WO2006108179A2

WO2006108179A2 - Two component dna pol iii replicases with modified beta-subunit binding motifs, and uses thereof

Info

Publication number: WO2006108179A2
Application number: PCT/US2006/013493
Authority: WO
Inventors: Lars-Erik Peters
Original assignee: Qiagen North American Holdings, Inc.
Priority date: 2005-04-07
Filing date: 2006-04-07
Publication date: 2006-10-12
Also published as: EP1874925A2; WO2006108179A3; JP2008535497A

Abstract

The invention provides modified DNA polymerase III replicases, and methods of using the same for various nucleic replication applications. The modified replicases comprise two components, particularly a polymerase component containing an α subunit, and a sliding clamp component containing a β subunit. The two component polymerases lack a clamp loader. The α subunits of the modified replicases have one or more mutations in one or more of their β-subunit binding sites. Consequently, the mutated α subunits have increased affinity for β-subunits, and the two component replicases exhibit increased processivity.

Description

TWO COMPONENT DNA POL III REPLICASES WITH MODIFIED BETA-SUBUNIT BINDING MOTIFS, AND USES THEREOF

STATEMENT OF RELATEDNESS

[001] This application is a continuation-in-part of U.S. application Serial No. 11/101 ,977 filed 7 April 2005, which is expressly incorporated herein in its entirety by reference. This application also claims the benefit under 35 U.S. C. 119(e) of U.S. Provisional Application Serial-No. 60/741 ,009, filed November 29, 2005.

FIELD

[002] The invention relates to DNA Pol III replicases, β-subunit binding motifs therein, modifications that alter β subunit association with DNA Pol III replicases, modifications that alter DNA Pol III replicase activity and thermostability, and to the processive enzymatic replication and amplification of nucleic acid molecules.

BACKGROUND OF THE INVENTION

[003] DNA polymerases have a number of applications in molecular biology, including but not limited to nucleic acid sequencing, nucleic acid quantification (Real Time PCR₁ NASBA), nucleic acid amplification (PCR, RDA, SDA), and reverse transcription of RNA into cDNA.

[004] A DNA polymerase III holoenzyme ("Pol III") was first purified and determined to be the principal replicase of the E. coli chromosome by Kornberg (Kornberg, A., 1982 Supplement to DNA Replication, Freeman Publications, San Francisco, pp 122-125, incorporated herein by reference). The holoenzyme is composed of 10 distinct subunits that form three separate functional components (see McHenry, et al,, J. Bio Chem., 252:6478-6484 (1977); Maki, et al., J. Biol. Chem., 263:6551-6559 (1988), each incorporated herein by reference). The three functional components are (i) the "core" (i.e. the polymerase), (ii) β (i.e., the sliding clamp), and (iii) the γ-complex (i.e., the clamp loader). Within the "core" are three subuhits: the α subunit (encoded by dnaE) represents the catalytic subunit with the DNA polymerase activity; the ε subunit (encoded by dnaQ, mutD) is the proofreading 3-5' exonuclease (Scheuermann, et al., Proc. Natl. Acad. Sci. USA, 81 :7747-7751 (1984); and DiFrancesco, et al., J. Biol. Chem., 259:5567-5573 (1984), each incorporated herein by reference), and the θ subunit (encoded by holE) stimulates ε (Studwell-Vaughan et al., J. Biol. Chem., 268:11785- 11791 (1993), incorporated herein by reference). The α subunit forms a tight 1:1 complex with ε (Maki, et al., J. Biol. Chem., 260:12987-12992 (1985) incorporated herein by reference), and θ forms a 1:1 complex with ε (Studwell-Vaughan et al., supra).

[005] The £. coll DNA Pol III replicase is highly efficient and completely replicates a uniquely primed bacteriophage single-strand DNA ("ssDNA") genome coated with the ssDNA binding protein ("SSB") at a speed of at least 500 nucleotides per second at 3O⁰C without dissociating from a 5 kb circular DNA even once (Fay, et al., J. Biol. Chem., 256:976-983 (1981); O'Donnell, et al., J. Biol. Chem., 260:12884-12889 (1985); and Mok, et al., J. Biol. Chem., 262:16644-16654 (1987), each incorporated herein by reference). Additionally, the E. coli DHA Pol III replicase exhibits high fidelity in DNA replication.

[006] DNA polymerase III replicases from a number of gram negative and gram positive bacteria, including thermophilic bacteria, have been described (for example, see Bullard et al., J.Biol.Chem., 277:13401-13408, 2002; and Bruck et al., J.Biol.Chem., 277:17334-17348, 2002; each incorporated herein by reference) and display the three component organization found in E.coli DNA Pol III. In Streptococcus pyogenes, for example, the DNA Pol III replicase is comprised of (i) the α subunit encoded by the polC gene (without epsilon and theta subunits), (ii) β-sliding clamp, and (iii) the τ/δ/δ¹- complex (i.e., the clamp loader) (Bruck I, O'Donnell M., J Biol Chem. 2000 Sep 15;275(37):28971-83, incorporated herein by reference).

[007] The literature has consistently taught that the three principal functional components of DNA polymerase III holoenzyme, i.e., the polymerase core (including the α subunit), the processivity clamp, and the clamp loader, are required for a functional, processive DNA replicase having rapid extension rates and low error rates characteristic of genomic replicases. (See, for example, U.S. Patent Nos. 6,555,349; 6,221,642; 5,668,004; 5,583,026; 6,677,146; and 6,238,905; see also O'Donnell, Bioessays, 14:105-111, 1992; O'Donnell et al., J. Biol. Chem., 260:12875-12883, 1985; McHenry, MoI. Microbiol., 49:1157-1165, 2003; McHenry, J. Biol. Chem., 266:19127-19130, 1991; Studwell et al., J. Biol. Chem., 265:1171-1178, 1990; Bullard et al., J. Biol. Chem., 277:13401-13408, 2002; and Bruck et al., J. Biol. Chem., 277:17334-17348, 2002; each incorporated herein by reference). The original reports by Kornberg et al. on E. coli DNA Pol III were the first to show that the polymerase activity of an isolated α subunit and isolated core polymerase could only be measured at very high enzyme concentrations using a "gap-filling" assay and produced a maximum extension rate of only 20 b/sec. This non-processive, slow synthesis, and the high protein concentration required to achieve it, precluded the use of an isolated α subunit or a DNA polymerase core subassembly in molecular biology applications. Many more recent reports on DNA Pol Ills from a variety of bacteria have further cemented the dogma that neither the α subunit nor the core complex of a DNA polymerase III holoenzyme can function alone in a processive mode with fast extension rates - a three component subassembly of the holoenzyme is required. For example, Bruck et al. (J. Biol. Chem., 275: 28971- 28983, 2000, incorporated herein by reference) have reported that the alpha subunit (polC) of Pol III from Streptococcus pyogenes does not function alone as a DNA polymerase, while Bullard et al., J. Biol. Chem., 277:13401-13408, 2002, and Bruck et al., J. Biol. Chem., 277:17334-17348, 2002 (each incorporated herein by reference), have reported that three components of Pol III are required for replicase activity in Thermus thermophilus and Aquifex aeolicus, respectively.

[008] The processivity of DNA Pol III replicases derives from β-sliding clamp association with the DNA polymerase core (alpha, epsilon, theta) in the holoenzyme, and β-sliding clamp association with DNA polymerase core on DNA has been thought to require a clamp loader - hence the three component dogma. The β subunit forms a dimer that encircles DNA and thereby tethers the associated core polymerase to its DNA substrate. Two sites in E.coli DNA Pol III alpha (Eco DnaE) have been reported to bind to β-sliding clamp. The first is an internal binding site located between residues 920-924 (QADMF) flanked upstream by the helix-hairpin-helix DNA binding domain and downstream by the OB-fold domain. A survey of other β-subunit binding proteins has suggested a consensus β-subunit binding motif, QL(S/D)LF. The second motif has the sequence QVELEF and is located at the carboxy-terminus of E. coli DnaE between the residues 1154 and 1159. This carboxy- terminus motif also exhibits high affinity for the E. coliτ subunit. See Lopez at al., PNAS 100: 14689- 14694, 2003; Dohrman et al., JMB 308, 228-239, 2005; Dalrymple et al., PNAS 98:11627-11632, 2001; Kim et al., JBC 271:20699-20704, 1996 (each incorporated herein by reference).

[009] Deletion and mutation analyses of the β-subunit binding sites ("β-binding sites") in E, coli DnaE have shown that while neither sequence is required for distributive polymerase (gap filling) activity, the internal site is required for efficient β-subunit binding activity ("β-binding activity") and for processive polymerase activity of the holoenzyme. See Dohrman et al., JMB 308, 228-239, 2005 (incorporated as a reference herein). The carboxy-terminal motif, in contrast, was shown, not to be required in vitro for binding to β subunit or for processive polymerase activity of the holoenzyme. Nevertheless, mutations in the motif were found to partially compromise the subunit's ability to complement a DnaE α mutant in the E coli chromosome, likely due to a perturbation in its association with τ. Additionally, replacement of the internal sequence, QADMF, with an E. coli consensus sequence, QLDLF, produced an α polymerase subunit with a greatly increased affinity for β. In contrast, replacement of the carboxy-terminus motif with the same consensus sequence produced an α subunit with marginally increased affinity for β subunit, but a 400 fold lower binding affinity for the τ subunit. Although the mutant α subunit with the internal consensus sequence showed greatly increased affinity for β in vitro, its specific polymerase activity, extension rate and processivity was not greater than that of the wild type α subunit when incorporated into a DNA polymerase III holoenzyme. The second mutant α subunit with β binding consensus sequence at the carboxy terminus, which showed a slightly higher affinity for beta, failed also to show any improvement in specific activity, processivity and extension rate in in vitro replication assays. Accordingly, the skilled artisan is taught that mutations increasing the affinity of β-binding sites do not improve the performance of the mutated DNA polymerase holoenzyme III versus the wild type enzyme in vitro.

[0010] These results suggest a very limited, non-essential contribution of the carboxy-terminus motif to processive DNA replication. However, competition between τ and β for binding to the carboxy- terminus of ct has been hypothesized to play a role in regulating the mode of DNA synthesis. Particularly, displacement of β by τ at the carboxy-terminus of α has been suggested to mediate the interruption of processive synthesis when DNA Pol III holoenzyme encounters a nick. Additionally, a nexus between β subunit interaction and DNA binding has been suggested. Particularly, the internal site is flanked by two potential DNA binding elements, suggesting α subunit binding to DNA may impact β subunit association, and vice versa. Accordingly, changes in the affinities of the carboxy- terminal beta binding motif is likely to impact the correct assembly of the DNA Pol III holoenzyme disassembly during and after completion of processive DNA replication, respectively.

[0011] The assembly and function of a Pol III replicase appears to depend upon the affinities of component subunits for one and other, and involves some degree of competition for binding to the same subunit sites. For example, the clamp loader complex (the delta subunit) and α subunits bind to the same pocket of the β sliding clamp, while τ and β subunits appear to compete for the carboxy- terminus site of α. In addition, other enzymes involved in DNA repair and replication, such as DNA ligase, MutS, DNA polymerase I, Il and IV, also compete with the alpha subunit in binding the same hydrophobic pocket on beta. Binding site mutations and changes in subunit affinities could prove detrimental to DNA Pol III replicase assembly and function, and mutants tested to date have proved capable only of compromising holoenzyme activity.

SUMMARY OF THE INVENTION

[0012] While previously reported deletion and mutation analyses of β-binding sites in E. coli DnaE have shed light on the mechanism of DNA Pol III holoenzyme action, there has been no readily apparent utility for β-binding site modifications in the rational design of recombinant DNA Pol 111 replicases, as modifications have only appeared capable of compromising the integrity and processive activity of holoenzyme. Additionally, given that repair-type polymerases require salt concentrations much higher than those supporting DNA Pol III activity in vitro, the use of β-sliding clamp in conjunction with repair-type polymerases has been precluded. All told, the identification and analysis of β-binding sites in Pol III α subunits has not found application in the design of recombinant DNA polymerases having higher processivity, specific activity and thermostability in PCR applications.

[0013] Contrary to previous reports, copending U.S. Patent Application Serial No. 11/101 ,977 establishes that bacterial dnaE encoded and polC encoded α subunits, and core polymerases containing the same, can function alone and in combination with a β-sliding clamp as minimal functional DNA Pol III replicases under appropriate conditions in vitro. Such single component (α or core alone) and two component (α or core, plus β-sliding clamp) Pol III replicases lack a Pol III clamp loader. Consequently competing interactions of various Pol III holoenzyme subunits for beta binding are irrelevant to assemble functional two component polymerase holoenzyme.

[0014] The present invention stems from the additional discovery that in such two component DNA Pol III replicase compositions, mutations may be introduced into one or more β-binding motifs of the α subunit constituent to increase affinity for β subunit and increase processivity of the replicase. This contrasts with reports on DNA Pol III holoenzyme, wherein mutations that increase the affinity of isolated α subunit for β subunit in vitro have not produced a corresponding increase in holoenzyme activity. In addition, as demonstrated herein, the tighter binding of beta to the alpha subunit in the present two component DNA Pol III replicases improves the thermostability of the complex in applications with elevated temperature stress such as PCR. Accordingly, the manipulation of β- binding sites has found utility in the novel context of two component DNA Pol III replicases.

[0015] Alpha subunits of the invention, which are derived through the modification of extant β-binding sites, as described herein, are referred to herein as "modified α subunits". Two component DNA Pol III replicases of the invention, which comprise such modified α subunits, are referred to herein as "modified Pol III replicases".

[0016] Modified Pol III replicases disclosed herein comprise first components, which first components comprise modified α subunits. In some preferred embodiments, the first components of modified Pol III replicases consist essentially of modified α subunits. In some preferred embodiments, the first components of modified Pol III replicases comprise one or more additional subunits of the core polymerase complex of a DNA Pol III replicase. However, all the modified Pol III replicases disclosed herein lack a clamp loader. [0017] Modified Pol III replicases disclosed herein also comprise second components. The second component of a modified Pol III replicase consists essentially of a processivity clamp. In a preferred embodiment, the processivity clamp consists essentially of a DNA Pol III β subunit.

[0018] The modified Pol III replicases used herein are functional replicases, as defined herein.

[0019] Modified α subunits are preferably derived from dnaE encoded α subunits of gram negative bacteria, or polC encoded α subunits of gram positive bacteria. Preferred modified α subunits include those derived from non-mesophilic bacteria, preferably extremophiles. Especially preferred for use in the invention are modified α subunits derived from thermophiles. Surprisingly, modified Pol III replicases containing modified α subunits derived from thermophiles exhibit increased thermostability under appropriate conditions to sustain repetitive DNA replication reactions in a temperature-cycled mode leading to the amplification of double stranded DNA molecules in vitro. Modified Pol III replicases comprising modified α subunits derived from thermophiles are referred to herein as "thermostable modified Pol III replicases".

[0020] In one aspect, the invention is directed to modified dnaE encoded α subunits and polC encoded α subunits, which comprise β-binding sites distinct from corresponding wildtype α subunits. Two β-binding sites have been previously identified E. coli DnaE, and a single β-binding site has been proposed for the PoIC α subunit of gram positive bacteria. Disclosed herein is the identification of a novel second β-binding site present in the PoIC α subunits of gram positive bacteria. This novel beta binding site is situated upstream from the helix-hairpin-helix DNA binding domain in the PoIC alpha subunits. The previously identified sites, as well as the novel β-binding site disclosed herein may be manipulated to produce the modified α subunits, and thus the first components of modified Pol III replicases, of the invention.

[0021] The β-binding sites of modified α subunits are distinguishable from their wildtype counterparts on the basis of sequence. The distinct β-binding sites confer to modified α subunits β-binding activity that is distinct from that of corresponding wildtype α subunits as exhibited in two component Pol III replicase compositions. Also provided are nucleic acids encoding the modified α subunits of the invention.

[0022] In one embodiment, the invention provides modified α subunits comprising one or more mutations one or more β-binding sites. In a preferred embodiment, the mutated β-binding site comprises or consists essentially of a sequence selected from the group consisting of GMMGLFS, QEAVPF, GLVGLFA, EEWPF, GALDAFG, TQNSLFG, GVKVII, GAFDFT, (S/A/G)LL(G/A/P/Q/N/S/T)LF(S/A/G), (S/A/GJQLIG/A/P/Q/N/S^LFCS/A/G), (8/AZG)NL(GZAZPyQZNZSZT)LF(SZAZG)₁ QL(GZAZL)L(PZAZG)F₁ G(LZA)(LZA)(GZA)LFG.

[0023] In one embodiment, the invention provides modified α subunits comprising one or more mutations in a β-binding site of polC encoded α subunits of gram positive bacteria. In a preferred embodiment, the one or more mutations are present in the internal β-binding site of dnaE encoded α subunits from gram negative bacteria. In another embodiment, the one or more mutations are present in the carboxy-terminus β-binding site of dnaE encoded α subunits from gram negative bacteria. In a preferred embodiment, the modified α subunits possess one or more mutations in the carboxy- terminus β-binding site and one or more mutations in the internal β-binding sites of DnaE from gram negative bacteria. The mutations may be the same or different.

[0024] In one embodiment, the invention provides modified α subunits comprising one or more mutations in a β-binding site of polC encoded α subunits of gram positive bacteria. In one embodiment, the one or more mutations are present in the carboxy-terminus β-binding site of PoIC. In another embodiment, the one or more mutations are present in the internal β-binding site of PoIC described herein. In another embodiment, one or more mutations are present in both the internal β- binding site and the carboxy-terminus β-binding site of PoIC. The mutations may be the same or different.

[0025] In one aspect, the invention is directed to modified Pol III replicases comprising modified α subunits disclosed herein. [0026] In one aspect, the invention is directed to the use of modified Pol III replicases in compositions and methods for nucleic acid replication, including methods of DNA amplification, such as PCR, and DNA sequencing.

[0027] Accordingly, provided herein are methods for replicating a nucleic acid molecule, which methods comprise subjecting the nucleic acid molecule to a replication reaction in a replication reaction mixture comprising a modified Pol III replicase disclosed herein.

[0028] In a preferred embodiment, the nucleic acid molecule replicated is a DNA molecule. In a further preferred embodiment, the DNA molecule is double stranded. In other embodiments, the DNA molecule is single stranded. In a further preferred embodiment, the double stranded DNA molecule is a linear DNA molecule. In other embodiments, the DNA molecule is non-linear, for example circular or supercoiled DNA.

[0029] In a preferred embodiment, the method for replicating a nucleic acid molecule is a sequencing method useful for sequencing a nucleic acid molecule, preferably DNA. In a preferred embodiment, the method involves subjecting the nucleic acid molecule to a sequencing reaction in a sequencing reaction mixture. The sequencing reaction mixture comprises a modified Pol III replicase disclosed herein. Preferably, the modified Pol III replicase lacks 3'-5' exonuclease activity capable of removing 3' terminal dideoxynucleotides in the sequencing reaction mixture. In a preferred embodiment, the modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus,

[0030] In another preferred embodiment, the method for replicating a nucleic acid molecule is an amplification method useful for amplifying a nucleic acid molecule, preferably DNA. In a preferred embodiment, the method involves subjecting the nucleic acid molecule to an amplification reaction in an amplification reaction mixture. The amplification reaction mixture comprises a modified Pol III replicase disclosed herein. Preferably, the modified Pol III replicase possesses 3'-5' exonuclease activity in the amplification reaction mixture. [0031] In a preferred embodiment, the amplification method is a thermocycling amplification method useful for amplifying a nucleic acid molecule, preferably DNA, which is preferably double stranded, by a temperature-cycled mode. In a preferred embodiment, the method involves subjecting the nucleic acid molecule to a thermocycling amplification reaction in an thermocycling amplification reaction mixture. The thermocycling amplification reaction mixture comprises a thermostable modified Pol III replicase. Preferably, the thermostable modified Pol III replicase possesses 3'-5' exonuclease activity in the thermocycling amplification reaction mixture. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a preferred embodiment, the thermocycling amplification reaction mixture further comprises thermostabilizers, as disclosed herein. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. In a further preferred embodiment, the thermocycling amplification reaction mixture further comprises DNA destabilizers, as disclosed herein.

[0032] In a preferred embodiment, the thermocycling amplification method is a PCR method, useful for amplifying a nucleic acid molecule, preferably DNA, which is preferably double stranded, by PCR. In a preferred embodiment, the method involves subjecting the nucleic acid molecule to PCR in a PCR reaction mixture. In a preferred embodiment, the PCR reaction mixture comprises a thermostable modified Pol III replicase disclosed herein. Preferably, the thermostable modified Pol III replicase possesses 3'-5' exonuclease activity in the PCR reaction mixture. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. In a preferred embodiment, the PCR reaction mixture further comprises thermostabilizers, as disclosed herein. In a further preferred embodiment, the PCR reaction mixture further comprises DNA destabilizers, as disclosed herein.

[0033] In a preferred embodiment, the PCR method is a fast PCR method. In a preferred embodiment, the method involves subjecting the nucleic acid molecule to fast PCR in a fast PCR reaction mixture. The fast PCR reaction mixture comprises a thermostable modified Pol III replicase disclosed herein. Preferably, the thermostable modified Pol ill replicase possesses 3^v-5' exonuclease activity in the fast PCR reaction mixture. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aqulfex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a preferred embodiment, the fast PCR reaction mixture further comprises thermostabilizers, as disclosed herein. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. In a further preferred embodiment, the fast PCR reaction mixture further comprises DNA destabilizers, as disclosed herein. The fast PCR methods are preferably two-step PCR methods that consist of repeated two- temperature cycles, with a first temperature for denaturation, and a second temperature for both primer annealing and primer extension.

[0034] In another preferred embodiment, the PCR method is a long range PCR method. In a preferred embodiment, the method involves subjecting the nucleic acid molecule to long range PCR in a long range PCR reaction mixture. The long range PCR reaction mixture comprises a thermostable modified Pol III replicase disclosed herein. Preferably, the thermostable modified Pol III replicase possesses 3'-5' exonuclease activity in the long range PCR reaction mixture. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. In a preferred embodiment, the long range PCR reaction mixture further comprises thermostabilizers, as disclosed herein. In a further preferred embodiment, the long range PCR reaction mixture further comprises DNA destabilizers, as disclosed herein.

[0035] In one embodiment, the primer used in a replication reaction herein is a DNA. In another embodiment, the primer used in a replication reaction herein is a RNA.

[0036] In one aspect, the invention provides replication reaction mixtures for nucleic acid replication, which mixtures comprise a modified Pol III replicase disclosed herein. Preferred replication reaction mixtures of the invention are useful for DNA replication. Replication reaction mixtures include, but are not limited to, charged reaction mixtures, which include template nucleic acid and primer. Replication reaction mixtures also include pre-reaction mixtures, which require addition of template nucleic acid and/or primer, and optionally dilution to provide for replication of template.

[0037] In a preferred embodiment, the replication reaction mixture is a sequencing reaction mixture useful for nucleic acid sequencing, preferably DNA sequencing. The sequencing reaction mixture comprises a modified Pol III replicase disclosed herein. Preferably, the modified Pol III replicase lacks 3'-5' exonuclease activity capable of removing 3' terminal dideoxynucleotides in the sequencing reaction mixture. In a preferred embodiment, the modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus.

[0038] In another preferred embodiment, the replication reaction mixture is an amplification reaction mixture useful for nucleic acid amplification, preferably DNA amplification. The amplification reaction mixture comprises a modified Pol III replicase disclosed herein. Preferably, the modified Pol III replicase possesses 3'-5' exonuclease activity in the amplification reaction mixture.

[0039] In a preferred embodiment, the amplification reaction mixture is a thermocycling amplification reaction mixture useful for amplifying nucleic acids, preferably DNA, which is preferably double stranded, by a temperature-cycled mode. Preferably, the thermocycling amplification reaction mixture comprises a thermostable modified Poi III replicase disclosed herein. Preferably, the thermostable modified Pol III replicase possesses 3'-5' exonuclease activity in the thermocycling amplification reaction mixture. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifβx, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. Preferably, the thermocycling amplification reaction mixture further comprises thermostabilizers disclosed herein. Preferably, the thermocycling amplification reaction mixture also comprises DNA destabilizers disclosed herein.

[0040] In a preferred embodiment, the thermocycling amplification reaction mixture is a PCR reaction mixture useful for amplifying nucleic acids, preferably DNA, which is preferably double stranded, by PCR. The PCR reaction mixture comprises a thermostable modified Pol III replicase disclosed herein. Preferably, the thermostable modified Pol III replicase possesses 3 -5' exonuclease activity in the PCR reaction mixture. In a preferred embodiment, the thermostable modified Pol Hi replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. Preferably, the PCR mixture further comprises thermostabilizers disclosed herein. Preferably, the PCR reaction mixture also comprises DNA destabilizers disclosed herein.

[0041] In a preferred embodiment, the PCR reaction mixture is a fast PCR reaction mixture useful for fast PCR methods. The fast PCR reaction mixture comprises a thermostable modified Pol III replicase disclosed herein. Preferably, the thermostable modified Pol III replicase possesses 3'-5' exonuclease activity in the fast PCR reaction mixture. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species. Thermus thermophilus, Thermus aquatlcus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. Preferably, the fast PCR reaction mixture further comprises thermostabilizers disclosed herein. Preferably, the fast PCR reaction mixture also comprises DNA destabilizers disclosed herein.

[0042] In another preferred embodiment, the PCR reaction mixture is a long range PCR reaction mixture useful for long range PCR methods. The long range PCR reaction mixture comprises a thermostable modified Pol III replicase disclosed herein. Preferably, the thermostable modified Pol III replicase possesses 3'-5' exonuclease activity in the long range PCR reaction mixture. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. Preferably, the long range PCR reaction mixture further comprises thermostabilizers disclosed herein. Preferably, the long range PCR reaction mixture also comprises DNA destabilizers disclosed herein.

[0043] In a preferred embodiment, a replication reaction mixture provided herein comprises an amount of modified Pol III replicase such that the reaction mixture or a charged reaction mixture derived therefrom has modified Pol III replicase activity that is capable of replicating the DNA template by extending the hybridized primer at a rate of greater than 100, more preferably greater than 150, more preferably greater than 200, more preferably greater than 250, more preferably greater than 300, more preferably greater than 350, more preferably greater than 400, more preferably greater than 450, more preferably greater than 500, more preferably greater than 550, more preferably greater than 600, more preferably greater than 650, more preferably greater than 700 nucleotides per second.

[0044] Preferably, β subunit is in a reaction mixture at a molar ratio (α:β) of between 1:1 and 1:10, preferably between 1:3 and 1 :5.

[0045] In a preferred embodiment, a replication reaction mixture provided herein comprises a zwitterionic buffer. In a preferred embodiment, the zwitterionic buffer has a pH between about pH 7.5- 8.9. In a preferred embodiment, the zwitterionic buffer comprises a combination of a weak organic acid and a weak organic base.

[0046] In a preferred embodiment, a thermocycling amplification reaction mixture provided herein comprises thermostabilizers (alternatively referred to herein as "stabilizers") that increase the thermostability of a modified Pol III replicase.

[0047] In a preferred embodiment, a thermocycling amplification reaction mixture provided herein comprises DNA destabilizers that reduce the temperature required for template denaturation.

[0048] In a preferred embodiment, a replication reaction mixture provided herein lacks CaCI₂.

[0049] In a preferred embodiment, a replication reaction mixture provided herein lacks a γ subunit and/or a τ subunit.

[0050] In one aspect, the invention provides nucleic acid replication reaction tubes, which comprise nucleic acid replication reaction mixtures disclosed herein. Tubes comprising a replication reaction mixture are tubes that contain a reaction mixture.

[0051] In a preferred embodiment, the nucleic acid replication reaction tubes are sequencing reaction tubes, which comprise a sequencing reaction mixture disclosed herein. [0052] In another preferred embodiment, the nucleic acid replication reaction tubes are amplification reaction tubes, which comprise an amplification reaction mixture disclosed herein.

[0053] In a preferred embodiment, the amplification reaction tubes are thermocycling amplification reaction tubes, which comprise a thermocycling amplification reaction mixture disclosed herein.

[0054] In a preferred embodiment, the thermocycling amplification reaction tubes are PCR reaction tubes, which comprise a PCR reaction mixture disclosed herein.

[0055] In a preferred embodiment, the PCR reaction tubes are fast PCR reaction tubes, which comprise a fast PCR reaction mixture disclosed herein.

[0056] In another preferred embodiment, the PCR reaction tubes are long range PCR reaction tubes, which comprise a long range PCR reaction mixture disclosed herein.

[0057] In one aspect, the invention provides a nucleic acid replication kit useful for nucleic acid replication, which kit comprises a modified Pol III replicase disclosed herein. In a preferred embodiment, the replication kit comprises a replication reaction mixture disclosed herein. The kit may provide modified Pol III replicase and a mixture which is free of modified Pol III replicase, and require addition of modified Pol III replicase prior to use to produce a reaction mixture disclosed herein. In a preferred embodiment, the replication kit is useful for DNA replication. Kits of the invention may further include information pamphlets and packaging materials.

[0058] In a preferred embodiment, the nucleic acid replication kit is a sequencing kit useful for nucleic acid sequencing, preferably DNA sequencing. The sequencing kit comprises a modified Pol III replicase disclosed herein. In a preferred embodiment, the modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilυs, Thermus aquaticus, or Aquifex aeolicus. [0059] In another preferred embodiment, the nucleic acid replication kit is an amplification kit useful for nucleic acid amplification, preferably DNA amplification. The amplification kit comprises a modified Pol III replicase disclosed herein. Preferably, the amplification kit comprises a nucleic acid amplification reaction mixture disclosed herein.

[0060] In a preferred embodiment, the amplification kit is a thermocycling amplification kit useful for amplifying nucleic acids, preferably DNA, which is preferably double stranded, by a temperature- cycled mode. The thermocycling amplification kit comprises a thermostable modified Pol III replicase disclosed herein. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. Preferably, the thermocycling amplification kit comprises a thermocycling amplification reaction mixture disclosed herein.

[0061] In a preferred embodiment, the thermocycling amplification kit is a PCR kit for amplifying nucleic acids, preferably DNA, which is preferably double stranded, by PCR. The PCR kit comprises a thermostable modified Pol III replicase disclosed herein. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. Preferably the PCR kit comprises a PCR reaction mixture disclosed herein.

[0062] In a preferred embodiment, the PCR kit is a fast PCR kit. The fast PCR kit comprises a thermostable modified Pol III replicase disclosed herein. In a preferred embodiment, the thermostable modified Pol III replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, from the species Thermus thermophilic, Thermus aquaticus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. Preferably the fast PCR kit comprises a fast PCR reaction mixture disclosed herein.

[0063] In another preferred embodiment, the PCR kit is a long range PCR kit. The long range PCR kit comprises a thermostable modified Pol III replicase disclosed herein. In a preferred embodiment, the thermostable modified Pol ill replicase comprises a modified dnaE encoded α subunit, preferably derived from the genus Thermus or Aquifex, preferably from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus. In a further preferred embodiment, the thermostable modified Pol III replicase comprises a modified polC encoded α subunit, preferably derived from the genus Thermotoga, Hydrogenobacter or Carboxydothermus, preferably from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans. Preferably the long range PCR kit comprises a long range PCR reaction mixture disclosed herein.

[0064] In a preferred embodiment, a nucleic acid replication kit provided herein comprises a nucleic acid replication reaction mixture, which replication reaction mixture comprises an amount of modified Pol III replicase such that the reaction mixture can be combined with template DNA, and a primer hybridizable thereto, and optionally appropriately diluted to produce a charged reaction mixture, wherein the modified Pol III replicase is capable of replicating the DNA template by extending the hybridized primer at a rate of greater than 100, more preferably greater than 150, more preferably greater than 200, more preferably greater than 250, more preferably greater than 300, more preferably greater than 350, more preferably greater than 400, more preferably greater than 450, more preferably greater than 500, more preferably greater than 550, more preferably greater than 600, more preferably greater than 650, more preferably greater than 700 nucleotides per second. Further, the charged reaction mixture preferably has a modified α subunit concentration between 1ng/μL to 100ng/μL, more preferably 2ng/μL to 50ng/μL, and most preferably 4ng/μL to 10ng/μL. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 shows an alignment of internal β subunit-binding site sequences for a variety of DNA polymerase III alpha subunits (DnaE) from gram negative bacteria. The second β-binding site sequence (β/τ-binding site) situated at the carboxy-terminus of DnaE subunits is not shown.

[0066] Figure 2 shows the helix-hairpin-helix DNA binding domain and the OB-fold domain in E. coli and Tth and their relation to the beta-binding sites, as described herein.

[0067] Figure 3 shows the helix-hairpin-helix DNA binding domain and the OB-fold domain in Tma and their relation to the beta-binding domains.

[0068] Figure 4 compares the wild-type C-terminal amino acid sequence of Tth alpha subunit against the amino acid sequence of a modified Tth alpha subunit in accordance with the present invention

[0069] Figure 5 shows comparative results for modified Pol III replicase and Pol III replicase in DNA amplification. Modified Pol III replicase is referred to as synthetic triple mutant α.

[0070] Figure 6 shows comparative results for modified Pol III replicase and Pol III replicase in DNA amplification. Modified Pol III replicase is referred to as synthetic triple mutant α.

[0071] Figure 7 shows comparative results for modified Pol III replicase and Pol III replicase in DNA amplification. Modified Pol III replicase is referred to as mutant α.

[0072] Figure 8 demonstrates improved processivity and product yield obtained with modified Pol III replicase in comparison with Pol III replicase in DNA amplification.

[0073] Figure 9 demonstrates improved thermostability obtained with modified Pol III replicase in comparison with Pol III replicase in DNA amplification. [0074] Figure 10 demonstrates improved thermostability obtained with modified Pol III replicase in comparison with Pol III replicase in DNA amplification.

DETAILED DESCRIPTION OF THE INVENTION

[0075] As used herein, a gene "of or "derived from" a particular bacterial genus or species does not mean directly of or directly derived from a particular bacterial genus or species. Rather, the phrases refer to correspondence of the particular gene to an endogenous gene of the particular bacterial genus or species.

[0076] As used herein, "thermostable" modified Pol III replicase refers to a modified Pol III replicase that is resistant to inactivation by heat. Modified Pol III replicases are DNA polymerases, which synthesize the formation of a DNA molecule complementary to a single-stranded DNA template by extending a primer in the 5' to 3' direction. As used herein, a thermostable modified Pol III replicase is more resistant to heat inactivation than a thermolabile modified Pol III replicase. However, a thermostable modified Pol III replicase is not necessarily totally resistant to heat inactivation, and, thus, heat treatment may reduce its DNA polymerase activity to some extent. A thermostable modified Pol III replicase typically will also have a higher optimum temperature for synthetic function than a thermolabile modified Pol III replicase. The components of thermostable modified Pol III replicases are typically isolated from thermophilic bacteria.

[0077] As used herein "thermolabile" modified Pol III replicase refers to a modified Pol III replicase which is not resistant to inactivation by heat. An example of a thermolabile DNA Polymerase is T5 DNA polymerase, the activity of which is totally inactivated by exposing the enzyme to a temperature of 90⁰C for 30 seconds. As used herein, a thermolabile modified Pol III replicase is less resistant to heat inactivation than is a thermostable modified Pol ill replicase. A thermolabile modified Pol III replicase typically is also likely to have a lower optimum temperature than a thermostable modified Pol III replicase. Thermolabile modified Pol III replicases are typically isolated from mesophilic bacteria. [0078] As used herein, the term "primer" may refer to a plurality of primers. "Primers" include oligonucleotide primers, which include DNA and RNA oligonucleotides.

Modified Pol III Replicases

[0079] Modified Pol III replicases of the invention are "functional" replicases, i.e., functional subassemblies of DNA polymerase III subunits. Functional refers to replicase activity, which can be characterized by primer extension rate. A modified Pol III replicase of the invention is characterized by its ability to perform in a primer extension assay at an extension rate of greater than 75 nucleotides per second. Especially preferred modified DNA Pol III replicases of the invention perform at an extension rate of greater than 100, more preferably greater than 150, more preferably greater than 200, more preferably greater than 250, more preferably greater than 300, more preferably greater than 350, more preferably greater than 400, more preferably greater than 450, more preferably greater than 500, more preferably greater than 550, more preferably greater than 600, more preferably greater than 650, more preferably greater than 700 nucleotides per second. Moreover, such preferred modified Pol III replicases are capable of performing at a preferred extension rate at a concentration of 2pmo!/μL or less.

[0080] Preferably, β subunit is in a reaction mixture at a molar ratio (α:β) ranging between 1 :1 and 1:10, preferably between 1:3 and 1:5.

[0081] Accordingly, modified DNA Pol III replicases derived from the E. coli DNA polymerase III α subunit, which is non-functional as demonstrated in the literature and confirmed in copending application Serial No. 11/101,977 filed 7 April 2005, are not included among the modified DNA Pol III replicases of the invention.

[0082] By "an E. coli DNA polymerase III α subunit" herein is meant the native DNA polymerase III α subunit of Escherichia coli. For example, see Maki et al., 1985, supra. By "modified" E. coli DNA polymerase III α subunit herein is meant an DNA polymerase III α subunit of Escherichia coli with at least one mutation in at a least one β subunit-binding site. [0083] The modified Pol III replicases of the invention comprise a first component, which comprises a modified Pol ill α subunit. In some preferred embodiments, the first component consists essentially of a modified Pol III α subunit. In some preferred embodiments, the first component comprises one or more additional subunits of the core polymerase complex of a DNA Pol III. The second component consists essentially of a processivity clamp. The processivity clamp comprises a DNA Pol III β subunit. In some preferred embodiments, the processivity clamp consists essentially of a DNA Pol III β subunit. The first and second components of a modified Pol III replicase are preferably from the same species. The modified Pol III replicases of the invention lack a clamp loader component. In some embodiments, a modified Pol III replicase comprises more than one first component, which may be the same or different.

[0084] In a preferred embodiment, the modified α subunit of a modified Pol III replicase used herein is derived from a dnaE encoded α subunit of a gram negative bacterium.

[0085] In another preferred embodiment, the modified α subunit of a modified Pol III replicase used herein is derived from a polC encoded α subunit of a gram positive bacterium.

[0086] In a preferred embodiment, the modified α subunit of a modified Pol III replicase is derived from a dnaE encoded or polC encoded α subunit of an extremophile bacterium taxonomically positioned at the base of the phylogentic tree eubacteria, such as the genera, but not limited to, Aquifex, Hydrogenobacter, Thermus, Carboxydothermus, Thermocrinis, Deinococcus and Thermotoga.

[0087] Especially preferred are modified α subunits derived from a bacterium or cyanobacterium selected from the group consisting of Aquifex aeolicus, Thermus thermophilus, Deinococcus radiurans, Thermus aquaticus, Thermotoga maritima, Thermoanaerobacter, Geobacillus stearothermophilus, Thermus flavus, Thermus ruber, Thermus brockianus, Thermotoga neapolitana and other species of the Thermotoga genus, Methanobacterium thermoautotrophicum, and species from the genera Thermocridis, Hydrogenobacter, Thermosynchecoccus, Carboxydothermus hydrogenoformans and mutants of these species. Especially preferred are Aquifex aeolicus, Aquifex pyogenes, Thermus thermophilic, Thermus aquaticus, Thermotoga neapolitana and Thermotoga maritima.

[0088] Extremophile bacteria are bacteria that grow and propagate under extreme environmental conditions out of the range of normal physiological conditions such as high radiation, extremely low humidity, high (above 6O⁰C) or low (below 1O⁰C) temperature and high osmotic pressure (salt concentration above 0.5 M).

[0089] Among the dnaE encoded and polC encoded α subunits of extremophile bacteria, those derived from thermophilic bacteria are especially preferred.

[0090] In an especially preferred embodiment, the modified α subunit of a modified Pol III replicase is derived from the dnaE encoded α subunit of Thermus thermophilus.

[0091] Modified dnaE encoded α subunits used in the invention preferably possess intrinsic zinc- dependent 3'-5' exonuclease activity.

[0092] In one embodiment, the first component of a modified Pol III replicase additionally comprises an ε subunit having 3'-5' exonuclease activity.

[0093] Examples of α subunits are found, for example, in U.S. Patent No. 6,238,905, issued May 29, 2001 ; U.S. Patent Application Serial No. 09/642,218, filed August 18, 2000; U.S. Patent Application Serial No. 09/716,964, filed November 21 , 2000; U.S. Patent Application Serial No. 09/151 ,888, filed September 11, 1998; and U.S. Patent Application Serial No. 09/818,780, filed March 28, 2001 , each of which is expressly incorporated herein by reference.

[0094] Included among the α subunits that may be modified for use in modified Pol III replicases herein are those characterized by the presence and arrangement of protein motifs as disclosed in copending application Serial No. 11/101,977. [0095] Also included among the α subunits that may be modified for use in modified Pol ill replicases herein are those purified by the methods disclosed in copending application Serial No. 11/101,977, as well as those identified as having desired properties by the assay methods disclosed in copending application Serial No. 11/101,977.

[0096] In one embodiment, the 3'-5' exonuclease activity of a modified Pol III replicase in a reaction mixture is conferred by an ε subunit. In another embodiment, the 3'-5' exonuclease activity of a modified Pol III replicase in a reaction mixture is conferred by a modified α subunit. The 3'-5' exonuclease activity of a modified α subunit may be modulated by manipulating zinc concentration, and may differ in different reaction mixtures, as described herein.

[0097] In one embodiment, a modified Pol III replicase does not exhibit significant 3'-5' exonuclease activity capable of removing 3' terminal dideoxyrtucleotides in a reaction mixture. In a preferred embodiment, the modified Pol III replicase lacking significant 3'-5' exonuclease activity comprises a modified α subunit derived from a dnaE encoded α subunit of a gram negative bacterium.

[0098] In one embodiment, the first component of a modified Pol III replicase includes additional subunits of a Pol III DNA polymerase core, such as θ.

[0099] In one embodiment, the first component of a modified Pol III includes an ε subunit encoded by a bacterial dnaQ gene. Examples of ε subunits are found, for example, in U.S. Patent Application Serial No. 09/642,218, filed August 18, 2000; U.S. Patent Application Serial No. 09/716,964, filed November 21 , 2000; U.S. Patent Application Serial No. 09/151,888, filed September 11, 1998; and U.S. Patent Application Serial No. 09/818,780, filed March 28, 2001.

[00100] Examples of β subunits are found, for example, in U.S. Patent Application Serial No. 09/642,218, filed August 18, 2000; U.S. Patent Application Serial No. 09/716,964, filed November 21, 2000; U.S. Patent Application Serial No. 09/151 ,888, filed September 11, 1998; and U.S. Patent Application Serial No. 09/818,780, filed March 28, 2001, each expressly incorporated herein in its entirety by reference. [00101] The first and second components of a modified Pol IiI replicase of the invention may be coincubated and allowed to form a two component polymerase in solution. The coincubation solution may be a reaction mixture, or, preferably, the components may be associated prior to addition to reaction mixture. In one embodiment, more than one first component is coincubated with a second component.

Modified Alpha Subunits

[00102] The modified α subunits herein are derived through the modification of extant β-binding sites, including those previously described in the art as well as the novel β-binding site for gram positive PoIC alpha subunits identified herein. Table 1 provides a representative list of internal beta-binding motifs in both gram negative DnaE and gram positive PoIC alpha subunits

TABLE 1 : Internal Beta binding Motifs of DNA Pol III α Subunits

[00103] As noted in the background, prior art publications define a consensus sequences, QL(S/D)LF for the internal DnaE protein β binding site. See, for example, see Dalrymple et al., PNAS 98:11627- 11632, 2001. With reference to Figure 1, aligning the amino acid sequences of the flanking domains allowed us to identify this internal site in DnaE proteins from a number of gram negative and gram positive bacteria. Surprisingly, the results of a sequence alignment revealed the lack of any primary sequence conservation within the β subunit binding motifs that would allow deviation of a consensus amino acid sequence for the DnaE internal β binding site. The only consistent features of the internal β binding motif in bacterial DnaE proteins is the presence of a C-terminal phenylalanine residue and two small amino acids (proline, glycine, serine or threonine) flanking the ^β binding motif on both sides. This surprising finding contradicts the teaching in the art. The amino acid sequences of β-binding sites for α subunits not included in Figure 1 may be identified by sequence comparison with those in the figure, as well as by a structural analysis that identifies the OB fold and helix-hairpin-helix domains (see Dohrmann et al., J MoI Biol. 2005 JuI 8;350(2):228-39). See also Figures 2 and 3. The β- binding site sequence at the carboxy-terminus of DnaE is well documented. See, for example, Kim et al., JBC 271:20690-20698, 1996.

Preferred Modified α Subunit β-binding Site Sequences of Thermophilic DNA POL III of Gram Negative Bacteria

[00104] Tth DNA Pol III

Additional Mutant Motifs for Improved β-clamp Binding:

[00107] (S/A/G)LL(G/A/P/Q/N/S/T)LF(S/A/G)

[00108] QL(G/A/L)L(P/A/G)F [00109] G(L/A)(L/A)(G/A)LFG

[00110] (S/A/G)QL(G/A/P/Q/N/S/T)LF(S/A/G)

[00111] (S/A/G)NL(G/A/P/Q/N/S/T)LF(S/A/G)

[00112] A number of studies have compared the affinities of various peptides, including peptides corresponding to various α-subunit β-binding site sequences, for β-subunit. Additional studies examining the affinities of α-subunits with mutations in the internal beta binding site, and the carboxy- terminus (τ-binding site) to β-subunit have been performed. These studies may inform the choice of β-binding site modification in the present invention. In particular, sequences showing higher affinity for the β-subunit than the extant β-binding sequence of an α subunit may be used to replace such extant β-binding sites to improve β subunit binding, including but not limited to point mutations, multiple mutations, and complete motif substitutions.

Nucleic Acid Replication

[00113] In one aspect, the invention provides methods for replicating a nucleic acid molecule, comprising subjecting the nucleic acid molecule to a replication reaction in a replication reaction mixture comprising a modified Pol III replicase.

[00114] "Nucleic acid replication" is a process by which a template nucleic acid molecule is replicated in whole or in part. Thus, the product of a nucleic acid replication reaction can be completely or partially complementary to the template nucleic acid molecule it is replicating. Nucleic acid replication is done by extending a primer hybridized to the template nucleic acid in the 5'-3' direction, incorporating nucleotides complementary to the bases of the template nucleic acid at each position in the extension product. The primer may be, for example, a synthetic oligonucleotide that hybridizes to a region of a single stranded DNA template. The primer may also be, for example, a portion of a single stranded DNA template that is complementary to a second region of the single stranded DNA template and can self-prime. Included within the scope of nucleic acid replication reactions are isothermal replication reactions, sequencing reactions, amplification reactions, thermocycling amplification reactions, PCR, fast PCR, and long range PCR.

[00115] The nucleic acid replicated in a nucleic acid replication reaction is preferably DNA, and replication preferably involves the DNA-dependent DNA polymerase activity of a modified Pol III replicase disclosed herein.

[00116] In a preferred embodiment, a replication reaction mixture comprises a zwitterionic buffer, comprising a combination of a weak organic acid, having a pK between about 7.0-8.5 (e.g., HEPES, DIPSO, TAPS, HEPBS, HEPPSO, TRICINE, POPSO, MOBS, TAPSO, TABS and TES) and a weak organic base, having a pK between about 6.8-8.5 (e.g., Tris, Bis-Tris-propane, imidazol, TMNO, 4- methyl imidazol, triethanolamine and diethanolamine), wherein the pH of the buffer is set by titration with organic base between about pH 7.5-8.9, and wherein the concentration of the organic acid is between about 10-4OmM, more preferably between about 20-3OmM.

[00117] In an especially preferred embodiment, a replication reaction mixture and modified Pol III replicase combination is selected from the following combinations: (i) HEPES-Bis-Tris-Propane (20 mM, pH 7.5) with a modified Pol III replicase comprising a modified dnaE encoded α subunit from the genus Thermus, preferably from the species Thermus thermophilus; and (ii) TAPS-Tris (2OmM, pH 8.7) with a modified Pol III replicase comprising a modified dnaE encoded α subunit from the genus Aquifex, preferably from the species Aquifex aeolicus.

[00118] In a preferred embodiment, a nucleic acid replication reaction mixture comprises one or more ions selected from the group consisting Of Zn²⁺, Mg²⁺, K⁺, and NH₄ ²⁺, which are included for optimum activity of the modified Pol III replicase in the reaction mixture. The ions are preferably titrated in preliminary assays to determine the optimum concentrations for optimum activity of the modified Pol III replicase in the reaction mixture. In a particularly preferred embodiment, the nucleic acid replication reaction mixture lacks Ca²⁺ ion. [00119] In some preferred embodiments, the nucleic acid replication reaction mixture includes potassium ions. Potassium ions are preferably titrated initially to determine the optimal concentration for the modified Pol III replicase being used. Generally, the K⁺ concentration of the replication reaction mixture is preferably between 0 and about 10OmM, more preferably between about 5-25mM. Potassium ion is preferably provided in the form of KCI, K₂SO₄, or potassium acetate. The particular counter anion provided with K⁺ can impact the activity of the modified Pol III replicase , and preliminary assays are preferably done in order to determine which counter anion best suits the particular modified Pol III replicase for the particular replication reaction. In general, sulfate or chloride counter anion is preferably used with a modified Pol III replicase derived from Aquifex aeolicus, with sulfate being preferred over chloride. Additionally, potassium ion is not preferred for use in a replication reaction mixture with a modified Pol III replicase derived from Thermus thermophilus.

[00120] In some preferred embodiments, the nucleic acid replication reaction mixture includes ammonium ions. Ammonium ions are preferably titrated initially to determine the optimal concentration for the modified Pol III replicase being used. Generally, the NH₄ ²⁺ concentration of the replication reaction mixture is preferably between 0 and about 15mM. Ammonium ion is preferably provided in the form of ammonium sulfate. Ammonium ions are preferably included in a replication reaction mixture with a modified Pol III replicase derived from Aquifex aeolicus. Additionally, ammonium ion is not preferred for use in a replication reaction mixture with a modified Pol III replicase derived from Thermus thermophilus.

[00121] In some preferred embodiments, the replication reaction mixture includes zinc ions. Zinc ions are preferably titrated initially to determine the optimal concentration for the modified Pol III replicase being used. Generally, the Zn²⁺ concentration in a replication reaction mixture is preferably between 0 and about 50μM, more preferably between about 5-15μM. Zinc ion is preferably provided in the form of a salt selected from the group consisting of ZnSO₄, ZnCI₂ and zinc acetate. The particular counter anion provided with Zn²⁺ can impact the activity of the modified Pol III replicase , and preliminary assays are preferably done in order to determine which counterion best suits the particular modified Pol III replicase for the particular replication reaction. In general, chloride or acetate counter anions are preferably used in a replication reaction mixture with a modified Pol III replicase derived from Thermus thermophilus, and sulfate counter anions are preferably used in a replication reaction mixture with a modified Pol III replicase derived from Aquifex aeolicus.

[00122] In general, Zn²⁺ is not preferred for use in sequencing reaction mixtures, as it can increase the 3'-5' exonuclease activity of a number of α subunits (e.g., Thermus thermophilus dnaE). The impact Of Zn²⁺ on the 3'-5' exonuclease activity of any particular minimal functional Pol III replicase, and its impact on sequencing reactions using the same, may be assessed using standard exonuclease activity assays that are well known in the art.

[00123] In some preferred embodiments, the replication reaction mixture includes magnesium ions. Magnesium ions are preferably titrated initially to determine the optimal concentration for the modified Pol III replicase being used. Generally, the Mg²⁺ concentration in a replication reaction mixture is preferably between 0 and about 15mM, more preferably between about 4-1 OmM. In general, isothermal nucleic acid replication reactions, including nucleic acid sequencing reactions, are more accommodating of Mg²⁺ concentrations at the higher end of the preferred concentration range. Magnesium ion is preferably provided in the form of a salt selected from the group consisting of MgCI₂, MgSO₄, and magnesium acetate. The particular counter anion provided with Mg²⁺ can impact the activity of the modified Pol III replicase , and preliminary assays are preferably done in order to determine which counterion best suits the particular modified Pol III replicase for the particular replication reaction. In general, acetate or chloride counter anions are preferably used with a modified Pol III replicase derived from Thermus thermophilus, with acetate being preferred over chloride. Additionally, sulfate counter anions are preferably used with a modified Pol III replicase derived from Aquifex aeolicus.

[00124] In an especially preferred embodiment, a replication reaction mixture for use with a modified Pol III replicase derived from Aquifex aeolicus comprises TAPS-Tris (2OmM, pH8.7), 25mM K₂SO₄, 1 OmM NH₄(OAc)₂, and 1OmM MgSO₄. [00125] In another especially preferred embodiment, a replication reaction mixture for use with a modified Pol III replicase derived from Thermus thermophilus comprises HEPES-Bis-Tris-Propane (2OmM, pH7.5), and 1OmM Mg(OAc)₂.

[00126] In one embodiment, a helicase is included in a replication reaction in order to lower the denaturation temperature required to provide single stranded nucleic acid template for replication.

[00127] In one embodiment, a replication reaction mixture provided herein lacks ATP.

[00128] In one embodiment, a replication reaction mixture provided herein lacks SSB, wherein SSB, if present in the replication reaction mixture, would reduce the DNA polymerase activity of the particular modified Pol III replicase used in the replication reaction mixture. In a preferred embodiment, a replication reaction mixture comprising a modified Pol III replicase , which modified Pol III replicase comprises an α subunit encoded by Streptococcus pyogenes polC lacks SSB.

[00129] In nucleic acid replication reactions herein, wherein the modified Pol III replicase used is derived from a thermophilic bacterium, the reaction mixture preferably has a pH from about 7.2-8.9. In some preferred embodiments, the reaction mixture has a Zn²⁺ concentration between 0 and about 50μM, more preferably between about 5-15μM. In some preferred embodiments, the reaction mixture has a Mg²⁺ concentration between 0 and about 15mM, more preferably between about 4-1OmM. In some preferred embodiments, the reaction mixture has a K⁺ concentration between 0 and about 10OmM, more preferably between about 5-25mM. In some preferred embodiments, the reaction mixture has an NH4²⁺ concentration between 0 and about 12mM, more preferably between about 5- 12mM. In some preferred embodiments, the reaction mixture has a combination of these cations in their preferred concentration ranges.

[00130] In nucleic acid replication reactions herein, the temperature at which primer extension is done is preferably between about 55°C-72°C, more preferably between about 60°C-68°C. [00131] In a preferred embodiment, the temperature at which primer annealing and primer extension are done in a thermocycling amplification reaction is between about 55°C-72°C, more preferably between about 60°C-68°C, more preferably between about 60°C-65°C, though the optimum temperature will be determined by primer length, base content, degree of primer complementarity to template, and other factors, as is well known in the art.

[00132] In a preferred embodiment, the temperature at which denaturation is done in a thermocycling amplification reaction is between about 86°C-95°C, more preferably between 87°C-93°C, with temperatures at the lower end of the range being preferred for use in combination with thermocycling amplification reaction mixtures that include DNA destabilizers, as disclosed herein. Preferred thermocycling amplification methods include polymerase chain reactions involving from about 10 to about 100 cycles, more preferably from about 25 to about 50 cycles, and peak temperatures of from about 86°C-95°C, more preferably 87°-93°C, with temperatures at the lower end of the range being preferred for use in combination with PCR reaction mixtures that include DNA destabilizers, as disclosed herein.

Nucleic Acid Amplification

[00133] In one aspect, the invention provides methods for amplifying a nucleic acid molecule, comprising subjecting the nucleic acid molecule to an amplification reaction in an amplification reaction mixture comprising a modified Pol III replicase disclosed herein. Preferably, the amplification reaction is done in an amplification reaction tube described herein.

[00134] Nucleic acid molecules may be amplified according to any of the literature-described manual or automated amplification methods. As used herein "amplification" refers to any in vitro method for increasing the number of copies of a desired nucleotide sequence. The nucleic acid amplified is preferably DNA, and amplification preferably involves the DNA-dependent DNA polymerase activity of a modified Pol III replicase described herein.

[00135] In one embodiment, nucleic acid amplification results in the incorporation of nucleotides into a DNA molecule or primer, thereby forming a new DNA molecule complementary to a nucleic acid template. The formed DNA molecule and its template can be used as templates to synthesize additional DNA molecules. As used herein, one amplification reaction may consist of many rounds of DNA replication. DNA amplification reactions include, for example, polymerase chain reactions ("PCR"). One PCR reaction may consist of 10 to 100 "cycles" of denaturation and synthesis of a DNA molecule. Such methods include, but are not limited to, PCR (as described in U.S. Pat. Nos. 4,683,195 and 4,683,202, which are hereby incorporated by reference), Strand Displacement Amplification ("SDA") (as described in U.S. Pat. No. 5,455,166, which is hereby incorporated by reference), and Nucleic Acid Sequence-Based Amplification ("NASBA") (as described in U.S. Pat. No. 5,409,818, which is hereby incorporated by reference). For example, amplification may be achieved by a rolling circle replication system which may even use a helicase for enhanced efficiency in DNA melting with reduced heat (see Yuzhakou et al., Cell 86:877-886 (1996) and Mok et al., J. Biol. Chem. 262:16558-16565 (1987), which are hereby incorporated by reference).

[00136] In a preferred embodiment, the temperature at which denaturation is done in a thermocycling amplification reaction is between about 86°C-95°C, more preferably between 87°C-93°C, with temperatures at the lower end of the range being preferred for use in combination with thermocycling amplification reaction mixtures that include DNA destabilizers, as disclosed herein. Preferred thermocycling amplification methods include polymerase chain reactions involving from about 10 to about 100 cycles, more preferably from about 25 to about 50 cycles, and peak temperatures of from about 86°C-93°C, more preferably 87°C-93°C, with temperatures at the lower end of the range being preferred for use in combination with PCR reaction mixtures that include DNA destabilizers, as disclosed herein. In an especially preferred embodiment, the thermostable modified Pol III replicase comprises a dnaE α subunit, preferably of the genus Thermus or Aquifex, preferably of the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus.

[00137] In a preferred embodiment, the amplification reaction mixture used in an amplification reaction involving one or more high temperature denaturation steps further comprises stabilizers that contribute to the thermostability of the modified Pol III replicase, as described and exemplified more fully herein. [00138] In a preferred embodiment, an amplification mixture provided herein lacks SSB, wherein SSB, if present in the replication reaction mixture, would inhibit the DNA polymerase activity of the particular modified Pol III replicase used in the replication reaction mixture.

[00139] In a preferred embodiment, a PCR reaction is done using a modified Pol III replicase with appropriate stabilizers to produce, in exponential quantities relative to the number of reaction steps involved, at least one target nucleic acid sequence, given (a) that the ends of the target sequence are known in sufficient detail that oligonucleotide primers can be synthesized which will hybridize to them and (b) that a small amount of the target sequence is available to initiate the chain reaction. The product of the chain reaction will be a discrete nucleic acid duplex with termini corresponding to the ends of the specific primers employed.

[00140] Any source of nucleic acid, in purified or nonpurified form, can be utilized as the starting nucleic acid, if it contains or is thought to contain the target nucleic acid sequence desired. Thus, the process may employ, for example, DNA or RNA, including messenger RNA, which DNA or RNA may be single stranded or double stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. A mixture of any of these nucleic acids may also be employed, or the nucleic acids produced from a previous amplification reaction using the same or different primers may be so utilized. The nucleic acid amplified is preferably DNA. The target nucleic acid sequence to be amplified may be only a fraction of a larger molecule or can be present initially as a discrete molecule, so that the target sequence constitutes the entire nucleic acid. It is not necessary that the target sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as a portion of the β-globin gene contained in whole human DNA or a portion of nucleic acid sequence due to a particular microorganism which organism might constitute only a very minor fraction of a particular biological sample. The starting nucleic acid may contain more than one desired target nucleic acid sequence which may be the same or different. Therefore, the method is useful not only for producing large amounts of one target nucleic acid sequence, but also for amplifying simultaneously multiple target nucleic acid sequences located on the same or different nucleic acid molecules. [00141] The nucleic acid(s) may be obtained from any source and include plasmids and cloned DNA or RNA, as well as DNA or RNA from any source, including bacteria, yeast, viruses, and higher organisms such as plants or animals. DNA or RNA may be extracted from, for example, blood or other fluid, or tissue material such as corionic villi or amniotic cells by a variety of techniques such as that described by Maniatis et al., Molecular Cloning: A Laboratory Manual, (New York: Cold Spring Harbor Laboratory) pp 280-281 (1982).

[00142] Any specific (i.e., target) nucleic acid sequence can be produced by the present methods. It is only necessary that a sufficient number of bases at both ends of the target sequence be known in sufficient detail so that two oligonucleotide primers can be prepared which will hybridize to different strands of the desired sequence and at relative positions along the sequence such that an extension product synthesized from one primer, when it is separated from its template (complement), can serve as a template for extension of the other primer into a nucleic acid of defined length. The greater the knowledge about the bases at both ends of the sequence, the greater the specificity of the primers for the target nucleic acid sequence, and, thus, the greater the efficiency of the process. It will be understood that the word primer as used hereinafter may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the fragment to be amplified. For instance, in the case where a nucleic acid sequence is inferred from protein sequence information a collection of primers containing sequences representing all possible codon variations based on degeneracy of the genetic code can be used for each strand. One primer from this collection will be homologous with the end of the desired sequence to be amplified.

[00143] In some alternative embodiments, random primers, preferably hexamers, are used to amplify a template nucleic acid molecule. In such embodiments, the exact sequence amplified is not predetermined.

[00144] In addition, it will be appreciated by one of skill in the art that one-sided amplification using a single primer can be done. [00145] Oligonucleotide primers may be prepared using any suitable method, such as, for example, the phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment diethylophosphoramidites are used as starting materials and may be synthesized as described by Beaucage et al., Tetrahedron Letters, 22:1859-1862 (1981), which is hereby incorporated by reference. One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,006, which is hereby incorporated by reference. It is also possible to use a primer which has been isolated from a biological source (such as a restriction endonuclease digest).

[00146] Preferred primers have a length of from about 15-100, more preferably about 20-50, most preferably about 20-40 bases. Notably, preferred primers for use herein are longer than those preferred for Pol I polymerases.

[00147] The target nucleic acid sequence is amplified by using the nucleic acid containing that sequence as a template. If the nucleic acid contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as the template, either as a separate step or simultaneously with the synthesis of the primer extension products. This strand separation can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means. One physical method of separating the strands of the nucleic acid involves heating the nucleic acid until it is completely (>99%) denatured. Typical heat denaturation may involve temperatures ranging from about 80⁰C to 105⁰C, preferably about 9O⁰C to about 98°C, still more preferably 93°C to 94°C, for times ranging from about 1 to 10 minutes. Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or the enzyme RecA, which has helicase activity and is known to denature DNA. The reaction conditions suitable for separating the strands of nucleic acids with helicases are described by Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLIII "DNA: Replication and Recombination" (New York: Cold Spring Harbor Laboratory, 1978), and techniques for using RecA are reviewed in C. Radding, Ann. Rev. Genetics, 16:405-37 (1982), which is hereby incorporated by reference. Preferred helicases for use in the present invention are encoded by dnaB. [00148] If the original nucleic acid containing the sequence to be amplified is single stranded, its complement is synthesized by adding oligonucleotide primers thereto. If an appropriate single primer is added, a primer extension product is synthesized in the presence of the primer, a modified Pol III replicase, and the four nucleotides described below. The product will be partially complementary to the single-stranded nucleic acid and will hybridize with the nucleic acid strand to form a duplex of unequal length strands that may then be separated into single strands, as described above, to produce two single separated complementary strands.

[00149] If the original nucleic acid constitutes the sequence to be amplified, the primer extension product(s) produced will be completely complementary to the strands of the original nucleic acid and will hybridize therewith to form a duplex of equal length strands to be separated into single-stranded molecules.

[00150] When the complementary strands of the nucleic acid are separated, whether the nucleic acid was originally double or single stranded, the strands are ready to be used as a template for the synthesis of additional nucleic acid strands. This synthesis can be performed using any suitable method. Generally, it occurs in a buffered aqueous solution. In some preferred embodiments, the buffer pH is about 8.5 to 8.9, notably different from the preferred pH ranges of Pol I enzymes. Preferably, a molar excess (for cloned nucleic acid, usually about 1000:1 primeπtemplate, and for genomic nucleic acid, usually about 10⁶ :1 primeπtemplate) of the two oligonucleotide primers is added to the buffer containing the separated template strands. It is understood, however, that the amount of complementary strand may not be known if the process herein is used for diagnostic applications, so that the amount of primer relative to the amount of complementary strand cannot be determined with certainty. As a practical matter, however, the amount of primer added will generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process.

[00151] Nucleoside triphosphates, preferably dATP, dCTP, dGTP, dTTP, and/or dUTP are also added to the synthesis mixture in adequate amounts. [00152] The newly synthesized strand and its complementary nucleic acid strand form a double- stranded molecule which is used in the succeeding steps of the process. In the next step, the strands of the double-stranded molecule are separated using any of the procedures described above to provide single-stranded molecules.

[00153] New nucleic acid is synthesized on the single-stranded molecules. Additional polymerase, nucleotides, and primers may be added if necessary for the reaction to proceed under the conditions described above. Again, the synthesis will be initiated at one end of the oligonucleotide primers and will proceed along the single strands of the template to produce additional nucleic acids.

[00154] The steps of strand separation and extension product synthesis can be repeated as often as needed to produce the desired quantity of the specific nucleic acid sequence. The amount of the specific nucleic acid sequence produced will increase in an exponential fashion.

[00155] When it is desired to produce more than one specific nucleic acid sequence from the first nucleic acid or mixture of nucleic acids, the appropriate number of different oligonucleotide primers are utilized. For example, if two different specific nucleic acid sequences are to be produced, four primers are utilized. Two of the primers are specific for one of the specific nucleic acid sequences and the other two primers are specific for the second specific nucleic acid sequence. In this manner, each of the two different specific sequences can be produced exponentially by the present process. Of course in instances where terminal sequences of different template nucleic acid sequences are the same, primer sequences will be identical to each other and complementary to the template terminal sequences.

[00156] Additionally, as mentioned above, in an alternative embodiment, random primers, preferably hexamers, are used to amplify a template nucleic acid molecule.

[00157] Additionally, one-sided amplification using a single primer may be done. [00158] The present invention can be performed in a step-wise fashion where after each step new reagents are added, or simultaneously, wherein all reagents are added at the initial step, or partially step-wise and partially simultaneously, wherein fresh reagent is added after a given number of steps. Additional materials may be added as necessary, for example, stabilizers. After the appropriate length of time has passed to produce the desired amount of the specific nucleic acid sequence, the reaction may be halted by inactivating the enzymes in any known manner or separating the components of the reaction.

[00159] Thus, in amplifying a nucleic acid molecule according to the present invention, the nucleic acid molecule is contacted with a composition preferably comprising a thermostable modified Pol III replicase in an appropriate amplification reaction mixture, preferably with stabilizers.

[00160] In one embodiment, the invention provides methods of amplifying large nucleic acid molecules, by a technique commonly referred to as "long range PCR" (Barnes, W. M., Proc. Natl. Acad. Sci. USA, 91:2216-2220 (1994) ("Barnes"); Cheng, S. et. al., Proc. Natl. Acad. Sci. USA, 91:5695-5699 (1994), which are hereby incorporated by reference). In one method, useful for amplifying nucleic acid molecules larger than about 5-6 kilobases, the composition with which the target nucleic acid molecule is contacted comprises not only a modified Pol III replicase, but also comprises a low concentration of a second DNA polymerase (preferably thermostable repair type polymerase, or a polC α subunit) that exhibits 3'-5' exonuclease activity ("exo+" polymerases), at concentrations of about 0.0002-200 units per milliliter, preferably about 0.002-100 units/mL, more preferably about 0.002-20 units/mL, even more preferably about 0.002-2.0 units/mL, and most preferably at concentrations of about 0.40 units/mL Preferred exo+polymerases for use in the present methods are Thermotoga maritima PoIC, Pfu/DEEPVENT or Tli/NENT™ (Barnes; U.S. Pat. No. 5,436,149, which are hereby incorporated by reference); thermostable polymerases from Thermotoga species such as Tma Pol I (U.S. Pat. No. 5,512,462, which is hereby incorporated by reference); and certain thermostable polymerases and mutants thereof isolated from Thermotoga neapolitana such as Tne(3'exo+). The PoIC product of Thermus thermophilus is also preferred. By using a modified Pol III replicase in combination with a second polymerase in the present methods, DNA sequences of at least 35-100 kilobases in length may be amplified to high concentrations with significantly improved fidelity.

[00161] For a discussion of long range PCR, see for example, Davies et al., Methods MoI Biol. 2002; 187:51-5, expressly incorporated herein by reference.

[00162] Preferably, the amplification methods of the invention include the use of stabilizers with two- modified Pol III replicase. The stabilizers are preferably included in amplification reaction mixtures and increase the thermostability of the modified Pol III replicase in these reaction mixtures.

[00163] Amplification reaction mixtures of the present invention may include up to 25% co-solvent (total for all co-solvents added to a reaction mix), up to 5% crowding agent (total for all crowding agents added to a reaction mix) and up to 2M oxide (total for all oxides added to a reaction mix).

[00164] In an especially preferred embodiment, an amplification reaction mixture for use with a modified Pol III replicase derived from Aquifex aeolicus comprises TAPS-Tris (2OmM, pH8.7), 25mM K₂SO₄, 1OmM NH₄(OAc)₂, 15μmol ZnSO₄, and 4mM MgSO₄.

[00165] In another especially preferred embodiment, an amplification reaction mixture for use with a modified Pol II! replicase derived from Thermus thermophilus comprises HEPES-Bis-Tris-Propane (2OmM, pH7.5), O.δμmol ZnCI₂ or Zn(OAc)₂, and 6mM Mg(OAc)₂.

[00166] In one embodiment, wherein one or more high temperature denaturation steps is done at less than 89⁰C, a thermocycling amplification method involves the use of a helicase in the thermocycling amplification reaction mixture, and preferably a helicase encoded by a bacterial dnaB gene. Helicases are preferably not used in thermocycling amplification methods involving one or more denaturation steps at or above 89°C.

[00167] In one embodiment, a nucleic acid replication method herein involves the use of a nucleic acid replication mixture that lacks ATP. [00168] In one embodiment, a nucleic acid replication method herein involves the use of a nucleic acid replication mixture that lacks SSB, wherein SSB, if present in the replication reaction mixture, would inhibit the DNA polymerase activity of the particular minimal functional Pol III replicase used in the replication reaction mixture.

Stabilizers

[00169] Preferably, a combination of at least two and more preferably at least three stabilizers is included in a thermocycling amplification reaction mixture. In preferred embodiments, the stabilizers include at least one co-solvent, such as a polyol (e.g. glycerol, sorbitol, mannitol, maltitol), at least one crowding agent, such as polyethylene glycol (PEG), ficoll, polyvinyl alcohol or polypropylene glycol, and a third component selected from the group consisting of sugars, organic quaternary amines, such as betaines, and their N-oxides and detergents. In particularly preferred embodirfients, the stabilizers include a co-solvent, a crowding agent, and a quaternary amine N-oxide, such as trimethylamine-N- oxide (TMNO) or morpholino-N-oxide. In further preferred embodiments, the reaction mixture further comprises a fourth stabilizer, most preferably a second polyol. Other preferred four stabilizer combinations include three different co-solvents, and a quaternary amine N-oxide.

[00170] Nucleic acid replication reactions employing high temperature denaturation steps may benefit from the inclusion of one or more stabilizers in the reaction mixture. Preferred stabilizers in accordance with the present invention include co-solvents such as polyols and crowding agents such as polyethylene glycols, typically with one or more oxides, sugars, detergents, betaines and/or salts. Combinations of the foregoing components are most preferred.

[00171] As used herein, "crowding polymeric agent" or "crowding agent" refers to macromolecules that at least in part mimic protein aggregation. Illustrative crowding agents for use in the present invention include polyethylene glycol (PEG), PVP, Ficol, and propylene glycol.

[00172] As used herein, "detergent" refers to any substance that lowers the surface tension of water and includes, but is not limited to, anionic, cationic, nonionic, and zwitterionic detergents. Illustrative detergents for use in the present invention include Tween 20, NP-40 and Zwittergent 3-10. [00173] As used herein, "polyol" refers to a polyhydric alcohols, i.e., alcohols containing three or more hydroxy! groups. Those having three hydroxyl groups (trihydric) are glycerols; those with more than three are called sugar alcohols, with general formula CH₂OH(CHOH)_nCH₂OH, where n may be from 2 to 5.

Table 2: Stabilizer Agents

Table 3: Preferred Stabilizer Combinations

Table 3 continued: Preferred Stabilizer Combinations

I Preferred Preferred | Preferred I Preferred I Preferred \

[00174] Embodiments of the present invention generally include combining at least two and more preferably at least three different stabilizers selected from Groups I-VII (see Table 2) together to facilitate temperature-based nucleic acid amplification. Preferred embodiments of the present invention include a combination of at least one member from Group Il with a member from Group III within the amplification reaction mixture, particularly where the member from Group Il is glycerol and/or sorbitol. Particularly preferred combinations include two different members of Group Il combined with one member from Group III and one member from Group VII.

Nucleic Acid Sequencing

[00175] In one aspect, the invention provides methods for sequencing a nucleic acid, preferably DNA, comprising subjecting the nucleic acid to a sequencing reaction in a sequencing reaction mixture comprising a modified Pol III replicase.

[00176] Preferably the modified Pol III replicases used lack 3'-5' exonuclease activity capable of removing 3' terminal dideoxynucleotides in the sequencing reaction mixture.

[00177] Accordingly, modified Pol III replicases comprising a polC encoded α subunit are generally not preferred for use in sequencing reactions, owing to their high level of zinc-independent 3'-5' exonuclease activity.

[00178] In a preferred embodiment, the modified Pol III replicase comprises a dnaE α subunit, preferably of the genus Thermus or Aquifex, preferably of the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus.

[00179] Notably, the 3'-5' exonuclease activity of dnaE α subunits used in the invention is generally capable of removing 3' terminal dideoxynucleotides, while the 3'-5' exonuclease activity of ε subunits is generally incapable of such terminal dideoxy nucleotide removal. Accordingly, modified Pol III replicases having 3'-5' exonuclease activity which is conferred by an ε subunit in a sequencing reaction mixture are generally useful in sequencing reactions herein. Moreover, undesirable dnaE α subunit 3'-5' exonuclease activity is preferably reduced or completely inhibited through chemical means (i.e., buffer conditions, more particularly, Zn²⁺ concentration and pH).

[00180] Nucleic acid molecules may be sequenced according to any of the literature-described manual or automated sequencing methods. Such methods include, but are not limited to, dideoxy sequencing methods ("Sanger sequencing"; Sanger, F., et al., J. MoI. Biol., 94:444-448 (1975); Sanger, F., et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977); U.S. Pat. Nos. 4,962,022 and 5,498,523, which are hereby incorporated by reference), as well as by PCR based methods and more complex PCR-based nucleic acid fingerprinting techniques such as Random Amplified Polymorphic DNA ("RAPD") analysis (Williams, J. G. K., et al., Nucl. Acids Res., 18(22):6531-6535, (1990), which is hereby incorporated by reference), Arbitrarily Primed PCR ("AP-PCR") (Welsh, J., et al., Nucl. Acids Res., 18(24):7213-7218, (1990), which is hereby incorporated by reference), DNA Amplification Fingerprinting ("DAF") (Caetano-Anolles et al., Bio/Technology, 9:553-557, (1991), which is hereby incorporated by reference), microsatellite PCR or Directed Amplification of Minisatellite-region DNA ("DAMD") (Heath, D.D., et al., Nucl. Acids Res., 21(24): 5782-5785, (1993), which is hereby incorporated by reference), and Amplification Fragment Length Polymorphism ("AFLP") analysis (Vos, P., et al., Nucl. Acids Res., 23(21):4407-4414 (1995); Lin, J. J., et al., FOCUS, 17(2):66-70, (1995), which are hereby incorporated by reference).

[00181] Once the nucleic acid molecule to be sequenced is contacted with the modified Pol III replicase in a sequencing reaction mixture, the sequencing reactions may proceed according to protocols disclosed above or others known in the art.

[00182] In an especially preferred embodiment, a sequencing reaction mixture for use with a modified Pol III replicase derived from Aquifex aeolicus comprises TAPS-Tris (2OmM, pH8.7), 25mM K₂SO₄, 1OmM NH₄(OAc)₂, and 1OmM MgSO₄. Preferably, the reaction mixture lacks zinc so as to limit the 3'- 5' exonuclease activity of the α subunit. [00183] In another especially preferred embodiment, a sequencing reaction mixture for use with a modified Pol III replicase derived from Thermus thermophilus comprises HEPES-Bis-Tris-Propane (2OmM, pH7.5), and 1OmM Mg(OAc)₂. Preferably, the reaction mixture lacks zinc so as to limit the 3'- 5' exonuclease activity of the α subunit.

Kits

[00184] In one aspect, the invention provides kits for nucleic acid replication utilizing a minimal Pol III disclosed herein. The kits comprise a modified Pol III replicase disclosed herein.

[00185] In a preferred embodiment, a nucleic acid amplification kit includes buffers and stabilizers, or buffers with stabilizers as described herein. Stabilizers are especially preferred in kits for thermocycling reactions using a thermostable modified PpI III replicase.

[00186] A nucleic acid sequencing kit according to the present invention comprises modified Pol III replicase and preferably dideoxynucleotide triphosphates. The sequencing kit may further comprise additional reagents and compounds necessary for carrying out standard nucleic sequencing protocols, such as pyrophosphatase, agarose or polyacrylamide media for formulating sequencing gels, and other components necessary for detection of sequenced nucleic acids (See U.S. Pat. Nos. 4,962,020 and 5,498,523, which are directed to methods of DNA sequencing).

[00187] A nucleic acid amplification kit preferably comprises a modified Pol III replicase and dNTPs. The amplification kit may further comprise additional reagents and compounds necessary for carrying out standard nucleic acid amplification protocols (See U.S. Pat. Nos.4,683,195 and 4,683,202, directed to methods of DNA amplification by PCR; incorporated herein by reference).

[00188] In one embodiment, a kit lacks ATP and ATP is not used in the nucleic acid replication reaction provided for by the kit.

[00189] In additional preferred embodiments, the nucleic acid replication kits of the invention may further comprise a second DNA polymerase having 3-5' exonuclease activity. Preferred are Pfu/DEEPVENT, TliNENT™, Tma, Tne(3'exo+), and mutants and derivatives thereof. Also preferred is PoIC.

[00190] Kits of the present invention may include information pamphlets.

Vectors and Host Cells

[00191] The present invention provides vectors containing the polynucleotide molecules of the invention, as well as host cells transformed with such vectors. Any of the polynucleotide molecules of the invention can be contained in a vector, which generally includes a selectable marker and an origin of replication. The vectors further include suitable transcriptional and/or translational regulatory sequences, such as those derived from microbial or viral molecules. Examples of such regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences. A promoter nucleotide sequence is operably linked to an encoding DNA sequence if the promoter nucleotide sequence directs the transcription of the encoding sequence.

[00192] Selection of suitable vectors for the cloning of a subunit molecules encoding the target a polypeptides of the invention will depend upon the host cell in which the vector will be transformed, and, where applicable, the host cell from which the target polypeptide is to be expressed. Suitable host cells have been discussed above, but include prokaryotes, yeast, and other like organisms. Specific examples include bacteria of the genera Escherichia, Bacillus and Salmonella, as well as members of the genera Pseudomonas, Streptomyces, and Staphylococcus; yeast from the genera Sacchoromyces, Pichia, and Kluveromyces.

[00193] The modified a subunits of the present invention may be recombinantly joined sequences encoding heterologous proteins or peptides, to generate fusion protein constructs. Such heterologous proteins or peptides may be included to allow for example, enhanced purification, increased secretion, or increased stability. For example, a nucleic acid sequence encoding a signal peptide (secretory leader) may be fused in-frame to the modified α subunit sequence so that the modified α subunit is translated as a fusion protein comprising the signal peptide. [00194] Modification of a modified α-subunit encoding polynucleotide molecule of the invention to facilitate insertion into a particular vector, ease of use in a particular expression system or host (for example, by modifying restriction sites), and the like, are known and are contemplated for use in the invention. Genetic engineering methods for the production of modified α-subunit polypeptides include the expression of the polynucleotide molecules in cell free expression systems, in cellular systems, in host cells, in tissues, and in animal models.

Antibodies

[00195] The novel polypeptides of the present invention, and segments thereof, may be used to raise polyclonal and monoclonal antibodies. Preferably, a peptide containing a modified β-binding site is used in preparation of an antibody. Methods for the design and production of antibodies are known in the art, see for example, Antibodies: A Laboratory Manual, Harlow and Land (eds.), 1988 Cold Spring Harbor Laboratory Press, Cold Spring Harbor , New York; Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analysis, Kennet et al (eds), 1980 Plenum Press, New York.

[00196] References cited herein are expressly incorporated herein in their entirety by reference.

EXPERIMENTAL

Example 1: PCR Comparison - Mutated vs. Non-mutated Two Component Pol III Systems

[00197] The following example is provided to illustrate the utility of amplifying target nucleic acid using a two component Tth DNA Pol III system, comprising a β subunit in combination with an α subunit having at least one amino acid mutation within one or more of its internal β-binding domains, as disclosed in Figure 4. The experiment compares a two component Tth DNA Pol III system having a mutated α subunit against a two component Tth DNA Pol III system having a non-mutated α subunit. The reaction mixtures comprise 20 mM Hepes-Bis-Tris Propane pH 7.5, 200 μM dNTPs, 200 nM human beta globin gene primer mix, 50 ng human genomic DNA template, at least one stabilizer combination illustrated in table 1 and 1, 1.25, 1.5 and 1.75 ug β subunit for the modified two . component assemblies and 1.6, 2.0, 2.4, 2.8 ug β subunit for the non-modofied two component DNA Pol III assemblies, respectively, As Figure 8 shows all reactions utilizing the modified two component polymerase yield more specific PCR product, even though the amount of the modified Tth Pol III polymerase per reaction is 1.6 fold lower than the amount of the unmodified Tth Pol III polymerase. This is indicates a better processivity and specific activity of the modified two component Tth Pol III polymerase.

Example 2: PCR Comparison - Mutated vs. Non-mutated Pol III Holoenzyme Systems

[00198] The following example is provided to illustrate an adverse amplification yield when an α subunit having a mutation in its internal β-binding site, as disclosed herein, is added to the TtH DNA Pol III Holoenzyme system, in comparison to the Tth DNA Pol III Holoenzyme system with a non- mutated α subunit. The reaction mixtures comprise 20 mM Hepes-Bis-Tris Propane pH 7.5, 200 μM dNTPs, 200 nM Uni-ori primer mix, 1 ng PBS plasmid DNA template, at least one stabilizer combination illustrated in Tables 2 and 3, 2-5 mg/mL β subunit, 0.1-0.3 mg/mL δ subunit, 0.1-0.3 mg/mL δ' subunit, and 0.2-0.5 mg/mL DNAX complex (Y subunit + T subunit) . In both the mutated and non-mutated TtH DNA Pol III Holoenzyme systems, the corresponding α subunit has a reaction mixture concentration of 2-5 mg/mL. A final reaction volume of 50 μl_ is obtained. The reaction is exposed to a plurality of temperature cycles ranging from 95°C to 4°C.

[00199] A higher amplified product yield is obtained for the Pol III Holoenzyme system comprising the non-mutated α subunit.

Example 3: Extension Rate Comparison - Mutated vs. Non-mutated Two Component Pol III Systems

[00200] The extension rates of two two-component TtH DNA Pol III systems are compared in a replication assay using primed single-stranded M13mp18 DNA (7.2 kb) as a template. The first two component system comprises an α subunit with a mutation in its internal β-binding site, as disclosed herein; the second has a non-mutated α subunit. In this assay, the time point at which each system completes the replication of the 7.2 kb long single-stranded template DNA is measured. The template size in bases divided by the reaction time in seconds required to complete replication provides the extension rate of the Two Component TtH DNA Pol III systems. The reaction mixtures comprise reaction buffer, dNTPs, primed M13 DNA template, and 2-5 mg/mL Tth β subunit. Each reaction mix comprises 2-5 mg/mL of corresponding α subunit.

[00201] A faster extension rate is exhibited by the two component TtH DNA Pol III system comprising a mutated α subunit.

Example 4:Two Component Pol III Amplification Comparison (Synthetic a vs. Mutated Synthetic α)

[00202] Purified non-mutated, native Tth DNA polymerase III alpha subunit (Tth nDnaE) was compared to purified mutated Tth DNA polymerase III alpha subunit (Tth muDnaE) in combination with purified Tth DNA polymerase III beta subunit. In this series of experiments, the concentration of the beta subunit (β) was held constant at 815 ng per reaction while two concentrations (250 ng/ reaction and 125 ng/reaction) of the two alpha subunits (α) were tested. Two targets, 3kb pBS2 and 5 kb lambda, were amplified using temperature cycling amplification protocols. The reaction mixtures and cycling conditions were set up as follows: [00203] pBS2 Reactions

[00204] 5kb Lambda Reactions

[00205] As can be seen in Figures 5 and 6, the mutated synthetic alpha/ beta combination yielded more amplification product for both the 3kb pBS2 and 5 kb lambda targets than did the synthetic alpha/beta combination.

Example 5: Two Component Pol III Extension Rate Comparison (Synthetic α vs. Mutated Synthetic α)

[00206] In this experiment, the comparison between the synthetic alpha and mutated synthetic alpha within the two component Pol III system was made. The two alphas were tested for their ability to generate the 5 kb lambda amplicon when combined with beta, in all cases, the beta to alpha protein concentration ratio was 4:1. The reactions and cycling conditions were set up as follows:

[00207] The reaction mixtures were cycled with extension times of either 40 seconds or 70 seconds. As can be seen from Figure 7, both the synthetic alpha / beta and mutated synthetic alpha/ beta combination yielded the full 5kb amplicon at 70 seconds extension time. However, only the mutated synthetic alpha/ beta combination yielded the 5 kb amplicon at 40 seconds extension time.

[00208] All citations herein are incorporated herein in their entirety by reference.

Claims

[00209] We claim:

1. A modified α subunit, comprising one or more mutations in the internal β-binding site sequence of a polC encoded α subunit from a gram positive bacterium.

2. The modified α subunit according to claim 1, further comprising one or more mutations in the carboxy-terminus β-binding site of said polC encoded α subunit.

3. A modified DNA Pol III replicase, comprising a first component, said first component comprising a modified α subunit.

4. The modified DNA Pol III replicase according to claim 3, wherein said modified α subunit is a modified dnaE encoded α subunit.

5. The modified DNA Pol III replicase according to claim 4, wherein said modified dnaE encoded α subunit is from the genus Thermus or Aquifex.

6. The modified DNA Pol III replicase according to claim 5, wherein said modified dnaE encoded α subunit is from the species Thermus thermophilus, Thermus aquaticus, or Aquifex aeolicus.

7. The modified DNA Pol III replicase according to claim 3, wherein said modified α subunit is a modified polC encoded α subunit.

8. The modified DNA Pol III replicase according to claim 7, wherein said modified polC encoded α subunit is from the genus Thermotoga, Hydrogenobacter or Carboxydothermus.

9. The modified Pol III replicase according to claim 8, wherein said modified polC encoded α subunit is from the species Thermotoga maritima, Thermotoga neapolitana, or Carboxydothermus hydrogenoformans.

10. A method for replicating a nucleic acid molecule, comprising subjecting a nucleic acid molecule to a replication reaction in a replication reaction mixture, which replication reaction mixture comprises a modified DNA Pol III replicase according to claim 3, wherein said nucleic acid molecule is replicated in said replication reaction in said replication reaction mixture by said modified DNA Pol III replicase.

11. A nucleic acid replication reaction mixture, comprising a modified DNA Pol III replicase according to claim 3.

12. A nucleic acid replication kit useful for nucleic acid replication, comprising a modified DNA Pol III replicase according to claim 3.

13. A nucleic acid replication reaction tube, comprising a modified DNA Pol III replicase according to claim 3.

14. A nucleic acid replication reaction mixture, comprising (i) a template DNA molecule, (ii) optionally a primer hybridizable to said template DNA molecule, (Hi) a modified Pol III replicase according to claim 3, and (iv) at least one complete copy of said template DNA molecule, wherein said complete copy of said template DNA molecule is the product of a replication reaction using said modified Pol III replicase.