Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
원문보기
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C12Q-001/68
C12P-019/34
G01N-015/06
C07H-021/04
출원번호
US-0099718
(2011-05-03)
등록번호
US-9212393
(2015-12-15)
발명자
/ 주소
Williams, Peter
출원인 / 주소
Life Technologies Corporation
인용정보
피인용 횟수 :
5인용 특허 :
348
초록▼
The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template syst
The present invention relates to a novel method for analyzing nucleic acid sequences based on real-time detection of DNA polymerase-catalyzed incorporation of each of the four nucleotide bases, supplied individually and serially in a microfluidic system, to a reaction cell containing a template system comprising a DNA fragment of unknown sequence and an oligonucleotide primer. Incorporation of a nucleotide base into the template system can be detected by any of a variety of methods including but not limited to fluorescence and chemiluminescence detection. Alternatively, microcalorimetic detection of the heat generated by the incorporation of a nucleotide into the extending template system using thermopile, thermistor and refractive index measurements can be used to detect extension reactions.
대표청구항▼
1. An apparatus for DNA sequencing comprising: (a) at least one reaction chamber including a DNA primer/template system which produces a detectable signal when a DNA polymerase enzyme incorporates an unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer strand;(b) a
1. An apparatus for DNA sequencing comprising: (a) at least one reaction chamber including a DNA primer/template system which produces a detectable signal when a DNA polymerase enzyme incorporates an unlabeled and unblocked deoxyribonucleotide monophosphate onto the 3′ end of the primer strand;(b) a system for introducing into, and evacuating from, said reaction chamber at least one reagent selected from the group consisting of: buffers, electrolytes, DNA template, DNA primer, deoxyribonucleotides, and polymerase enzymes;(c) a system for converting said detectable signal into an electrical signal based on an electrical potential generated from said detectable signal in the at least one reaction chamber; and(d) a system for measuring the electrical signal generated by the detectable signal produced in the at least one reaction chamber. 2. The apparatus of claim 1, wherein said at least one reaction chamber further comprises said deoxyribonucleotide monophosphate, and wherein said deoxyribonucleotide monophosphate is not chemically modified. 3. The apparatus of claim 1, further comprising (e) a voltmeter configured to measure said electrical signal. 4. The apparatus of claim 1, wherein said at least one chamber further includes a solid support, wherein said primer/template system comprises a primer and a template, and wherein said template is tethered to said solid support. 5. The apparatus of claim 4, wherein said template comprises a linker moiety for tethering to said solid support. 6. The apparatus of claim 1, wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity. 7. The apparatus of claim 1, wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand by said DNA polymerase. 8. The apparatus of claim 1, wherein said apparatus provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand. 9. A method of DNA sequencing comprising: (a) providing a primer/template system comprising a template sequence hybridized to a primer oligonucleotide in the presence of a DNA polymerase;(b) contacting said primer/template system with a single type of deoxyribonucleotide under conditions that produce a detectable signal when said DNA polymerase incorporates an unlabeled and unblocked deoxyribonucleotide onto the 3′ end of said primer oligonucleotide, wherein the contacting occurs in a reaction chamber;(c) converting, with a device, said detectable signal into an electrical signal based on an electrical potential generated across said device by said detectable signal, wherein the converting occurs in the reaction chamber; and(d) detecting the electrical signal generated by the detectable signal produced in the reaction chamber. 10. The method of claim 9, further comprising (e) measuring said electrical signal with a voltmeter. 11. The method of claim 9, further comprising (e) flushing said reaction chamber with dNTP free buffer. 12. The method of claim 11, further comprising repeating steps (b) through (e). 13. The method of claim 11, further comprising repeating steps (b) through (e) until the complete nucleotide sequence of said template sequence is determined. 14. The method of claim 9, wherein said deoxyribonucleotide is not chemically modified. 15. The method of claim 9, wherein said reaction chamber further includes a solid support, and wherein said template sequence is tethered to said solid support. 16. The method of claim 15, wherein said template sequence comprises a linker moiety for tethering to said solid support. 17. The method of claim 9, wherein said DNA polymerase lacks 5′ to 3′ exonuclease activity. 18. The method of claim 9, wherein the amplitude of said electrical signal corresponds to the number of nucleotides added to said primer strand by said DNA polymerase. 19. The method of claim 9, wherein said electrical signal provides real-time detection of incorporation of said deoxyribonucleotide monophosphate onto said primer strand by said DNA polymerase.
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이 특허에 인용된 특허 (348)
Hiatt Andrew C. ; Rose Floyd, 3' protected nucleotides for enzyme catalyzed template-independent creation of phosphodiester bonds.
Fung Steven (Palo Alto CA) Woo Sam L. (Redwood City CA) Smith Lloyd M. (South Pasadena CA), Amino-derivatized phosphite and phosphate linking agents, phosphoramidite precursors, and useful conjugates thereof.
Goodman Thomas C. (Mountain View CA) Becker Martin (Palo Alto CA) Ullman Edwin F. (Atherton CA) Rose Samuel (Mountain View CA), Amplification method for polynucleotide assays.
J. Leighton Read ; Stephen P.A. Fodor ; Lubert Stryer ; Michael C. Pirrung ; Paul D. Hoeprich, Jr., Apparatus for forming polynucleotides or polypeptides.
McGall Glenn Hugh ; Miyada Charles Garrett ; Cronin Maureen T. ; Tan Jennifer Dee ; Chee Mark S., Arrays of modified nucleic acid probes and methods of use.
Chee Mark ; Cronin Maureen T. ; Fodor Stephen P. A. ; Huang Xiaohua X. ; Hubbell Earl A. ; Lipshutz Robert J. ; Lobban Peter E. ; Morris MacDonald S. ; Sheldon Edward L., Arrays of nucleic acid probes on biological chips.
Bitler Joel R. (Allentown PA) Bodnar Michael W. (Macungie PA) Booth Raymond H. (Walnutport PA) Roman Daniel J. (Bethlehem PA) Schneider Fred J. (Northampton PA) Seitzer Philip W. (Bethlehem PA) Wilki, Article carrying a distinctive mark.
Goodman Myron F. (4719 Alminar Ave. LaCanada CA 91011) Reha-Krantz Linda J. (10044 87th Avenue Edmonton ; Alberta CAX T6E 2N9 ), Chain-terminating nucleotides for DNA sequencing methods.
Cheng Jing ; Sheldon ; III Edward L. ; Wu Lei ; O'Connell James P., Channel-less separation of bioparticles on a bioelectronic chip by dielectrophoresis.
Baldarelli Richard ; Branton Daniel ; Church George ; Deamer David W. ; Akeson Mark ; Kasianowicz John, Characterization of individual polymer molecules based on monomer-interface interactions.
Church George ; Deamer David W. ; Branton Daniel ; Baldarelli Richard ; Kasianowicz John, Characterization of individual polymer molecules based on monomer-interface interactions.
Ju, Jingyue; Li, Zengmin; Tong, Anthony; Russo, James J., Combinatorial fluorescence energy transfer tags and their applications for multiplex genetic analyses.
GuoFan Hong CN; Wei-hua Huang CN, DNA polymerase having ability to reduce innate selective discrimination against fluorescent dye-labeled dideoxynucleotides.
Caldwell Karin D. (Salt Lake City UT) Chu Tun-Jen (Salt Lake City UT) Pitt William G. (Orem UT), DNA sequencing using fluorescence background electroblotting membrane.
Overbeck James W. ; Flowers Peter T. ; Montagu Jean I. ; Mace Myles L. ; Honkanen Peter, Depositing fluid specimens on substrates, resulting ordered arrays, techniques for deposition of arrays.
Hung, Su-Chun; Glazer, Alexander N.; Mathies, Richard A., Detection of nucleotide sequence variation through fluorescence resonance energy transfer label generation.
Cherukuri Satyam Choudary ; Demers Robert Richard ; Fan Zhong Hui Hugh ; Levine Aaron W. ; McBride Sterling Edward ; Zanzucchi Peter John, Device for selective distribution of liquids.
Evans Steven,GBX ; Mamone Joseph Anthony ; Davis Maria ; Connolly Bernard A.,GBX, Dideoxynucleotide-triphosphate utilization by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus.
Klimash June W. ; Brothers ; II Herbert M. ; Swanson Douglas R. ; Yin Rui ; Spindler Ralph ; Tomalia Donald A. ; Hsu Yong ; Cheng Roberta C., Disulfide-containing dendritic polymers.
Mathies Richard A. (Contra Costa County CA) Peck Konan (Contra Costa County CA) Stryer Lubert (Santa Clara County CA), High sensitivity fluorescent single particle and single molecule detection apparatus and method.
Ambrose W. Patrick ; Grace W. Kevin ; Goodwin Peter M. ; Jett James H. ; Orden Alan Van ; Keller Richard A., High throughput analysis of samples in flowing liquid.
Pirrung Michael C. (Durham NC) Read J. Leighton (Palo Alto CA) Fodor Stephen P. A. (Palo Alto CA) Stryer Lubert (Stanford CA), Large scale photolithographic solid phase synthesis of an array of polymers.
Pirrung Michael C. (Durham NC) Read J. Leighton (Palo Alto CA) Fodor Stephen P. A. (Palo Alto CA) Stryer Lubert (Stanford CA), Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof.
Nikiforov Theo ; Karn Jonathan,GBX ; Goelet Philip, Ligase/polymerase-mediated genetic bit analysis of single nucleotide polymorphisms and its use in genetic analysis.
Zanzucchi Peter John ; Cherukuri Satyam Choudary ; McBride Sterling Edward ; Demers Robert R. ; Levine Aaron W. ; Thaler Barry Jay ; Quinn Robert Leon ; Braun Paul Leonard ; Chiang William ; Fan Zhon, Liquid distribution system.
Mehta, Tammy Burd; Kopf-Sill, Anne R.; Parce, J. Wallace; Chow, Andrea W.; Bousse, Luc J.; Knapp, Michael R.; Nikiforov, Theo T.; Gallagher, Steve, Manipulation of microparticles in microfluidic systems.
Stavrianopoulos Jannis G. (New York NY) Kirtikar Dollie (Elmhurst NY) Johnston Kenneth H. (New York NY) Thalenfeld Barbara E. (New York NY), Method and structures employing chemically-labelled polynucleotide probes.
Green Ronald J.,CAX ; Chi Vrijmoed,CAX ; Gilchrist Rodney D.,CAX ; Dee Gregory,CAX ; Stevens John K.,CAX, Method and system for DNA sequence determination and mutation detection with reference to a standard.
Boyce-Jacino Michael T. ; Rogers Yu-Hui ; Goelet Philip, Method for analyzing the nucleotide sequence of a polynucleotide by oligonucleotide extension on an array.
Batz Hans-Georg,DEX ; Hansen Henrik Frydenlund,DKX ; Orum Henrik,DKX ; Koch Troels,DKX ; Schuster Gary B. ; Armitage Bruce A. ; Ly Danith, Method for determining a nucleic acid.
Holzrichter John F. (Berkeley CA) Siekhaus Wigbert J. (Berkeley CA), Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electro.
Jett James H. (Los Alamos NM) Keller Richard A. (Los Alamos NM) Martin John C. (Los Alamos NM) Moyzis Robert K. (Los Alamos NM) Ratliff Robert L. (Los Alamos NM) Shera E. Brooks (Los Alamos NM) Stewa, Method for rapid base sequencing in DNA and RNA.
Jett James H. (Los Alamos NM) Keller Richard A. (Los Alamos NM) Martin John C. (Los Alamos NM) Posner Richard G. (Los Alamos NM) Marrone Babetta L. (Los Alamos NM) Hammond Mark L. (Los Alamos NM) Sim, Method for rapid base sequencing in DNA and RNA with two base labeling.
Cocuzza Anthony J. (Wilmington DE) Hobbs ; Jr. Frank W. (Wilmington DE) Zagursky Robert J. (Wilmington DE) Straus Neil A. (North York CAX), Method for sequencing DNA using biotin-strepavidin conjugates to facilitate the purification of primer extension product.
Kilger Christian,DEX ; Paabo Svante,DEX, Method for the uncoupled, direct, exponential amplification and sequence of DNA molecules with the addition of a second thermostable DNA polymerase and its application.
Drmanac Radoje T. (Beograd YUX) Crkvenjakov Radomir B. (Beograd YUX), Method of determining an ordered sequence of subfragments of a nucleic acid fragment by hybridization of oligonucleotide.
Williams, Peter; Taylor, Thomas J.; Williams, Daniel J. B.; Gould, Ian; Hayes, Mark A., Method of determining the nucleotide sequence of Oligonucleotides and DNA molecules.
Williams, Peter; Taylor, Thomas J.; Williams, Daniel J. B.; Gould, Ian; Hayes, Mark A., Method of determining the nucleotide sequence of oligonucleotides and DNA molecules.
Williams, Peter; Taylor, Thomas J.; Williams, Daniel J. B.; Gould, Ian; Hayes, Mark A., Method of determining the nucleotide sequence of oligonucleotides and DNA molecules.
Williams, Peter; Taylor, Thomas J.; Williams, Daniel J. B.; Gould, Ian; Hayes, Mark A., Method of determining the nucleotide sequence of oligonucleotides and DNA molecules.
Williams,Peter; Hayes,Mark A.; Rose,Seth D.; Bloom,Linda B.; Reha Krantz,Linda J.; Pizziconi,Vincent B., Method of determining the nucleotide sequence of oligonucleotides and DNA molecules.
Burke Thomas J. (Madison WI) Thompson David V. (Monona WI) Spurgeon Sandra L. (Madison WI), Method of using a TAQ DNA polymerase without 5′-3′-exonuclease activity.
Prober James M. (Wilmington DE) Dam Rudy J. (Landenberg PA) Robertson ; Jr Charles W. (Rockland DE) Hobbs ; Jr. Frank W. (Wilmington DE) Trainor George L. (Glen Mills PA), Method, system and reagents for DNA sequencing.
Van Ness Jeffrey ; Tabone John C. ; Howbert J. Jeffry ; Mulligan John T., Methods and compositions for determining the sequence of nucleic acid molecules.
Van Ness, Jeffrey; Tabone, John C.; Howbert, J. Jeffry; Mulligan, John T., Methods and compositions for determining the sequence of nucleic acid molecules.
Sosnowski Ronald G. ; Butler William F. ; Tu Eugene ; Nerenberg Michael I. ; Heller Michael J. ; Edman Carl F., Methods and procedures for molecular biological analysis and diagnostics.
Schueller, Olivier J. A.; Duffy, David C.; Rogers, John A.; Brittain, Scott T.; Whitesides, George M., Methods for fabricating microfluidic structures.
Faris Sadeg M. (24 Pocantico River Rd. Pleasantville NY 10570) Jain Kanti (18 Algonquian Trail Briarcliff Manor NY 10510), Microchannel plate technology.
Richard A. Mathies ; Pankaj Singhal ; Jin Xie ; Alexander N. Glazer, Microfabricated capillary electrophoresis chip and method for simultaneously detecting multiple redox labels.
Ekstrm Bjrn (Upsala SEX) Jacobson Gunilla (Upsala SEX) Ohman Ove (Upsala SEX) Sjdin Hakan (Upsala SEX), Microfluidic structure and process for its manufacture.
Anderson Charles W. ; Bargeron C. Brent ; Benson Richard C. ; Carlson Micah A. ; Fraser Allan B. ; Groopman John D. ; Ko Harvey W. ; Kohler David R. ; Phillips Terry E. ; Strickland Paul T., Miniature immuno-optical rapid analyte sensor platform.
Ward David C. (Guilford CT) Langer Pennina R. (Monsey NY) Waldrop ; III Alexander A. (Charlottesville VA), Modified nucleotides and methods of preparing and using same.
Ward David C. (Guilford CT) Langer Pennina R. (Monsey NY) Waldrop ; III Alexander A. (Charlottesville VA), Modified nucleotides and polynucleotides and complexes form therefrom.
Ward David C. (Guilford CT) Langer Pennina R. (Monsey NY) Waldrop ; III Alexander A. (Charlottesville VA), Modified polynucleotides and methods of preparing same.
Heller Michael J. (Encinitas CA) Tu Eugene (San Diego CA) Butler William F. (Carlsbad CA), Molecular biological diagnostic systems including electrodes.
Laurence R. Gilbert ; James M. Jonza ; Andrew J. Ouderkirk ; Carl A. Stover ; Michael F. Weber ; William W. Merrill, Multilayer polymer film with additional coatings or layers.
Dale R. Pfost ; Sheila H. DeWitt ; Chao Lin ; Rolf E. Swenson ; Robert D. Juncosa ; Gary J. Schnerr ; Sheldon M. Kugelmass, Multiple fluid sample processor and system.
Yang Won S. (6-405 ; Donga APT. ; 26 Chang-dong ; Dobong-ku ; Seoul KRX) Kim Yoon O. (865-2 Daerim-dong ; Youngdungpo-ku ; Seoul KRX), Non-invasive method and apparatus for measuring blood glucose concentration.
Muehlegger, Klaus; Hoeltke, Hans-Joachim; Birkner, Christian; Von Der Eltz, Herbert, Nucleotides labelled with an infrared dye and their use in nucleic acid detection.
Pease R. Fabian (Stanford CA) McGall Glenn (Mountain View CA) Goldberg Martin J. (San Jose CA) Rava Richard P. (Palo Alto CA) Fodor Stephen P. A. (Palo Alto CA) Goss Virginia (Santa Barbara CA) Strye, Printing molecular library arrays using deprotection agents solely in the vapor phase.
Pease R. Fabian ; McGall Glenn ; Goldberg Martin J. ; Rava Richard P. ; Fodor Stephen P. A. ; Goss Virginia ; Stryer Lubert ; Winkler James L., Printing oligonucleotide arrays using deprotection agents solely in the vapor phase.
Mullis Kary B. (Kensington CA) Erlich Henry A. (Oakland CA) Arnheim Norman (Woodland Hills CA) Horn Glenn T. (Emeryville CA) Saiki Randall K. (Richmond CA) Scharf Stephen J. (Berkeley CA), Process for amplifying, detecting, and/or-cloning nucleic acid sequences.
Gelfand David H. (Oakland CA) Stoffel Susanne (El Cerrito CA) Lawyer Frances C. (Oakland CA) Saiki Randall K. (Richmond CA), Purified thermostable enzyme.
Hunkapiller Michael W. (San Carlos CA) Connell Charles R. (Redwood City CA) Mordan William J. (Sunnyvale CA) Lytle John D. (San Jose CA) Bridgham John A. (Hillsborough CA), Real time scanning electrophoresis apparatus for DNA sequencing.
Engelhardt Dean (New York NY) Rabbani Elazar (New York NY) Kline Stanley (Brooklyn NY) Stavrianopoulos Jannis G. (New York NY) Kirtikar Dollie (Elmhurst NY), Saccharide specific binding system labeled nucleotides.
Goodwin Peter M. ; Jett James H. ; Keller Richard A. ; Van Orden Alan K. ; Machara Nicholas P., Single molecule identification using selected fluorescence characteristics.
Hamilton Alan Lewis,GBX ; West Richard Martin,GBX ; Cummins William Jonathan,GBX ; Briggs Mark Samuel Jonathan,GBX ; Bruce Ian Edward,IEX, Squarate dyes and their use in fluorescent sequencing method.
Lepp Cyrus A. (Stoughton MA) Mussatto James J. (Fairfax Station VA) Ziegenmeyer James M. (Lexington MA), System for identification of cells by electrophoresis.
Slater Michael R. ; Huang Fen ; Hartnett James R. ; Bolchakova Elena ; Storts Douglas R. ; Otto Paul ; Miller Katharine M. ; Novikov Alexander ; Velikodvorskaya Galina A.,RUX, Thermophilic DNA polymerases from Thermotoga neapolitana.
Barnes Wayne M. (223 Renaldo Dr. Chesterfield MO 63017), Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of primer extension.
David Harrow Gelfand ; Lisa Vivian Kalman ; Fred Lawrence Reichert ; Christopher Lim Sigua ; Thomas W. Myers, Thermostable DNA polymerases incorporating nucleoside triphosphates labeled with fluorescein family dyes.
Fodor Stephen P. A. (Palo Alto CA) Stryer Lubert (Stanford CA) Pirrung Michael C. (Durham NC) Read J. Leighton (Palo Alto CA), Very large scale immobilized polymer synthesis.
Williams, Peter; Williams, Daniel J. B.; Bloom, Linda; Taylor, Thomas J., Method of determining the nucleotide sequence of oligonucleotides and DNA molecules.
Williams, Peter; Williams, Daniel J. B.; Bloom, Linda; Taylor, Thomas J., Method of determining the nucleotide sequence of oligonucleotides and DNA molecules.
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