United States Patent
`Birch et al.
`
`[19]
`
`[54] NUCLEICACIDAMPLIFICATION USINGA
`REVERSIBLY INACTIVATED
`THERMOSTABLE ENZYME
`
`[75] Inventors: David Edward Birch, Berkeley;
`Walter Joseph Laird, Pinole; Michael
`Anthony Zoccoli, Moraga, all of Calif.
`[73] Assignee: Roche Molecular Systems, Inc.,
`Branchburg, N.J.
`
`[*] Notice:
`
`The term of this patent shall not extend
`beyond the expiration date of Pat. No.
`5,677,152.
`
`[21] Appl. No.: 680,283
`[22] Filed:
`Jul. 11, 1996
`Related U.S. Application Data
`[60] Provisional application No. 60/002,673, Aug. 25, 1995.
`[51] Int. Cl." .............................. C12P 19/34; C12N 9/00;
`C12N 9/99
`[52] U.S. Cl. ......................... 435/91.2, 435/183; 435/184;
`435/810
`[58] Field of Search .................................... 435/91.2, 810,
`435/183, 184
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5,262,525 11/1993 Bonnaffe et al. ....................... 530/411
`5,338,671
`8/1994 Scalice et al. ......................... 435/91.2
`OTHER PUBLICATIONS
`Atassi et al. Reaction of Proteins with Citraconic Anhydride,
`Methods in Enzymology, vol. XXV, pp. 546–553, 1972.
`Goldberger and Anfinsen, May, 1962, “The Reversible
`Masking of Amino Groups in Ribonuclease and Its Possible
`Usefulness in the Synthesis of the Protein” Biochemistry
`1(3):401–405.
`
`US005773258A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,773,258
`*Jun. 30, 1998
`
`Hunter and Ludwig, Sep., 1962, “The Reaction of Imi
`doesters With Proteins and Related Small Molecules” Imi
`doesters With Proteins 84:3491—3504.
`Habeeb, 1966, “Determination of Free Amino Groups in
`Proteins by Trinitrobenzenesulfonic Acid” Analytical Bio
`chemistry 14:328–336.
`Bailey et al., 1967, “Liver Enzyme Changes in the Devel
`oping Rats” Proceedings of the Biochemical Society
`103.78p–79p.
`Marzotto et al., 1967, “Acetcacetylation of Ribonuclease A”
`Biochemical and Biophysical Research Communications
`26(5):517–521.
`Marzotto et al., 1968, “Reversible Acetoacetylation of
`Amino Groups in Proteins” Biochimica et Biophysica Acta
`154:450–456.
`Braunitzer et al., Feb., 1968, “Tetrafluorbernsteinsaure—an
`hydrid, ein neues Reagens zur Spezifischen und reversiblen
`Maskierung der Aminogruppen in Proteinen” Hoppe-Sey
`ler’s Z. Physiol. Chem. 349; 265.
`Dixon and Perham, 1968, “Reversible Blocking of Amino
`Groups with Citraconic Anhydride” Biochem. J.
`109:312–314.
`(List continued on next page.)
`Primary Examiner—Kenneth R. Horlick
`Assistant Examiner—Joyce Tung
`Attorney, Agent, or Firm—George W Johnston; Dennis P.
`Tramaloni; Douglas A. Petry
`[57]
`ABSTRACT
`The present invention provides methods for the amplifica
`tion of nucleic acids using a reversibly inactivated thermo
`stable enzyme. The reversibly inactivated enzyme is the
`result of a chemical modification of the protein which
`inactivates the enzyme. The activity of the inactivated
`enzyme is recovered by an incubation of the reaction mix
`ture at an elevated temperature prior to, or as part of, the
`amplification reaction. Non-specific amplification is reduced
`because the reaction mixture does not support the formation
`of extension products prior to the activating incubation.
`13 Claims, 5 Drawing Sheets
`
`citraconic anhydride
`
`cis-aconitic anhydride
`
`A scheme for the reversible reaction of citraconic anhydride
`With lysine residues
`
`NH,
`çH.
`CH3
`ÇH2
`ÇHe
`orio 'o - gº 3–
`.
`.
`Citraconic
`anhydride
`
`H H
`
`MYR 1029
`Myriad Genetics, Inc. et al. (Petitioners) v. The Johns Hopkins University (Patent Owner)
`IPR For USPN 7,824,889
`
`Page 1 of 20
`
`

`

`5,773,258
`Page 2
`
`OTHER PUBLICATIONS
`Habeeb and Atassi, 1970, “Enzymatic and Immunochemical
`Properties of Lysozyme—Evaluation of Several Amino
`Group Reversible Blocking Reagents” Biochemistry
`9(25):4939–4944.
`Atassi and Habeeb, 1972, “Reaction of Proteins with Cit
`raconic Anhydride” Methods in Enzymology 25(Part
`B):546–553.
`Shetty and Kinsella, 1980, “Ready Separation of Proteins
`from Nucleoprotein Complexes by Reversible Modifieda
`tion of Lysine Residues” Biochem. J. 191:269–272.
`Rozovskaya et al., 1981, “Modification of Escherichia coli
`RNA Polymerase by Diethyl Procarbonate” Molecular biol
`ogy 15(1):61–66.
`Naithani and Gattner, Dec., 1982, “Preparation and Proper
`ties of Citraconylinsulins.” Hoppe-Seyler’s Physiol. Chem.
`363:1443–1448.
`
`de la Escalera and Palacian, 1989, “Dimethylmaleic Anhy
`dride, a Specific Reagent for Protein Amino Groups” Bio
`chem. Cell. Biol. 67:63–66.
`
`Nieto and Palacian, 1989, “Effects of Temperature and pH
`on the Regeneration of the Amino Groups of Ovalbumin
`After Modification with Citraconic and Dimethylmaleic
`Anhydrides” Biochimica et Biophysica Acta 749:204–210.
`Palacian et al., 1990, Dicarboxylic Acid Anhydrides as
`Dissociating Agents of Protein–Containing Structures
`Molecular and Cellular Biochemistry 97:101–111.
`Lundblad, R.L., Chemical Reagents for Protein Modifica
`tion, second edition, Boca Raton, Florida, CRC Press, 1991,
`Chapter 10, entitled “The Modification of Lysine”.
`
`Page 2 of 20
`
`

`

`U.S. Patent
`
`Jun. 30, 1998
`
`Sheet 1 of 5
`
`5,773,258
`
`Fig. 1
`
`citraconic anhydride
`
`fº
`
`O’ SO SO
`
`cis-aconitic anhydride
`H.Q
`
`A. "
`
`O’ SO SO
`
`A scheme for the reversible reaction of citraconic anhydride
`with lysine residues
`
`NH2
`ÇH2
`CH
`|
`2
`J.S. #9
`CH2
`o o 'o -º-; 3–
`-
`it
`C| raconic
`anhydride
`
`CH3
`
`H H
`
`O
`Q
`C-OH -?
`Hºc
`Hºc OH
`Ç-H
`HAC-Q
`C=O
`C=O
`—º- i
`t
`N-H
`+
`N–H
`ÇH2
`ÇHe
`QH2
`QH2
`ÇH2
`ÇH2
`ÇH2 Q
`ÇH2 Q
`––N–C–C– —N–C–C–
`H H
`H H
`
`NHe
`CH2
`CH2
`ÇH2
`QH2 Q
`—N-C-C-
`
`Q
`ç-OH
`+ HAC-Q
`ç-H
`ç-OH
`O
`
`Page 3 of 20
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`

`

`U.S. Patent
`
`Jun. 30, 1998
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`Sheet 2 of 5
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`5,773,258
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`Tag
`
`Ta.HS
`
`cn £2 of ee cy
`
`ZEaseaes
`
`~~Target
`
`
`
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`

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`U.S. Patent
`U.S. Patent
`
`Jun. 30, 1998
`Jun. 30, 1998
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`Sheet 3 of 5
`Sheet 3 of 5
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`5,773,258
`5,773,258
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`U.S. Patent
`
`Jun. 30, 1998
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`Sheet 4 of 5
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`5,773,258
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`
`
`*-alget
`
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`Page 6 of 20
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`

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`U.S. Patent
`
`Jun. 30, 1998
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`Sheet 5 of 5
`
`5,773,258
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`tit & £it #8%
`& 3% tig 48%
`gººd isºgg Bºis 43395660484.3396,8048.4336
`
`
`
`Page 7 of 20
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`

`

`1
`NUCLEIC ACID AMPLIFICATION USING A
`REVERSIBLY INACTIVATED
`THERMOSTABLE ENZYME
`
`This application claims the benefit of U.S. provisional
`Application No. 60/002,673, filed Aug. 25, 1995.
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`This invention relates generally to the field of nucleic acid
`chemistry. More specifically, it relates to methods of ampli
`fying nucleic acid sequences and to methods of reducing
`non-specific amplification.
`2. Description of the Related Art
`The polymerase chain reaction (PCR) process for ampli
`fying nucleic acid sequences is well known in the art and
`disclosed in U.S. Pat. Nos. 4,683,202; 4,683,195; and 4,965,
`188; each incorporated herein by reference. Commercial
`vendors, such as Perkin Elmer, Norwalk, Conn., market
`PCR reagents and publish PCR protocols.
`In each cycle of a PCR amplification, a double-stranded
`target sequence is denatured, primers are annealed to each
`strand of the denatured target, and the primers are extended
`by the action of a DNA polymerase. Specificity of amplifi
`cation depends on the specificity of primer hybridization.
`Primers are selected to be complementary to, or substan
`tially complementary to, sequences occurring at the 3' end of
`each strand of the target nucleic acid sequence. Under the
`elevated temperatures used in a typical PCR, the primers
`hybridize only to the intended target sequence. However,
`amplification reaction mixtures are typically assembled at
`room temperature, well below the temperature needed to
`insure primer hybridization specificity. Under such less
`stringent conditions, the primers may bind non-specifically
`to other only partially complementary nucleic acid
`sequences (or even to other primers) and initiate the syn
`thesis of undesired extension products, which can be ampli
`fied along with the target sequence. Amplification of the
`non-specific primer extension products can compete with
`amplification of the desired target sequences and can sig
`nificantly decrease the efficiency of the amplification of the
`desired sequence. Problems caused by non-specific ampli
`fication are discussed further in Chou et al., 1992, Nucleic
`Acids Research 2007):1717–1723, incorporated herein by
`reference.
`Non-specific amplification can be reduced by reducing the
`formation of extension products from primers bound to
`non-target sequences prior to the start of the reaction. In one
`method, referred to as a “hot-start” protocol, one or more
`critical reagents are withheld from the reaction mixture until
`the temperature is raised sufficiently to provide the neces
`sary hybridization specificity. In this manner, the reaction
`mixture cannot support primer extension during the time that
`the reaction conditions do not insure specific primer hybrid
`ization.
`Hot-start methods can be carried out manually by opening
`the reaction tube after the initial high temperature incubation
`step and adding the missing reagents. However, manual
`hot-start methods are labor intensive and increase the risk of
`contamination of the reaction mixture. Hot-start methods
`which use a heat labile material, such as wax, to separate or
`sequester reaction components are described in U.S. Pat. No.
`5,411,876, incorporated herein by reference, and Chou et al.,
`1992, supra. In these methods, a high temperature pre
`reaction incubation melts the heat labile material, thereby
`allowing the reagents to mix.
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`Another method of reducing the formation of extension
`products from primers bound to non-target sequences prior
`to the start of the reaction relies on inhibition of the DNA
`polymerase using a compound which non-covalently binds
`to the DNA polymerase a heat-reversible manner. U.S. Pat.
`No. 5,338,671, incorporated herein by reference, describes
`the use of antibodies specific for a thermostable DNA
`polymerase to inhibit the DNA polymerase activity. The
`antibodies must be incubated with the DNA polymerase in
`a buffer at room temperature prior to the assembly of the
`reaction mixture in order to allow formation of the antibody
`DNA polymerase complex. Antibody inhibition of DNA
`polymerase activity is inactivated by a high temperature
`pre-reaction incubation. A disadvantage of this method is
`that the production of antibodies specific to the DNA poly
`merase is expensive and time-consuming, especially in large
`quantities. Furthermore, the addition of antibodies to a
`reaction mixture may require redesign of the amplification
`reaction.
`The formation of extension products can also be inhibited
`by the addition of a compound which non-covalently binds
`to the primers in a heat-reversible manner, thereby prevent
`ing the primers from hybridization to any sequence, target or
`otherwise. For example, single-stranded binding protein
`added to a reaction mixture will bind the primers, thereby
`preventing primer hybridization and inhibiting primer exten
`sion. Improvements in the yield of PCR products using gene
`32 protein are described in Schwarz et al., 1990, Nucleic
`Acids Research 18(4):10, incorporated herein by reference.
`Non-specific amplification can be reduced by degrading
`extension products formed from primers bound to non-target
`sequences prior to the start of the reaction, such as using the
`methods described in copending U.S. Ser. No. 07/960,362,
`now allowed, which is incorporated herein by reference. The
`degradation of newly-synthesized extension products is
`achieved by incorporating into the reaction mixture duTP
`and UNG, and incubating the reaction mixture at 45°-60° C.
`prior to carrying out the amplification reaction. A disadvan
`tage of this method is that the degradation of extension
`product competes with the formation of extension product
`and the elimination of non-specific primer extension product
`is likely to be less complete.
`Conventional techniques of molecular biology, protein
`chemistry, and nucleic acid chemistry, which are within the
`skill of the art, are fully explained fully in the literature. See,
`for example, Sambrook et al., 1989, Molecular Cloning—A
`Laboratory Manual, Cold Spring Harbor Laboratory, Cold
`Spring Harbor, N.Y.; Oligonucleotide Synthesis (M. J. Gait,
`ed., 1984); Nucleic Acid Hybridization (B. D. Hames and S.
`J. Higgins, eds., 1984); Chemical Reagents for Protein
`Modification (CRC Press); and a series, Methods in Enzy
`mology (Academic Press, Inc.), all of which are incorporated
`herein by reference. All patents, patent applications, and
`publications mentioned herein, both supra and infra, are
`incorporated herein by reference.
`
`SUMMARY OF THE INVENTION
`The present invention provides methods and reagents for
`amplifying nucleic acid using a primer-based amplification
`reaction which provide a simple and economical solution to
`the problem of non-specific amplification. The methods use
`a reversibly inactivated thermostable enzyme which can be
`reactivated by incubation in the amplification reaction mix
`ture at an elevated temperature. Non-specific amplification
`is greatly reduced because the reaction mixture does not
`support primer extension until the temperature of the reac
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`

`3
`tion mixture has been elevated to a temperature which
`insures primer hybridization specificity.
`One aspect of the present invention relates to reversibly
`inactivated thermostable enzymes which are produced by a
`reaction between a thermostable enzyme which catalyzes a
`primer extension reaction and a modifier reagent. The reac
`tion results in a significant, preferably essentially complete,
`reduction in enzyme activity. Incubation of the modified
`enzyme in an aqueous buffer at alkaline pH at a temperature
`which is less than about 25° C. results in essentially no
`increase in enzyme activity in less than about 20 minutes.
`Incubation of the modified enzyme in an aqueous buffer,
`formulated to pH 8–9 at 25°C., at a temperature greater than
`about 50° C. results in at least a two-fold increase in primer
`extension activity in less than about 20 minutes. The revers
`ibly inactivated thermostable enzymes of the invention, in
`their active state, either catalyze primer extension or are
`necessary for primer extension to occur. Preferred enzymes
`include thermostable DNA polymerases and ligases.
`Preferred modifier reagents are dicarboxylic acid anhy
`drides of the general formula:
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`4
`wherein incubation of the modified enzyme in an aqueous
`buffer, formulated to pH 8–9 at 25° C., at a temperature
`greater than about 50° C. results in at least a two-fold
`increase in enzyme activity in less than about 20 minutes;
`and
`(b) incubating the resulting mixture of step (a) at a
`temperature which is greater than about 50° C. for a time
`sufficient to reactivate the enzyme and allow formation of
`primer extension products.
`As a preferred method, the present invention provides a
`method for the amplification of a target nucleic acid con
`tained in a sample, comprising:
`(a) contacting the sample with an amplification reaction
`mixture containing a primer complementary to the target
`nucleic acid and a modified thermostable enzyme, wherein
`the modified thermostable enzyme is produced by a reaction
`of a mixture of a thermostable enzyme and a dicarboxylic
`acid anyhydride of the general formula:
`
`where R1 and R2 are hydrogen or organic radicals, which
`may be linked, or of the general formula:
`
`where R1 and R2 are organic radicals, which may be linked,
`and the hydrogen are cis. The organic radical may be directly
`attached to the ring by a carbon-carbon bond or through a
`carbon-hereoatom bond, such as a carbon-oxygen, carbon
`nitrogen, or carbon-sulphur bond. The organic radicals may
`also be linked to each other to form a ring structure as in, for
`example, 3,4,5,6-tetrahydrophthalic anhydride.
`Preferred reagents include maleic anhydride; substituted
`maleic anhydrides such as citraconic anhydride, cis-aconitic
`anhydride, and 2,3-dimethylmaleic anhydride; exo-cis-3,6
`endoxo-A"-tetrahydropthalic anhydride; and 3,4,5,6
`tetrahydrophthalic anhydride. In particular, citraconic anhy
`dride and cis-aconitic anhydride are preferred for the
`preparation of reversibly inactivated DNA polymerases for
`use in PCR amplifications.
`Another aspect of the present invention relates to methods
`for carrying out a nucleic acid amplification reaction using
`a reversibly-inactivated thermostable enzyme of the present
`invention. The present invention provides methods for the
`amplification of a target nucleic acid contained in a sample
`comprising the steps of:
`(a) contacting the sample with an amplification reaction
`mixture containing a primer complementary to the target
`nucleic acid and a modified thermostable enzyme, wherein
`the modified thermostable enzyme is produced by a reaction
`of a mixture of a thermostable enzyme which catalyzes a
`primer extension reaction and a modifier reagent, wherein
`the reaction is carried out at alkaline pH at a temperature
`which is less than about 25°C., wherein the reaction results
`in a chemical modification of the enzyme which results in
`essentially complete inactivation of enzyme activity, and
`
`25
`
`where R1 and R2 are hydrogen or organic radicals, which
`may be linked, or of the general formula:
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`where R, and R2 are organic radicals, which may be linked,
`and the hydrogen are cis, wherein the reaction results in
`essentially complete inactivation of enzyme activity; and
`(b) incubating the resulting mixture of step (a) at a
`temperature which is greater than about 50° C. for a time
`sufficient to reactivate the enzyme and allow formation of
`primer extension products.
`Preferred embodiments of the methods use reversibly
`modified enzymes modified using the preferred modifier
`reagents. In some embodiments of the invention, the incu
`bation step, step (b), is carried out prior to the start of the
`amplification reaction. In other embodiments, the incubation
`which results in reactivation of the enzyme is an integral step
`in the amplification process. For example, the denaturation
`step carried out in each PCR cycle can function simulta
`neously to reactivate a modified DNA polymerase.
`In a preferred embodiment of the invention, the amplifi
`cation reaction is a polymerase chain reaction (PCR) and a
`reversibly-inactivated thermostable DNA polymerase is
`used. The reaction mixture is incubated prior to carrying out
`the amplification reaction at a temperature which is higher
`than the annealing temperature of the amplification reaction.
`Thus, the DNA polymerase is inactivated until the tempera
`ture is above the temperature which insures specificity of the
`amplification reaction, thereby reducing non-specific ampli
`fication.
`Another aspect of the invention relates to amplification
`reaction mixtures which contain a reversibly-inactivated
`thermostable enzyme of the present invention along with
`reagents for carrying out the amplification reaction. In a
`preferred embodiment, the amplification reaction mixture
`contains oligonucleotide primers for carrying out a PCR.
`Another aspect of the invention relates to kits which
`comprise a reversibly inactivated thermostable enzyme of
`the invention and one or more amplification reagents.
`
`Page 9 of 20
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`5,773.258
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`15
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`5
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 shows the structures of citraconic anhydride,
`cis-aconitic anhydride, and 2,3-dimethylmaleic anhydride,
`and the reaction between citraconic anhydride and lysine.
`FIG. 2 shows the results of amplifications carried out
`using citraconylated Taq DNA polymerase as described in
`Example 4.
`FIG. 3 shows the results of amplifications carried out
`using citraconylated DNA polymerases as described in
`Example 6.
`FIG. 4 shows the results of varying the pre-reaction
`incubation time in amplifications carried out using citraco
`nylated and cis-aconitylated DNA polymerases as described
`in Example 9.
`FIG. 5 shows the results of varying the amplification cycle
`number in amplifications carried out using citraconylated
`and cis-aconitylated DNA polymerases as described in
`Example 10.
`DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`To aid in understanding the invention, several terms are
`defined below.
`The terms “nucleic acid” and “oligonucleotide” refer to
`primers, probes, and oligomer fragments to be detected, and
`shall be generic to polydeoxyribonucleotides (containing
`2-deoxy-D-ribose), to polyribonucleotides (containing
`D-ribose), and to any other type of polynucleotide which is
`an N glycoside of a purine or pyrimidine base, or modified
`purine or pyrimidine base. There is no intended distinction
`in length between the terms “nucleic acid” and
`“oligonucleotide”, and these terms will be used interchange
`ably. These terms refer only to the primary structure of the
`molecule. Thus, these terms include double- and single
`stranded DNA, as well as double- and single-stranded RNA.
`Oligonucleotide can be prepared by any suitable method. A
`review of synthesis methods is provided in Goodchild, 1990,
`Bioconjugate Chemistry 1(3):165–187, incorporated herein
`by reference.
`The term “hybridization” refers the formation of a duplex
`structure by two single-stranded nucleic acids due to
`complementary base pairing. Hybridization can occur
`between fully complementary nucleic acid strands or
`between “substantially complementary” nucleic acid strands
`that contain minor regions of mismatch. Conditions under
`which only fully complementary nucleic acid strands will
`hybridize are referred to as “stringent hybridization condi
`tions” or “sequence-specific hybridization conditions”.
`Stable duplexes of substantially complementary sequences
`can be achieved under less stringent hybridization condi
`tions. Those skilled in the art of nucleic acid technology can
`determine duplex stability empirically considering a number
`of variables including, for example, the length and base pair
`concentration of the oligonucleotides, ionic strength, and
`incidence of mismatched base pairs, following the guidance
`provided by the art (see, e.g., Sambrook et al., 1989, supra).
`Generally, stringent hybridization conditions are selected
`to be about 5° C. lower than the thermal melting point (Tm)
`for the specific sequence at a defined ionic strength and pH.
`The Tm is the temperature (under defined ionic strength and
`pH) at which 50% of the base pairs have dissociated.
`Relaxing the stringency of the hybridization conditions will
`allow sequence mismatches to be tolerated; the degree of
`mismatch tolerated can be controlled by suitable adjustment
`of the hybridization conditions.
`
`6
`The term “primer” refers to an oligonucleotide, whether
`natural or synthetic, capable of acting as a point of initiation
`of DNA synthesis under conditions in which synthesis of a
`primer extension product complementary to a nucleic acid
`strand is induced, i.e., in the presence of four different
`nucleoside triphosphates and an agent for polymerization
`(i.e., DNA polymerase or reverse transcriptase) in an appro
`priate buffer and at a suitable temperature. Oligonucleotide
`analogues, such as “peptide nucleic acids”, can act as
`primers and are encompassed within the meaning of the term
`“primer” as used herein. A primer is preferably a single
`stranded oligodeoxyribonucleotide. The appropriate length
`of a primer depends on the intended use of the primer but
`typically ranges from 6 to 50 nucleotides. Short primer
`molecules generally require cooler temperatures to form
`sufficiently stable hybrid complexes with the template. A
`primer need not reflect the exact sequence of the template
`nucleic acid, but must be sufficiently complementary to
`hybridize with the template.
`The term “primer extension” as used herein refers to both
`to the synthesis of DNA resulting from the polymerization of
`individual nucleoside triphosphates using a primer as a point
`of initiation, and to the joining of additional oligonucle
`otides to the primer to extend the primer. As used herein, the
`term “primer extension” is intended to encompass the liga
`tion of two oligonucleotides to form a longer product which
`can then serve as a target in future amplification cycles. As
`used herein, the term “primer” is intended to encompass the
`oligonucleotides used in ligation-mediated amplification
`processes which are extended by the ligation of a second
`oligonucleotide which hybridizes at an adjacent position.
`Primers can incorporate additional features which allow
`for the detection or immobilization of the primer but do not
`alter the basic property of the primer, that of acting as a point
`of initiation of DNA synthesis. For example, primers may
`contain an additional nucleic acid sequence at the 5' end
`which does not hybridize to the target nucleic acid, but
`which facilitates cloning of the amplified product. The
`region of the primer which is sufficiently complementary to
`the template to hybridize is referred to herein as the hybrid
`izing region.
`The terms “target region” and “target nucleic acid” refers
`to a region or subsequence of a nucleic acid which is to be
`amplified. The primer hybridization site can be referred to as
`the target region for primer hybridization.
`As used herein, an oligonucleotide primer is “specific” for
`a target sequence if the number of mismatches present
`between the oligonucleotide and the target sequence is less
`than the number of mismatches present between the oligo
`nucleotide and non-target sequences which may be present
`in the sample. Hybridization conditions can be chosen under
`which stable duplexes are formed only if the number of
`mismatches present is no more than the number of mis
`matches present between the oligonucleotide and the target
`sequence. Under such conditions, the oligonucleotide can
`form a stable duplex only with a target sequence. Thus, the
`use of target-specific primers under suitably stringent ampli
`fication conditions enables the specific amplification of
`those target sequences which contain the target primer
`binding sites. The use of sequence-specific amplification
`conditions enables the specific amplification of those target
`sequences which contain the exactly complementary primer
`binding sites.
`The term “non-specific amplification” refers to the ampli
`fication of nucleic acid sequences other than the target
`sequence which results from primers hybridizing to
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`5,773.258
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`sequences other than the target sequence and then serving as
`a substrate for primer extension. The hybridization of a
`primer to a non-target sequence is referred to as “non
`specific hybridization”, and can occur during the lower
`temperature, reduced stringency pre-reaction conditions.
`The term “thermostable enzyme” refers to an enzyme that
`is relatively stable to heat. The thermostable enzymes can
`withstand the high temperature incubation used to remove
`the modifier groups, typically greater than 50° C., without
`suffering an irreversible loss of activity. Modified thermo
`stable enzymes usable in the methods of the present inven
`tion include thermostable DNA polymerases and thermo
`stable ligases.
`The term “thermostable DNA polymerase” refers to an
`enzyme that is relatively stable to heat and catalyzes the
`polymerization of nucleoside triphosphates to form primer
`extension products that are complementary to one of the
`nucleic acid strands of the target sequence. The enzyme
`initiates synthesis at the 3' end of the primer and proceeds in
`the direction toward the 5' end of the template until synthesis
`terminates. Purified thermostable DNA polymerases are
`described in U.S. Pat. No. 4,889,818; U.S. Pat. No. 5,352,
`600; U.S. Pat. No. 5,079,352; PCT/US90/07639; PCT/
`US 91/05753; PCT/US91/0703; PCT/US 91/07076;
`co-pending U.S. patent application Ser. No. 08/062,368;
`WO 92/09689; and U.S. Pat. No. 5,210,036; each incorpo
`rated herein by reference.
`An enzyme “derived” from an organism herein refers to
`an enzyme which is purified from the organism or a recom
`binant version of an enzyme which is purified from the
`organism, and includes enzymes in which the amino acid
`sequence has been modified using techniques of molecular
`biology.
`The term “reversibly inactivated”, as used herein, refers to
`an enzyme which has been inactivated by reaction with a
`compound which results in the covalent modification (also
`referred to as chemically modification) of the enzyme,
`wherein the modifier compound is removable under appro
`priate conditions. The reaction which results in the removal
`of the modifier compound need not be the reverse of the
`modification reaction. As long as there is a reaction which
`results in removal of the modifier compound and restoration
`of enzyme function, the enzyme is considered to be revers
`ibly inactivated.
`The term “reaction mixture” refers to a solution contain
`ing reagents necessary to carry out a given reaction. An
`“amplification reaction mixture”, which refers to a solution
`containing reagents necessary to carry out an amplification
`reaction, typically contains oligonucleotide primers and a
`DNA polymerase or ligase in a suitable buffer. A “PCR
`reaction mixture” typically contains oligonucleotide
`primers, a thermostable DNA polymerase, d\TP's, and a
`divalent metal cation in a suitable buffer. A reaction mixture
`is referred to as complete if it contains all reagents necessary
`to enable the reaction, and incomplete if it contains only a
`subset of the necessary reagents. It will be understood by
`one of skill in the art that reaction components are routinely
`stored as separate solutions, each containing a subset of the
`total components, for reasons of convenience, storage
`stability, and to allow for independent adjustment of the
`concentrations of the components depending on the
`application, and, furthermore, that reaction components are
`combined prior to the reaction to create a complete reaction
`mixture.
`The methods of the present invention involve carrying out
`an amplification reaction using a heat-activated thermo
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`stable enzyme, wherein the active enzyme is required for
`primer extension. Prior to the high temperature incubation
`which activates the enzyme, the amplification reaction mix
`ture does not support primer extension and no extension
`products, non-specific or otherwise, are formed. Following
`the high temperature incubation which reactivates the
`enzyme, the amplification reaction is maintained at elevated
`temperatures which insure reaction specificity. Thus, primer
`extension products are formed only under conditions which
`insure amplification specificity.
`In the methods of the present invention, the heat-activated
`enzyme, in its active state, catalyzes the primer extension
`reaction. For use in a typical amplification reaction, e.g., a
`PCR, the heat-activated thermostable enzyme possesses, in
`its active state, DNA polymerase activity. For use in ligase
`mediated amplification systems, the heat-activated thermo
`stable enzyme possesses, in its active state, DNA ligase
`activity.
`In a ligase-meditated amplification system, an “extension
`product” is formed by the ligation of a first oligonucleotide
`(herein encompassed by the term “primer”) to a second
`oligonucleotide which hybridizes adjacent to the 3' end of
`the first oligonucleotide. The second oligonucleotide may be
`hybridized immediately adjacent to the primer, in which
`case only ligation is required, or may be hybridized one or
`more bases away from the primer, in which case polymerase
`activity is required to extend the primer prior to ligation. In
`either case, the joining of two oligonucleotides which
`hybridize to adjacent regions of the target DNA is intended
`to be herein encompassed by the term “primer extension”.
`Reversibly Inactivated Thermostable Enzymes
`The reversibly inactivated thermostable enzymes of the
`invention are produced by a reaction between the enzyme
`and a modifier reagent, which results in a reversible chemi
`cal modification of the enzyme, which results in the loss of
`all, or nearly all, of the enzyme activity. The modification
`consists of the covalent attachment of the modifier group to
`the protein. The modifier compound is chosen such that the
`modification is reversed by incu