EP1141402A1 - Technique de detection de sequences nucleotidiques specifiques par incorporation de polymerase et de nucleotides - Google Patents

Technique de detection de sequences nucleotidiques specifiques par incorporation de polymerase et de nucleotides

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Publication number
EP1141402A1
EP1141402A1 EP99964072A EP99964072A EP1141402A1 EP 1141402 A1 EP1141402 A1 EP 1141402A1 EP 99964072 A EP99964072 A EP 99964072A EP 99964072 A EP99964072 A EP 99964072A EP 1141402 A1 EP1141402 A1 EP 1141402A1
Authority
EP
European Patent Office
Prior art keywords
nucleotides
primer
target dna
rna
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99964072A
Other languages
German (de)
English (en)
Other versions
EP1141402A4 (fr
Inventor
Alonso Castro
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of California
Original Assignee
University of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California filed Critical University of California
Publication of EP1141402A1 publication Critical patent/EP1141402A1/fr
Publication of EP1141402A4 publication Critical patent/EP1141402A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates generally to detection of nucleic acid sequences, and, more particularly, to the selective incorporation of fluorescent markers to detect nucleic acid sequences.
  • the rapid and efficient detection of specific nucleic acid sequences in biological samples plays a central role in a variety of fields, including molecular biology, biotechnology, immunology, medical diagnosis, forensic analysis, and quality control of food products.
  • One of the most commonly used techniques for the detection of specific nucleic acid sequences is the Southern blot. This is a hybridization technique in which the fragments to be interrogated have been size- separated by gel electrophoresis and transferred from the gel to a nylon nitrocellulose filter. A radioactive probe is then added to the filter so that hybridization takes place. After washing away the excess probe, the band containing the target nucleic acid is detected by exposing an x-ray film to the filter.
  • Southern blotting suffers from some limitations: it involves a series of manually intensive procedures that cannot be run unattended and cannot be readily automated. The process for separating the fragments by gel electrophoresis and subsequently detecting the bands by autoradiography are time- consuming tasks that are susceptible to poor quantitative accuracy and poor reproducibility.
  • PCR polymerase chain reaction
  • Amplification products are usually detected by dyes that stain nucleic acids or by hybridization with sequence-specific probes. Amplification methods, however, may introduce ambiguities resulting from contamination or from variability in amplification efficiency. Therefore, there is a need for robust analytical methods that provide accurate quantitation and molecular weight estimates for target DNA or RNA segments.
  • the present invention includes a method for identifying a target DNA or RNA sequence.
  • a primer having a 3'-hydroxyl group at one end and having a sequence of nucleotides sufficiently homologous to hybridize with an identifying sequence of nucleotides in the target DNA or RNA is selected.
  • the primer is hybridized to the identifying sequence of nucleotides and a reporter molecule is synthesized on the target sequence by extending the primer by progressively binding nucleotides to the primer that are complementary to the corresponding nucleotides of the DNA or RNA sequence, where the complementary nucleotides include nucleotides labeled with a fluorophore. Fluorescence emitted by fiuorophores on individual reporter molecules is detected to identify the target DNA or RNA sequence.
  • FIGURES 1A-1 E schematically depict the process of the present invention.
  • FIGURE 2 graphically depicts the experimental results for the detection of a specific sequence of pUC19 DNA at the single-molecule level of sensitivity according to one embodiment of the present invention.
  • FIGURE 3 graphically depicts results for a control experiment run under identical conditions as those corresponding to the experimental results shown in FIGURE 2, except that the target was replaced by lambda DNA.
  • FIGURE 4 graphically depicts a simulation of single molecule fluorescence signals from a reporter molecule according to a second embodiment of the present invention.
  • a new method enables the direct detection of specific nucleic acid sequences in biological samples.
  • the basis of the approach is to monitor for the presence of a specific nucleic acid sequence of bacterial, human, plant or other origin.
  • the nucleic acid sequence may be a DNA or RNA sequence, and may be characteristic of a specific taxonomic group, a specific physiological function, or a specific genetic trait.
  • the method consists of synthesizing in vitro a fluorescent nucleic acid reporter molecule using a relatively short sequence of the target as a template as shown in Figures 1 A-1 E.
  • a DNA target ( Figure 1 A) is denatured according to well known processes to form a single stranded DNA target ( Figure 1 B).
  • a short oligonucleotide primer that is specific and complementary to the target is then hybridized to the single stranded DNA target.
  • a suitable polymerase and free nucleotides are added to the sample.
  • One of these oligonucleotides is at least partially labeled with a fluorophore.
  • the primer binds to an identifying sequence of the target, ( Figure 1 C) and the polymerase will incorporate the labeled and unlabeled nucleotides ( Figure 1 D) to reconstruct the target's complementary sequence as shown in Figure 1 E.
  • the labeled nucleotide concentration is kept below that of the unlabeled nucleotides, most of the labeled nucleotides will be incorporated into the reporter DNA molecule. Nonetheless, some free (i.e., unbound) labeled nucleotides will remain in the reaction mixture, but fluorescence from each synthesized reporter molecule will be much stronger than that of the free nucleotide background over the single-molecule detection time.
  • the sample is analyzed in a single molecule detection apparatus, as are well known and described in the art. Detection of the synthesized reporter molecule signifies the presence of the target being sought.
  • the fluorescent signal from the reporter molecule is much larger than that of the background fluorescence originating from free labeled nucleotides, since the reaction is allowed to proceed until the reporter molecule is hundreds or thousands of bases long.
  • the new method described here combines the advantages of flow-based analytical systems (system automation, speed, reproducibility) with the unsurpassed sensitivity of single-molecule detection.
  • the sensitivity of this method allows for the direct detection of specific genes without the need for using amplification methods such as PCR and exhibits advantages over current methodologies in terms of sensitivity, speed and per-assay-cost.
  • the non-radioactive approach for the ultrasensitive detection of specific sequences described here has applications in a wide variety of fields, such as gene identification, gene mapping, medical diagnostics, and biotechnology.
  • Primer design should be specific to the target being sought. Primers are typically 15-30 nucleotides long. Primer lengths greater than 15 nucleotides ensure that they will not anneal specifically to non-target nucleic acid. Generally, primer sequences have the following characteristics:
  • a proper temperature is selected for the hybridization of dNTP to extend the primer along the target DNA molecule. If the temperature is too low, nonspecific annealing will increase.
  • An optimal hybridization temperature may be predicted for a given primer/target pair with available software routines, e.g., PRIMER, developed by The Whitehead Institute for Biomedical Research. For this example, the optimal temperature for Taq DNA polymerase activity is 72° C.
  • Optional Add “STOP" solution to terminate enzymatic activity. If the reaction is not stopped, and the target is of suitable size, the amount of incorporated dye and, therefore, the reporter fluorescence intensity, will be proportional to the size of the fragment.
  • a suitable immobilization group e.g., biotin
  • a single-molecule detection apparatus such as a variation of that described in References 2 and 3 or U.S. Patent 5,209,834, issued May 11 , 1993, is used to detect fluorescence from the reporter molecule.
  • Suitable flow cytometer apparatus and methods for single molecule detection are found in U.S. Patent 5,558,998, issued September 24, 1996, and U.S. Patent Application 09/169,025, filed October 9, 1998, both incorporated by reference.
  • reaction conditions such as initial nucleotide concentration and temperature, it may or may not be necessary to remove unincorporated labeled nucleotide as explained in the Procedure section.
  • the reaction mixture was diluted 1000-fold to 50 mL.
  • Another way to avoid detecting interfering free nucleotides is to perform "single-molecule electrophoresis" as described in Reference 3 and in

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Cette invention a trait à une technique permettant une détection rapide et efficace d'une séquence cible d'ADN ou d'ARN. On sélectionne, dans le cadre de cette invention, une amorce possédant un groupe 3'-hydroxy sur l'une de ses extrémités et pourvue d'une séquence nucléotidique suffisamment homologue avec une séquence d'identification de nucléotides dans l'ADN ciblé. On hybride l'amorce à la séquence d'identification de nucléotides sur la séquence d'ADN ou d'ARN et on synthétise une molécule reporter sur la séquence cible par fixation progressive de nucléotides complémentaires à l'amorce, ces nucléotides complémentaires comportant des nucléotides marqués à l'aide d'un fluorophore. La fluorescence émise par les fluorophores sur les molécules du reporter est détectée aux fins de l'identification de la séquence d'ADN ou d'ARN cible.
EP99964072A 1998-12-18 1999-12-03 Technique de detection de sequences nucleotidiques specifiques par incorporation de polymerase et de nucleotides Withdrawn EP1141402A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US11313998P 1998-12-18 1998-12-18
US113139P 1998-12-18
PCT/US1999/028612 WO2000037680A1 (fr) 1998-12-18 1999-12-03 Technique de detection de sequences nucleotidiques specifiques par incorporation de polymerase et de nucleotides

Publications (2)

Publication Number Publication Date
EP1141402A1 true EP1141402A1 (fr) 2001-10-10
EP1141402A4 EP1141402A4 (fr) 2004-10-06

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP99964072A Withdrawn EP1141402A4 (fr) 1998-12-18 1999-12-03 Technique de detection de sequences nucleotidiques specifiques par incorporation de polymerase et de nucleotides

Country Status (5)

Country Link
EP (1) EP1141402A4 (fr)
JP (1) JP2002533097A (fr)
AU (1) AU2038500A (fr)
CA (1) CA2354682A1 (fr)
WO (1) WO2000037680A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7875440B2 (en) 1998-05-01 2011-01-25 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
US6780591B2 (en) 1998-05-01 2004-08-24 Arizona Board Of Regents Method of determining the nucleotide sequence of oligonucleotides and DNA molecules
AU2002216035A1 (en) * 2000-11-13 2002-05-21 Gnothis Holding Sa Detection of nucleic acid polymorphisms
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
JP4508632B2 (ja) * 2003-12-25 2010-07-21 キヤノン株式会社 核酸検出方法及び液組成物
US7981604B2 (en) 2004-02-19 2011-07-19 California Institute Of Technology Methods and kits for analyzing polynucleotide sequences
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids
US10344328B2 (en) 2017-11-17 2019-07-09 Ultima Genomics, Inc. Methods for biological sample processing and analysis
US11499962B2 (en) 2017-11-17 2022-11-15 Ultima Genomics, Inc. Methods and systems for analyte detection and analysis

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) * 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US6004744A (en) * 1991-03-05 1999-12-21 Molecular Tool, Inc. Method for determining nucleotide identity through extension of immobilized primer
US5518900A (en) * 1993-01-15 1996-05-21 Molecular Tool, Inc. Method for generating single-stranded DNA molecules

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BAINS M A ET AL: "FLOW CYTOMETRIC QUANTITATION OF SEQUENCE-SPECIFIC MRNA IN HEMOPOIETIC CELL SUSPENSIONS BY PRIMER-INDUCED IN SITU (PRINS) FLUORESCENT NUCLEOTIDE LABELING" EXPERIMENTAL CELL RESEARCH, SAN DIEGO, CA, US, vol. 208, no. 1, 1993, pages 321-326, XP002916199 ISSN: 0014-4827 *
CASTRO A ET AL: "SINGLE-MOLECULE DETECTION OF SPECIFIC NUCLEIC ACID SEQUENCES IN UNAMPLIFIED GENOMIC DNA" ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. COLUMBUS, US, vol. 69, no. 19, 1 October 1997 (1997-10-01), pages 3915-3920, XP000720832 ISSN: 0003-2700 *
CASTRO A ET AL: "SINGLE-MOLECULE ELECTROPHORESIS" ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. COLUMBUS, US, vol. 67, no. 18, 15 September 1995 (1995-09-15), pages 3181-3186, XP000532342 ISSN: 0003-2700 *
GOODWIN P M ET AL: "SINGLE-MOLECULE DETECTION IN LIQUIDS BY LASER-INDUCED FLUORESCENCE" ACCOUNTS OF CHEMICAL RESEARCH, AMERICAN CHEMICAL SOCIETY. WASHINGTON, US, vol. 29, 1996, pages 607-613, XP002942658 ISSN: 0001-4842 & SCHECKER J A ET AL: "FLOW-BASED CONTINUOUS DNA SEQUENCING VIA SINGLE MOLECULE DETECTION OF ENZYMATICALLY CLEAVED FLUORESCENT NUCLEOTIDES" PROCEEDINGS OF THE SPIE, SPIE, BELLINGHAM, VA, US, vol. 2386, 1995, pages 4-12, XP009015211 ISSN: 0277-786X *
RIGLER R: "Fluorescence correlations, single molecule detection and large number screening - Applications in biotechnology" JOURNAL OF BIOTECHNOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 41, no. 2, 31 July 1995 (1995-07-31), pages 177-186, XP004036934 ISSN: 0168-1656 *
See also references of WO0037680A1 *

Also Published As

Publication number Publication date
EP1141402A4 (fr) 2004-10-06
JP2002533097A (ja) 2002-10-08
CA2354682A1 (fr) 2000-06-29
AU2038500A (en) 2000-07-12
WO2000037680A1 (fr) 2000-06-29

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