EP0467953A4 - Improved primer extension reactions - Google Patents

Improved primer extension reactions

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Publication number
EP0467953A4
EP0467953A4 EP19900906516 EP90906516A EP0467953A4 EP 0467953 A4 EP0467953 A4 EP 0467953A4 EP 19900906516 EP19900906516 EP 19900906516 EP 90906516 A EP90906516 A EP 90906516A EP 0467953 A4 EP0467953 A4 EP 0467953A4
Authority
EP
European Patent Office
Prior art keywords
dna polymerase
agent
extension
pyrophosphate
stranded
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Withdrawn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
EP19900906516
Other languages
English (en)
Other versions
EP0467953A1 (fr
Inventor
Stanley Dr. Tabor
Charles C. Richardson
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.)
Harvard College
Original Assignee
Harvard College
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Filing date
Publication date
Application filed by Harvard College filed Critical Harvard College
Publication of EP0467953A1 publication Critical patent/EP0467953A1/fr
Publication of EP0467953A4 publication Critical patent/EP0467953A4/en
Withdrawn legal-status Critical Current

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • 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
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • This invention relates to methods for performing a primer extension reaction, such as a DNA sequencing reaction, or a polymerase chain reaction.
  • a primer extension reaction an oligonucleotide primer having homology to a single-stranded template DNA, e.g., genomic DNA, is caused to anneal to the template DNA.
  • the annealed mixture is then provided with a DNA polymerase in the presence of nucleoside triphosphates under conditions in which the DNA polymerase extends the primer to form a complementary DNA strand to the template DNA.
  • the primer is extended in the presence of a chain-terminating agent, e.g., a dideoxynucleoside triphosphate, to cause base-specific termination of the primer extension.
  • the invention features a solution or kit for use in extension of an oligonucleotide primer having a first single-stranded region on a template molecule having a second single-stranded region, the first and second regions being homologous.
  • the solution or kit includes a first 0 agent able to cause extension of the first single stranded region of the primer on the second single-stranded region of the template in a reaction mixture, and a second agent able to reduce the level of pyrophosphate in the reaction mixture below the level 5 produced during extension in the absence of the second agent.
  • solution any aqueous and/or buffered liquid containing the components described above. These components are present in the solution at 0 concentrations sufficient to perform their desired function.
  • the first agent is present at a concentration sufficient to reduce the level of pyrophosphate in the solution.
  • kit is meant a container which holds one or more of the components of ____> the solution separately.
  • the first and second agents are held in separate containers in solutions adapted to be mixed together.
  • extension of the oligonucleotide primer is meant performing a reaction in which an 0 oligonucleotide primer having a single-stranded region is annealed, or naturally occurs in the annealed state, with another nucleic acid molecule which acts as a template upon which the oligonucleotide primer can be extended by addition of nucleoside triphosphates to form nucleic acid homologous to the template nucleic acid.
  • extension entails providing a DNA polymerase or RNA polymerase to covalently add nucleotides to the primer.
  • a reaction mixture is any solution or solid phase suitable for performing an extension reaction. Generally, it is a liquid buffer containing nucleoside or deoxynucleoside triphosphates and metal ions required for an extension reaction.
  • the mixture may also contain any standard buffering agents and, for a DNA sequencing reaction, one or more dideoxynucleoside triphosphates, or an equivalent chain-terminating agent.
  • reducing the level of pyrophosphate is meant that the amount of pyrophosphate in the reaction mixture is reduced to an amount which has little or no significant effect on the extension of the primer on the template. That is, the level of pyrophosphate is low enough to reduce pyrophosphorolysis to an insignificant level (less than 10% the level of pyrophosphorolysis in the presence of 300 ⁇ M pyrophosphate). Preferably, the level of pyrophosphate is reduced to below 25 ⁇ M, even more preferably to below 5 ⁇ M.
  • This phase is meant to include use of an agent, such as a pyrophosphatase, which acts to prevent the build-up of pyrophosphate, as well as remove it from a solution.
  • homologous is meant that the two single-stranded regions are able to form sufficient non-covalent bonds between their respective nucleotides to form a -stable double-stranded structure under conditions normally used for annealing nucleic acids, and for performing a primer extension reaction.
  • the first agent is a DNA polymerase, most preferably chosen from Klenow, Taq polymerase, a T7-type DNA polymerase (i.e., a polymerase similar to that in a phage in which the DNA polymerase requires host thioredoxin as a subunit, e.g., T7 DNA polymerase or the DNA polymerase of T3, ⁇ I, ⁇ II, H, W31, gh-1, Y, AA1122, or Sp6), T4 DNA polymerase, T5 DNA polymerase, ⁇ 29 DNA polymerase and reverse transcriptase;
  • the second agent is an enzyme, most preferably -a pyrophosphatase, for example, a pyrophosphatase resistant to heating at between 60°C and 95°C.
  • the invention features an improved method for extending an oligonucleotide primer having a first single-stranded region on a template molecule having a second single-stranded region, including providing a first agent able to cause extension of the primer on the template.
  • the improvement is provision of a second agent able to reduce the amount of pyrophosphate below the amount produced during extension in the absence of the second agent.
  • the method includes the steps of providing at least one or two oligonucleotide primers having single-stranded regions and at least one or two template molecules having single-stranded regions, and annealing the single-stranded regions of the primers and the templates to form an annealed mixture.
  • the resulting annealed mixture is provided with the first and second agents to cause extension of the primers.
  • the annealed mixture may also be provided with a dideoxynucleoside triphosphate.
  • the method may further include the step - 5 - of separating the primers from the templates after their extension, and repeating the steps of providing primers, extending the primers, and separating the primers.
  • the invention features a method for amplifying DNA, including performing a polymerase chain reaction in the presence of an agent able to reduce the amount of pyrophosphate in the reaction below the amount produced during a polymerase chain reaction in the absence of the agent.
  • the agent is a pyrophosphatase.
  • the invention features a method for amplifying DNA including providing a solution of ⁇ 29 DNA polymerase, a DNA to be amplified, and an agent able to reduce the amount of pyrophosphate in the solution below that amount produced in the absence of the agent.
  • pyrophosphorolysis where an oligonucleotide chain is reduced in length, is detrimental to a primer extension reaction.
  • the pyrophosphorolysis is caused by the availability of pyrophosphate.
  • a polymerase chain reaction as described by Cetus (European Patent Application 0,258,017) and by Saiki et al., 239 Science 487, 1988, is inhibited by addition of pyrophosphate even at very low concentrations.
  • This pyrophosphorolysis can be prevented by providing an agent, for example, a pyrophosphatase, capable of removing pyrophosphate.
  • One way to inhibit pyrophosphorolysis is to break down any pyrophosphate that is generated during a polymerase reaction, by adding the enzyme pyrophosphatase. Even trace addition of a pyrophosphatase (one thousanth the molar ratio of DNA polymerase molecules in a solution) to a primer extension reaction completely stabilizes oligonucleotide fragments produced in a polymerase reaction, by preventing pyrophosphorolysis.
  • the agent should be added at a concentration sufficient to either catalyze the hydrolysis of pyrophosphate in the reaction mixture at a rate that will prevent accumulation of pyrophosphate to a level that will lead to pyrophosphorolysis, or prevent accumulation of pyrophosphate in any other manner. The amount of agent needed is readily determined by standard techniques.
  • a pyrophosphatase used in this invention be resistant to heating at high temperatures, since high temperatures are used in a polymerase chain reaction, for example, temperatures between 95°C to 100°C, although temperatures between 65°C and 95°C are also commonly used. Thus, it is advantageous to provide a pyrophosphate resistant to heating at 65°C to 95°C.
  • Such a pyrophosphatase can be readily obtained from any bacterium that is naturally able to grow and flourish at high temperatures, e.g., Thermus a ⁇ uaticus. Most bacteria have naturally-occurring pyrophosphatases, and those existing in natural environments at high temperatures will therefore be suitable sources of this enzyme.
  • a pyrophosphatase in a polymerase chain reaction allows the reaction to run to completion—that is, to cause depletion of all the provided deoxynucleoside triphosphates.
  • diagnostic techniques which make use of a polymerase chain reaction to be automated.
  • Assay for progress of the reaction can entail measurement of the generation of phosphate or the generation of DNA from the deoxynucleoside triphosphates (for example, by acid precipitation), both of which are simple and quick assays, instead of the necessity to run a gel to detect the product of the polymerase chain reaction.
  • Example 1 PCR Reaction with Pyrophophatase
  • Trx-F DNA termed M13 Trx-F (the actual DNA used is not critical in this invention) was amplified by provision of a forward and reverse primer using a polymerase chain reaction as follows. This method is generally described in Saiki et. el., supra. Trx-F DNA at a concentration of 0.4 picomoles was mixed with l ⁇ l Tris (1M, pH 8.5), lO ⁇ l magnesium chloride (15 mM) , 6.7 ⁇ l of four deoxynucleoside triphosphates (3 mM) , lO ⁇ l of forward primer (10 picomole; from
  • pyrophosphatase used without purification, or used after purification on an FPLC mono Q column.
  • Another source of pyrophosphatase is Worthington yeast inorganic pyrophosphatase without further purification.
  • 0.001 units of yeast inorganic pyrophosphate (4ng) are suitable in a reaction as described above. This amount may of course be considerably greater, and may be less.
  • the range of concentrations is readily determined by routine expe imentation. The concentration need only be enough to lower the -level of pyrophosphate below about 5-50uM.
  • pyrophosphate inhibited the polymerase chain reaction at levels of 25 ⁇ M or - 9 - greater.
  • Example 2 Preparation of Heat Resistant Pyrophosphatase This is an example of purification of an inorganic pyrophosphatase from cells of Ther us a ⁇ uaticus. Cells of T. aquaticus were obtained from the American Type Culture Collection. 10 liters of cells were grown at 70°C using the growth medium of Chien et al. 127 J. Bacteriol. 1550 (1976).
  • the cells were harvested (-20 gm) , resuspended in 40 ml of 10% sucrose, 50 mM Tris KC1, ?H 7.5, 5 M EDTA; lysed by three passages through a French press, and cell debris removed by centrifugation at 30,000 rpm, for 60 min in a Beckman 50Ti rotor.
  • the supernatant was treated with streptomycin sulfate to remove DNA.
  • 4 ml of a 40% streptomycin solution was added to 40 ml supernatant, mixed for 30 min., and centrifuged for 30 min at 8,000 rpm. The resulting supernatant was then treated with ammonium sulfate.
  • the pellet was resuspended in 20 ml 20 mM Tris-HCl pH 7.5, 1 mM EDTA, 10% glycerol, 10 mM 2-mercaptoethanol (Buffer A) and then dialyzed overnight against 2 liters of Buffer A.
  • the dialysate was passed over a DEAE DE52 column (100 ml) equilibrated in Buffer A, washed with 300 ml of Buffer A + 50 mM NaCl, and then run in a liter gradient of buffer A containing from 50 mM to 500 mM NaCl.
  • the pyrophophatase eluted at buffer A containing 125 mM NaCl.
  • the eluate (60 mL) was diaiyzed against 2 liters of 20 mM KU>0 4 pH 7.4, 1 mM EDTA, 10 mM 2-mercaptoethanol, 10% glycerol (Buffer B) and loaded onto a phosphocellulose column (100 ml) equilibrated in buffer B. All of, the pyrophosphatase activity flowed through the column. This flow-through was then diaiyzed against 20 -mM Tris HC1 pH 7.0, 1 mM EDTA, 10% glycerol (Buffer C), and applied to an FPLC monoQ column in buffer C.
  • This pyrophosphatase activity was not affected by 40 cycles of a polymerase chain reaction, with each cycle containing a 95°C, 1 min. heating step. Further, the pyrophosphatase did not hydrolyze dNTPs, nor was it inhibited by dNTPs in the reaction mixture. The pyrophosphatase activity was assayed generally as described by Chen et al. 28 Anal. Chem. 1756 (1956), and Josse, 241 J, ⁇ iol. Chem. 1938 (1966).
  • enzymes which use a protein primer rather than -a DNA primer e.g.,* ⁇ 29 DNA polymerase which polymerizes double stranded DNA
  • enzymes which use a protein primer rather than -a DNA primer can be used to amplify DNA without need for denaturing heating steps or reannealing steps.
  • Inclusion of a pyrophosphatase, or its equivalent, in such an amplification reaction will enhance the yield of DNA amplified in this system.

Abstract

On a mis au point un kit ou une solution destiné à être utilisé dans l'extension d'une amorce d'oligonucléotide comportant une première région monocaténaire sur une molécule modèle ayant une seconde région monocaténaire homologue à ladite première région monocaténaire, comprenant un premier agent capable de provoquer l'extension de ladite première région monocaténaire de l'amorce, sur ladite seconde région monocaténaire du modèle dans un mélange de réaction, ainsi qu'un second agent capable de réduire la quantité de pyrophosphate se trouvant dans le mélange de réaction, en dessous de la quantité produite pendant l'extension, en l'absence dudit second agent.
EP19900906516 1989-04-12 1990-04-10 Improved primer extension reactions Withdrawn EP0467953A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US33675189A 1989-04-12 1989-04-12
US336751 1989-04-12

Publications (2)

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EP0467953A1 EP0467953A1 (fr) 1992-01-29
EP0467953A4 true EP0467953A4 (en) 1992-06-17

Family

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EP19900906516 Withdrawn EP0467953A4 (en) 1989-04-12 1990-04-10 Improved primer extension reactions

Country Status (8)

Country Link
EP (1) EP0467953A4 (fr)
JP (1) JP2997043B2 (fr)
KR (1) KR920700294A (fr)
AU (1) AU638246B2 (fr)
CA (1) CA2050276C (fr)
HU (1) HUT61054A (fr)
LT (1) LTIP1519A (fr)
WO (1) WO1990012111A1 (fr)

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5198543A (en) * 1989-03-24 1993-03-30 Consejo Superior Investigaciones Cientificas PHI29 DNA polymerase
US5001050A (en) * 1989-03-24 1991-03-19 Consejo Superior Investigaciones Cientificas PHφ29 DNA polymerase
FR2674254B1 (fr) * 1991-03-20 1995-10-06 Univ Reims Champagne Ardenne Detection non radioactive de la presence d'un acide nucleique determine dans un echantillon biologique.
US5256555A (en) 1991-12-20 1993-10-26 Ambion, Inc. Compositions and methods for increasing the yields of in vitro RNA transcription and other polynucleotide synthetic reactions
FI923911A (fi) * 1992-09-01 1994-03-02 Vsevolod Kiselev DNA-molekylers in vitro-syntes
DE4336266A1 (de) * 1993-10-23 1995-04-27 Boehringer Mannheim Gmbh Stabilisierte flüssige Mischungen für die Markierung von Nukleinsäuren
CA2222744C (fr) * 1995-05-31 2008-03-25 Amersham Life Science, Inc. Adn-polymerases thermostables
US5665551A (en) * 1995-09-13 1997-09-09 Roche Molecular Systems, Inc. Purified nucleic acid encoding a thermostable pyrophosphatase
DE19612779A1 (de) * 1996-03-29 1997-10-02 Boehringer Mannheim Gmbh Verfahren zur spezifischen Vervielfältigung von langen Nukleinsäuren durch PCR
WO1998015655A1 (fr) * 1996-10-07 1998-04-16 The Perkin-Elmer Corporation Reaction d'extension d'amorce utilisant une paire d'enzymes d'un meme substrat pour consommer le pyrophosphate
US6291164B1 (en) 1996-11-22 2001-09-18 Invitrogen Corporation Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate
GB0110501D0 (en) 2001-04-30 2001-06-20 Secr Defence Brit Amplification process
CA2540875A1 (fr) * 2003-09-30 2005-04-14 Perkinelmer Las, Inc. Compositions et procedes de genotypage de polymorphismes d'un nucleotide simple
EP3004378B1 (fr) 2013-05-24 2017-12-20 Illumina Cambridge Limited Sequençage pyrophosphorolytique utilisant des nanopores
CN117015616A (zh) * 2021-01-14 2023-11-07 合成Dna技术公司 用于独特分子标识符标记的珠子的生产和定量的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0258017A2 (fr) * 1986-08-22 1988-03-02 F. Hoffmann-La Roche Ag Enzyme thermostable purifiée et procédé d'amplification, de détection et/ou de clonage de séquences d'acide nucléique à l'aide de cette enzyme
WO1989009283A1 (fr) * 1988-03-25 1989-10-05 Edward David Hyman Procede a base de pyrophosphate de mise en sequence d'acides nucleiques

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683202A (en) * 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0258017A2 (fr) * 1986-08-22 1988-03-02 F. Hoffmann-La Roche Ag Enzyme thermostable purifiée et procédé d'amplification, de détection et/ou de clonage de séquences d'acide nucléique à l'aide de cette enzyme
WO1989009283A1 (fr) * 1988-03-25 1989-10-05 Edward David Hyman Procede a base de pyrophosphate de mise en sequence d'acides nucleiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9012111A1 *

Also Published As

Publication number Publication date
AU638246B2 (en) 1993-06-24
CA2050276C (fr) 2003-03-11
EP0467953A1 (fr) 1992-01-29
WO1990012111A1 (fr) 1990-10-18
KR920700294A (ko) 1992-02-19
AU5438290A (en) 1990-11-05
JP2997043B2 (ja) 2000-01-11
LTIP1519A (en) 1995-06-26
HUT61054A (en) 1992-11-30
HU903562D0 (en) 1992-03-30
JPH04506002A (ja) 1992-10-22
CA2050276A1 (fr) 1990-10-13

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