 , —P 3 O 10 , phosphate analog, or —OH. Additionaly, a primer extension method is provided employing the above propargylethoxyamino nucleosides.

Unnatural polymerase substrates that can sustain enzymatic synthesis of double stranded nucleic acids from a nucleic acid template and methods of use

Nucleotide analogs that can sustain the enzymatic synthesis of double-stranded nucleic acid from a nucleic template are described. The nucleotide analogs include: (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine, uracil and their analogs; (ii) a label attached to the base or analog of the base via a cleavable linker; (iii) a deoxyribose; and (iv) one or more phosphate groups. The linker and/or the label inhibits template directed polymerase incorporation of a further nucleotide substrate onto an extended primer strand. In addition, cleavage of the linker leaves a residue attached to the base which is not present in the natural nucleotide and which does not inhibit extension of the primer strand. The nucleotide analogs can therefore be used as reversible terminators in sequencing by synthesis methods without blocking the 3' hydroxyl group. Methods of sequencing DNA using the substrates are also described.

Einen starren linker enthaltende nukleotide

Energieübertragungsfarbstoffe mit verbesserter fluoreszenz

Energieübertragungsfarbstoffe mit verbesserter fluoreszenz

4,7-dichlorrhodaminfarbstoffe

Fluoreszierende nukleobasekonjugate mit anionische linker

Propargylethoxyamino nucleotide

Synthese von markierten oligonucleotiden auf festphasenträgern

Mobilitaetsveraendernde cyaninfarbstoffe

Atropisomers of asymmetric xanthene fluorescent dyes and methods of DNA sequencing and fragment analysis

Atropisomeric energy-transfer dye compounds are disclosed. A variety of molecular biology applications utilize atropisomeric xanthene fluorescent dyes as labels for substrates such as nucleotides, nucleosides, polynucleotides, polypeptides and carbohydrates. Methods include DNA sequencing, DNA fragment analysis, PCR, SNP analysis, oligonucleotide ligation, amplification, minisequencing, and primer extension.

Atropisomers of asymmetric xanthene fluorescent dyes and method of dna sequencing and fragment analysis

Atropisomeric energy-transfer dye compounds are disclosed. A variety of molecular biology applications utilize atropisomeric xanthene fluorescent dyes as labels for substrates such as nucleotides, nucleosides, polynucleotides, polypeptides and carbohydrates. Methods include DNA sequencing, DNA fragment analysis, PCR, SNP analysis, oligonucleotide ligation, amplification, minisequencing, and primer extension.

4,7-dichlororhodaminfarbstoffe als molekulare sonden

Verfahren zur dna-sequenzierung unter verwendung von 4,7-dichlorrhodaminfarbstoffen

Substituierte propargylethoxyamido-nukleoside

Kettenabbrechende nukleinsäure-sequenziermethode mit 2'-desoxyuridin-5'-triphosphat

Fluorescent nucleobase conjugates having anionic linkers

Chain-termination type nucleic acid sequencing method including 2'-deoxyuridine-5'-triphosphate

A chain-termination type DNA sequencing method is disclosed wherein deoxynucleotide 2'-deoxythymidine-5'-triphosphate is replaced by 2'-deoxyuridine-5'-triphosphate, or analogs thereof Kits for performing the method are also provided.

Polymer particles, nucleic acid polymer particles and methods of making and using the same

The disclosure relates to methods of making polymer particles, said methods including the steps of: making an aqueous gel reaction mixture; forming an emulsion having dispersed aqueous phase micelles of gel reaction mixture in a continuous phase; adding an initiator oil comprising at least one polymerization initiator to the continuous phase; and performing a polymerization reaction in the micelles. Further, the initiator oil is present in a volume % relative to a volume of the aqueous gel reaction mixture of between about 1 vol % to about 20 vol %. The disclosure also relates to methods of making nucleic acid polymer particles having the same method steps and wherein the aqueous gel reaction mixture includes a nucleic acid fragment, such as a primer.

Nucleotide transient binding for sequencing methods

Provided herein are compositions and systems for use in polymerase-dependent, nucleotide transient-binding methods. The methods are useful for deducing the sequence of a template nucleic acid molecule and single nucleotide polymorphism (SNP) analyses. The methods rely on the fact that the polymerase transient-binding time for a complementary nucleotide is longer compared to that of a non-complementary nucleotide. The labeled nucleotides transiently-binds the polymerase in a template-dependent manner, but does not incorporate. The methods are conducted under any reaction condition that permits transient binding of a complementary or non-complementary nucleotide to a polymerase, and inhibits nucleotide incorporation.

Mutant RB69 DNA polymerase

Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides.

Polymer particles, nucleic acid polymer particles and methods of making and using the same

The disclosure relates to methods of making polymer particles, said methods including the steps of: making an aqueous gel reaction mixture; forming an emulsion having dispersed aqueous phase micelles of gel reaction mixture in a continuous phase; adding an initiator oil comprising at least one polymerization initiator to the continuous phase; and performing a polymerization reaction in the micelles. Further, the initiator oil is present in a volume % relative to a volume of the aqueous gel reaction mixture of between about 1 vol % to about 20 vol %. The disclosure also relates to methods of making nucleic acid polymer particles having the same method steps and wherein the aqueous gel reaction mixture includes a nucleic acid fragment, such as a primer.