EP1071812A4 - Technique, solution reactive et kits pour le sequen age de l'adn - Google Patents

Technique, solution reactive et kits pour le sequen age de l'adn

Info

Publication number
EP1071812A4
EP1071812A4 EP99904129A EP99904129A EP1071812A4 EP 1071812 A4 EP1071812 A4 EP 1071812A4 EP 99904129 A EP99904129 A EP 99904129A EP 99904129 A EP99904129 A EP 99904129A EP 1071812 A4 EP1071812 A4 EP 1071812A4
Authority
EP
European Patent Office
Prior art keywords
polymerase
solution
substituent
labeled
labeled dideoxynucleotides
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Withdrawn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
EP99904129A
Other languages
German (de)
English (en)
Other versions
EP1071812A1 (fr
Inventor
Carl W Fuller
Joseph A Mamone
Bernard F Mcardle
Kristine M Hujer
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.)
Global Life Sciences Solutions USA LLC
Original Assignee
Amersham Pharmacia Biotech Inc
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 Amersham Pharmacia Biotech Inc filed Critical Amersham Pharmacia Biotech Inc
Publication of EP1071812A1 publication Critical patent/EP1071812A1/fr
Publication of EP1071812A4 publication Critical patent/EP1071812A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • 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
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to buffer solutions for use in DNA sequencing and to the use of such solutions.
  • DNA sequencing by the Sanger, or chain termination method has been in common use for some years.
  • the sample to be sequenced may be split into four portions, and each portion may be hybridized to a suitable primer and the primer extended using a DNA polymerase and deoxynucleotides.
  • the incorporation of different dideoxynucleotides in the reaction mixture terminates the chain extension reaction at consecutive positions so that a collection of DNA fragments is obtained each differing by one nucleotide.
  • T7 DNA polymerase or E.coli DNA polymerase is used in such a procedure.
  • thermostable DNA polymerases which incorporate dideoxynucleotides (ddNTPs) as efficiently as deoxynucleotides, thereby enabling the concentration of the former to be reduced, greatly facilitating the sequencing process (see European Patent No. 655506 Bl).
  • ddNTPs dideoxynucleotides
  • Thermostable Pol I family DNA polymerases in which a phenylalanine in the nucleotide binding domain has been replaced by tyrosine and the exonuclease activity reduced or deleted are particularly advantageous.
  • Such polymerases have been marketed by the ABD division of the Perkin-Elmer Corporation under the trademark AMPLITAQ FS and by Amersham Life Science under the trademark THERMO_SEQUENASE - these are both mutated Thermus aquaticus enzymes.
  • results obtained from sequencing reactions when using substituent-labeled, e.g., dye- labeled, ddNTPs and thermostable Pol I family DNA polymerases can be improved by adding a particular concentration of manganese and tartaric acid or an equivalent metal ion buffer.
  • the improved results are observed as less variation in band intensity (peak height) than is usually obtained when using the same dye-labeled ddNTPs without manganese ions.
  • the present invention provides a solution that contains manganese ion at a concentration of between 0.5mM to 3.0mM and a metal ion buffer at a concentration of 5mM to 50mM.
  • concentration of manganese is preferably between 0.5mM and l.OmM, or between 0.5mM and 2.0mM, or between 0.5mM and 2.5mM, with about 0.8mM being most preferable in the absence of a metal ion buffer, and about 1.5 mM being most preferable in the presence of a metal ion buffer.
  • the manganese concentration in the solution is less than 3.0mM in a polymerization reaction mixture.
  • concentration of the metal ion buffer is preferably between 5mM and 50mM.
  • the solution will preferably be pH buffered to a pH between 6 and 8.5, for example about pH
  • pH buffer refers to a material which regulates the concentration of hydronium ion, H 3 O + (or dissociated protons), in solution.
  • a pH buffer regulates the concentration of hydronium ion or dissociated protons in solution by resisting changes in the concentration of the ion in response to dilutions or to additions or subtractions of acids or bases from the solution. Buffers for regulating pH (as distinct from metal ion buffers) which lack primary amino groups, such as tertiary amine buffers are preferred pH buffering agents.
  • the buffer compound is preferably one which does not interfere with with a sequencing reaction in which the solution is to be used.
  • the solution is an aqueous solution.
  • substituted-labeled dd ⁇ TP or "substituent-labeled dideoxynucleotide triphosphate” or "substituent-labeled dideoxynucleotide” is meant a 2'-deoxyribonucleotide analog which has a covalently attached detectable group and which lacks a functional 3'-hydroxyl group, so that it terminates chain elongation catalyzed by D ⁇ A polymerase.
  • the substituent detectable group provides an additional atom or group to the dd ⁇ TP structure, and therefore does not consist of the replacement of an atom in the dd ⁇ TP structure with a radioactive isotope, e.g., 32 P.
  • the substituent label is a dye label, more preferably a fluroescent dye label.
  • Dye labels are chemical groups which are detectable spectrophotometrically, preferably be the emission or reflection of light of characteristic wavelengths. Such nucleotides and appropriate dye labels are well known in the art, for example, as described in Lee at al., 1992, Nucl. Acids Res.
  • metal ion buffer is meant a material which regulates the concentration of free metal ion, such as Mn 2+ , in solution.
  • a buffer regulates the concentration of a species (e.g., a metal ion) in solution by resisting changes in the concentration of the free ion in response to dilutions or to additions or subtractions of that ion from the solution.
  • a buffer can, for example, be a dicarboxylic acid, e.g., an alkyldicarboxylic acid such as tartaric acid, where alkyl is a straight or branched chain of 1,2,3,4,5 . 6,7, or 8 carbon atoms.
  • dicarboxylic acids include oxalic acid, malonic acid, succinic acid, maleic acid, glutaric acid, adipic acid, fumaric acid, glutamic acid, aspartic acid, and phthalic acid.
  • metal ion buffers include, for example, citric acid, EDTA (ethylenediaminetetraacetic acid), nitrilotriacetic acid, diethenetriaminepentaacetic acid, N-hydroxyethyliminodiacetic acid, 2-( ⁇ - morpholino)ethanesulfonic acid, dithiothreitol, and NN-bishydroxyethylglycine).
  • a metal ion buffer may be used in the presence of a pH buffer (i.e., a compound which regulates the concentration of free H + in solution).
  • the manganese ion will normally come from a salt, for example manganese sulphate (MnSO 4 ), manganese chloride (MnCl 2 ), or manganese acetate.
  • dicarboxylic acid is meant any lower alkyl, hydroxyalkyl, or aminoalkyl compound containing two carboxylic acid groups, such as oxalic acid, malonic acid, succinic acid, maleic acid, tartaric acid, glutaric acid, adipic acid, fumaric acid, phthalic acid, glutamic acid, or aspartic acid.
  • the invention provides a kit for D ⁇ A sequencing which includes reagents necessary for D ⁇ A sequencing including manganese and a metal ion buffer, preferably at the concentrations specified above or in concentrations readily diluted to those specified above.
  • the kit includes a Pol I family D ⁇ A polymerase that contains a tyrosine in the nucleotide binding domain of the polymerase at a position analogous to that occupied by phenylalanine in unmutated D ⁇ A polymerase (e.g. at position 667 in Thermus aquaticus 5 DNA polymerase I).
  • the DNA polymerase is a thermostable polymerase, preferably a Thermus aquaticus polymerase that has been mutated to replace the phenylalanine at position 667 with tyrosine and in which the exonuclease activity has been substantially removed, e.g. less than 1% of exonuclease activity remains.
  • the kit also includes dye-labeled ddNTPs, e.g. ddATP, ddCTP, ddGTP and ddTTPs.
  • the kit for DNA sequencing will preferably contain, as a first component, a metal ion buffer together with a buffering agent for regulating the pH to between pH 6 and pH 9, preferably about pH 8, deoxynucleotides and dye-labeled ddNTPs, and, as a second component, manganese ion together with a buffering agent for regulating the pH to between pH 3 to pH 7, preferably about pH 6, for example 2-(N-morpholino)ethanesulfonic acid.
  • a DNA polymerase may also be included in the kit, preferably a DNA Pol I polymerase and most preferably a thermostable DNA polymerase, that contains a tyrosine in the dideoxy binding domain at a position analogous to that occupied by phenylalanine in unmutated polymerase.
  • the kit may also contain a pyrophosphatase, for example, an inorganic pyrophosphatase such as that from Thermoplasma acidophilwn.
  • the polymerase and pyrophosphatase will conveniently be included in the first component of the kit.
  • the kit may also contain sources of other metal ions, for example magnesium and conveniently salts of such metal ions. These additional optional metal ions will conveniently be in the second component of the kit.
  • the concentration of the manganese ions and metal ion buffer will be such that they can be mixed directly to give the concentrations required for the sequencing reaction or they can be diluted readily to give the required concentrations. While the second component containing the manganese ions is buffered to about pH 6, the mixture resulting from adding the two components together will have a pH of about 8, e.g. pH 7 to pH 9.
  • the present invention provides a method for sequencing DNA which comprises performing a DNA sequencing reaction in the presence of dye-labeled ddNTPs and manganese, at a concentration of between 0.5mM and 3mM, and preferably also in the 6 presence of a metal ion buffer, for example tartaric acid, at a concentration of between 5mM and 50mM, in the reaction mixture.
  • a metal ion buffer for example tartaric acid
  • the present invention provides a solution containing a metal ion buffer together with a buffering agent for regulating the pH to about pH 8, deoxynucleotides, and dye-labeled ddNTPs.
  • a buffering agent for regulating the pH to about pH 8, deoxynucleotides, and dye-labeled ddNTPs.
  • the solution also contains a thermostable Pol I family DNA polymerase, more preferably a polymerase which has a tyrosine substituted for a phenylalanine in the nucleotide binding site.
  • Figure 1 shows DNA sequencing results from an automated fluorescent DNA sequencing apparatus (ABI model 373 instrument) for an exemplary sequencing in which the polymerization step was carried out in the presence of manganese and a metal ion buffer.
  • Figure 2 shows DNA sequencing results corresponding to the results shown in Fig. 1, except that manganese was not included in the reaction mix.
  • Figure 3 shows the structure of the four dye labeled dideoxynucleotides utilized in the examples below. Each of the structures is identified with a Roman numeral, which is referenced in the solution preparation in Example 1.
  • the volume was made up to 10 ml with deionized H 2 O.
  • 4 ⁇ l reagent mix A, 4 ⁇ l reagent mix B, 200 ng M13mp 18 DNA, 5 pmole of primer (M13 - 40 Forward 5'-GTTTTCCCAGTCACGAC), and deionized water to a total volume of 20 ⁇ l were mixed together and subjected to 25 cycles of 96°C 30 seconds, 50°C 15 seconds, and 60°C 4 minutes in a thermal cycler. After cycling, 4 ⁇ l of a solution which contained 1.5 M sodium acetate, 250 mM EDTA was added. The solution was mixed and 4 volumes (lOO ⁇ l) of ethanol added. The DNA was precipitated by incubation on ice for 15-20 minutes followed by centrifugation.

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

Abstract

Cette invention concerne des solutions qui permettent d'améliorer l'efficacité des réactions de séquençage de l'ADN grâce à l'emploi de ddNTP marqués par un substituant, tel qu'un colorant, et une ADN-polymérase thermostable. La solution comprend une concentration efficace d'ion manganèse et un composé tampon d'ions métal. L'invention porte également sur l'utilisation de ces solutions.
EP99904129A 1998-01-23 1999-01-19 Technique, solution reactive et kits pour le sequen age de l'adn Withdrawn EP1071812A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US1238598A 1998-01-23 1998-01-23
US12385 1998-01-23
PCT/US1999/001084 WO1999037810A1 (fr) 1998-01-23 1999-01-19 Technique, solution reactive et kits pour le sequençage de l'adn

Publications (2)

Publication Number Publication Date
EP1071812A1 EP1071812A1 (fr) 2001-01-31
EP1071812A4 true EP1071812A4 (fr) 2002-05-15

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP99904129A Withdrawn EP1071812A4 (fr) 1998-01-23 1999-01-19 Technique, solution reactive et kits pour le sequen age de l'adn

Country Status (3)

Country Link
EP (1) EP1071812A4 (fr)
JP (1) JP2002510466A (fr)
WO (1) WO1999037810A1 (fr)

Families Citing this family (8)

* 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
US7501245B2 (en) 1999-06-28 2009-03-10 Helicos Biosciences Corp. Methods and apparatuses for analyzing polynucleotide sequences
CA2382063A1 (fr) * 1999-09-17 2001-03-22 Shiv Kumar Terminateurs d'acides nucleiques a charge modifiee
AU1312502A (en) * 2000-10-11 2002-04-22 Pe Corp Ny Fluorescent nucleobase conjugates having anionic linkers
US7169560B2 (en) 2003-11-12 2007-01-30 Helicos Biosciences Corporation Short cycle methods for sequencing polynucleotides
CA2557177A1 (fr) 2004-02-19 2005-09-01 Stephen Quake Procedes et kits pour analyser des sequences de polynucleotides
US7666593B2 (en) 2005-08-26 2010-02-23 Helicos Biosciences Corporation Single molecule sequencing of captured nucleic acids

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0655506A1 (fr) * 1994-10-17 1995-05-31 President And Fellows Of Harvard College Polymérase d'ADN ayant les nucléotiden modifiées pour le site d'adhésion de l'ADN
US5871929A (en) * 1996-07-23 1999-02-16 Barnes; Wayne M. Suppression of pyrophosphorolysis in DNA sequencing and in other applications involving DNA replication

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0655506A1 (fr) * 1994-10-17 1995-05-31 President And Fellows Of Harvard College Polymérase d'ADN ayant les nucléotiden modifiées pour le site d'adhésion de l'ADN
US5871929A (en) * 1996-07-23 1999-02-16 Barnes; Wayne M. Suppression of pyrophosphorolysis in DNA sequencing and in other applications involving DNA replication

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of WO9937810A1 *
TABOR S ET AL: "DNA SEQUENCE ANALYSIS WITH A MODIFIED BACTERIOPHAGE T7 DNA POLYMERASE", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 265, no. 14, 15 May 1990 (1990-05-15), pages 8322 - 8328, XP000307483, ISSN: 0021-9258 *

Also Published As

Publication number Publication date
WO1999037810A1 (fr) 1999-07-29
JP2002510466A (ja) 2002-04-09
EP1071812A1 (fr) 2001-01-31

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