WO2007143436A2 - Nouvelle adn polymérase issue de spirochaeta thermophila - Google Patents

Nouvelle adn polymérase issue de spirochaeta thermophila Download PDF

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WO2007143436A2
WO2007143436A2 PCT/US2007/069837 US2007069837W WO2007143436A2 WO 2007143436 A2 WO2007143436 A2 WO 2007143436A2 US 2007069837 W US2007069837 W US 2007069837W WO 2007143436 A2 WO2007143436 A2 WO 2007143436A2
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seq
dna
dna polymerase
polymerase
enzyme
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PCT/US2007/069837
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WO2007143436A3 (fr
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Guolu Hu
Elena Garnova
Haiguang Xiao
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Ge Healthcare Bio-Sciences Corp.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present invention relates to novel DNA polymerases obtainable from the thermophilic organism Spirochaeta thermophila, to certain deletions and mutants of this enzyme, to genes and vectors encoding the wild type and mutant polymerases and their use in strand displacement activity, polymerase chain reaction, DNA sequencing and as reverse transcriptases.
  • DNA polymerases are a family of enzymes involved in DNA repair and replication.
  • DNA polymerases have been isolated from E.coli (e.g. E.coli DNA polymerase I and the Klenow fragment thereof) and T4 DNA polymerase and more recently thermostable DNA polymerases have been isolated (e.g. from T. aquaticus, U.S. Patent 4,889,818, and from T. litoralis).
  • Thermostable DNA polymerases have been suggested (U.S. Patent 4,683,195) for use in amplifying existing nucleic acid sequences in amounts that are large compared to that originally present.
  • the polymerase chain reaction (PCR) and strand displacement amplification (SDA) are two methods of amplifying nucleic acid sequences.
  • PCR is based on the hybridization of oligonucleotide primers to specific sequences on opposite strands of the target DNA molecule, and subsequent extension of these primers with a DNA polymerase to generate two new strands of DNA which themselves can serve as a template for a further round of hybridization and extension.
  • the product of one cycle serves as the template for the next cycle such that at each repeat of the cycle the amount of the specific sequence present in the reaction doubles leading to an exponential amplification process.
  • PCR relies on a process of temperature cycling to promote the amplification reaction. This temperature cycling is necessary to separate the strands of DNA formed in one cycle of the reaction to allow hybridization of the oligonucleotides required to initiate the next cycle. DNA strand separation is usually achieved by melting the DNA at temperatures in the range 90-100 0 C followed by cooling to a lower temperature to allow oligonucleotide hybridization followed by extension or ligation, depending on the reaction process. This cycling process may be repeated 20-50 times, again depending on the process and the degree of amplification required.
  • thermocyclers which can operate by a wide variety of mechanical, electrical or hydraulic means, but serve a common purpose in heating and cooling a small container or a number of such containers in which the amplification reaction is performed.
  • thermocyclers In order for the amplification reactions to proceed with the desired efficiency and specificity it is necessary to perform the temperature cycling process within strictly defined and reproducible limits of temperature and time. Failure of the temperature cycling apparatus to achieve the required conditions will result in the partial or total failure of the amplification reaction. These strict requirements on time and temperature of cycling can impose severe restrictions if the handling of large numbers of reactions is required. If it is desired to perform several hundred or more reactions simultaneously use of conventional thermocyclers would be extremely expensive in terms of the capital investment required in equipment, and would in any case be prone to variations between individual thermocyclers.
  • RNA primer In reverse transcription/polymerase chain reaction (RT/PCR), a DNA primer is hybridized to a strand of the target RNA molecule, and subsequent extension of this primer with a reverse transcriptase generates a new strand of DNA, which can serve as a template for PCR.
  • Preparation of the DNA template is preferably carried out at an elevated temperature to avoid early termination of the reverse transcriptase reaction caused by RNA secondary structure. There is a lack of efficient reverse transcriptases that act at elevated temperatures, e.g. above 50 0 C.
  • SDA differs from PCR in being an isothermal amplification process, i.e. all reactions occur at the same temperature without the need for elevated temperature to melt DNA strands. This is made possible by adoption of a reaction scheme which uses the ability of certain DNA polymerases when extending along a DNA template strand to displace any DNA molecules already hybridized to the template. In SDA this strand displacement is used to separate the double stranded DNA produced during the reaction process and hence to maintain continuous amplification of the target DNA sequence (Walker, G. T., Little, M.C., Nadeau, J.G. and Shank D.D. (1992) Proc. Natl Acad. Sci. USA 89:392-396).
  • SDA is therefore in principle more suited to use with large numbers of samples than PCR as the isothermal process, which is performed at temperatures of 37°C to 60 0 C, does not require stringent precautions to be taken to avoid evaporation and can be performed with simple temperature control equipment, for example in a standard laboratory incubator.
  • DNA polymerases e.g. Sequenase, Klenow, Taq, etc, have also been extensively used in DNA sequencing, see for example "Molecular Cloning: A Laboratory Manual” (Sambrook, Fritsch, and Maniatis, 2nd edition, Cold Spring Harbor laboratory Press, 1989).
  • the present invention provides a DNA polymerase from Spirochaeta thermophila. This enzyme is useful for procedures requiring strand-displacing DNA synthesis such as SDA, for DNA sequencing, reverse transcription and polymerase chain reaction. Included within the scope of the present invention are various mutants (deletion and substitution) that retain the ability to replicate DNA with substantially the same efficiency as the native Spirochaeta thermophila polymerase.
  • Fig. 1 is the DNA sequence from Spirochaeta thermophila encoding a full length novel DNA polymerase (SEQ ID NO:1).
  • Fig. 2 is the amino acid sequence encoding the full length DNA polymerase from Spirochaeta thermophila (SEQ ID NO:2). Translation is of the open reading frame spanning SEQ ID NO:1 as shown in Figure 1, encoding a native polymerase.
  • Figure 3 is a DNA sequence encoding the DNA polymerase from Spirochaeta thermophila, containing Y74C, F73 IY and E745R mutations (SEQ ID NO:3).
  • Figure 4 is the amino acid sequence of the DNA polymerase from Spirochaeta thermophila, containing Y74C, F731Y and E745R mutations (SEQ ID NO:4).
  • Figure 5 is DNA sequence encoding a truncated version of a DNA polymerase from Spirochaeta thermophila, containing F731Y and E745R mutations (SEQ ID NO:5).
  • Figure 6 is the amino acid sequence of the truncated version of DNA polymerase from Spirochaeta thermophila, containing F731Y and E745R mutations (SEQ ID NO:6).
  • Figure 7 is DNA sequence encoding a preferred, truncated version of a DNA polymerase from Spirochaeta thermophila, containing F731Y and E745R mutations (SEQ ID NO:7).
  • Figure 8 is an alignment of several DNA polymerase protein sequences. From the top, Spirochaeta thermophila (SEQ ID NO:2), NCBI AARl 1879 (SEQ ID NO:8), Pseudomonas aeruginosa UCBPP-PA14 (SEQ ID NO:9), Pseudomonas aeruginosa PAOl (SEQ ID NO: 10), Methylococcus capsulatus str.
  • the present invention provides a DNA polymerase or fragment thereof having the DNA polymerase activity of Spirochaeta thermophila and having at least 80% amino acid homology, preferably at least 90% homology, most preferably at least 96% homology to at least a contiguous 40 amino acid sequence shown in Fig. 2 (SEQ ID NO:2).
  • Fig. 2 represents the translation of the open reading frame of DNA sequence encoding a DNA polymerase from Spirochaeta thermophila (Fig. 1) (SEQ ID NO:1) potentially encoding the native polymerase.
  • amino acid homology means the amino acid identity of the parent enzyme or conservative amino acid changes thereto.
  • the DNA polymerase can be encoded by a full-length nucleic acid sequence or any portion of the full-length nucleic acid sequence, so long as a functional activity of the polypeptide is retained.
  • the amino acid sequence will be substantially similar to the sequence shown in Fig. 2, or fragments thereof.
  • a sequence that is substantially similar will preferably have at least 80% identity (more preferably at least 90% and most preferably 98-100%) to the sequence of Fig. 2.
  • identity is meant a property of sequences that measures their similarity or relationship. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.
  • the enzyme of the present invention has a molecular weight of approximately
  • a DNA polymerase or fragment thereof having the DNA polymerase activity of Spirochaeta thermophila means a DNA polymerase or fragment thereof (as hereinafter defined) which has the ability to replicate DNA with substantially the same efficiency as the enzyme encoded by the SEQ ID NO: 1.
  • substantially the same efficiency is meant at least 80% and preferably at least 90% of the efficiency of the enzyme encoded by SEQ ID NO:1 to incorporate deoxynucleotides.
  • the invention also encompasses a stable enzyme composition which comprises a purified DNA polymerase from Spirochaeta thermophila in a buffer.
  • the DNA polymerases of the present invention are preferably in a purified form.
  • the DNA polymerase is isolated from a majority of host cell proteins normally associated with it; preferably the polymerase is at least 10% (w/w), e.g. at least 50% (w/w) , of the protein of a preparation, even more preferably it is provided as a homogeneous preparation, e.g. homogeneous solution.
  • the DNA polymerase is a single polypeptide on an SDS polyacrylamide gel. Buffers around neutral pH (5-9) such as 5-100 rnM TrisHC 1 , phosphate or MES are suitable for use in the current invention.
  • the present invention also provides a gene encoding a polymerase of the present invention.
  • Fig. 1 represents the DNA sequence of a full length native polymerase from Spirochaeta thertnophila (SEQ ID NO:1).
  • the entire amino acid sequence of the polymerase is not required for enzymatic activity.
  • the exonuclease domain of the enzyme has been deleted to give an enzyme of molecular weight of approximately 70,000 daltons which retains enzyme activity.
  • This exonuclease-free enzyme is analogous to the Klenow fragment of E. coli DNA polymerase I.
  • the present invention also provides fragments of the polymerase which retain the DNA polymerase activity of Spirochaeta thermophila but have one or more amino acids deleted, preferably from the amino-terminus, while still having at least 80% amino acid homology to at least a 40 contiguous amino acid sequence shown in Fig. 2 (SEQ ID NO: 2) .
  • the present invention provides a DNA polymerase which corresponds to the DNA polymerase from Spirochaeta thermophila in which up to one third of the amino acid sequence at the amino-terminus has been deleted.
  • a DNA polymerase which corresponds to the DNA polymerase from Spirochaeta thermophila in which up to one third of the amino acid sequence at the amino-terminus has been deleted.
  • fragments of Spirochaeta thermophila having N-terminal deletions to give 630 amino acids (See Fig. 6) (SEQ ID NO:6) have been found to retain enzyme activity. It is preferred that the 5'-3' exonuclease activity of the DNA polymerase is removed or reduced. This may be achieved by deleting the amino acid region of the enzyme responsible for this activity, e.g. by deleting up to one third of the amino acid sequence at the amino terminus (Fig.
  • the enzyme may have conservative amino acid changes compared with the native enzyme which do not significantly influence enzyme activity. Such changes include substitution of like charged amino acids for one another or amino acids with small side chains for other small side chains, e. g. ala for val. More drastic changes may be introduced at non-critical regions where little or no effect on polymerase activity is observed by such a change.
  • the modification of the dNTP binding site for the dNTP substrate in DNA polymerase obtainable from Spirochaeta thermophila by the inclusion of a polar, hydroxyl containing amino acid residue at a position near the binding site increases the efficiency of the polymerase to incorporate a dideoxynucleotide.
  • the polar, hydroxyl containing amino acid is tyrosine. It has also been found that replacing the phenylalanine at the position corresponding to 731 of the native enzyme with tyrosine improves the incorporation of dideoxynucleotides when the enzyme is used for sequencing.
  • a polymerase from Spirochaeta thermophila in which the exonuclease activity has been deleted e.g. by point mutation or deletion and which has the phenylalanine at the position corresponding to 731 of the native enzyme replaced by an amino acid which increases the efficiency of the enzyme to incorporate dideoxynucleotides at least 20 fold compared to the wild type enzyme, e.g. tyrosine, is a particularly preferred enzyme for use in sequencing.
  • this modified enzyme has from between 592 and 634 amino acids, for example 612 to 634 amino acids and preferably 630 amino acids.
  • the DNA polymerases of the present invention can be constructed using standard techniques familiar to those who practice the art.
  • mutagenic PCR primers can be designed to incorporate the desired Phe to Tyr amino acid change (FY mutation in one primer). Deletion of the exonuclease function is carried out by PCR to remove the amino terminus, or standard techniques of site directed mutagenesis to generate point mutations.
  • Improved expression of the DNA polymerases of the present invention can be achieved by introducing silent codon changes (i.e., the amino acid encoded is not changed). Such changes can be introduced by the use of mutagenic PCR primers.
  • Silent codon changes such as the following increase protein production in E. coli: substitution of the codon GAG for GAA; substitution of the codon AGG, AGA, CGG or CGA for CGT or CGC; substitution of the codon CTT, CTC, CTA, TTG or TTA for CTG; substitution of the codon ATA for ATT or ATC; substitution of the codon GGG or GGA for GGT or GGC.
  • Figure 7 provides a nucleic acid sequence encoding the N-terminal truncated Spirochaeta thermophila DNA polymerase enzyme, with F731Y, E745R mutations, that incorporates such silent codon substitutions.
  • the sequence of this clone was generated based on analysis performed using a software program: E. coli codon usage analysis 2.0 (by Morris Madura).
  • the DNA polymerases of the present invention are suitably used in SDA, preferably in combination with a thermostable restriction enzyme. Accordingly, the present invention provides a composition which comprises a DNA polymerase of the present invention in combination with a thermostable restriction enzyme, for example BsoBI from Bacillus stearothermophilus.
  • a thermostable restriction enzyme for example BsoBI from Bacillus stearothermophilus.
  • the invention also features a kit or solution for SDA comprising a DNA polymerase of the present invention in combination and a thermostable restriction enzyme.
  • the polymerases of the present invention are also useful in methods for generating and amplifying a nucleic acid fragment via a strand displacement amplification (SDA) mechanism.
  • the method generally comprises: a) specifically hybridizing a first primer 5' to a target nucleic acid sequence, the first primer containing a restriction enzyme recognition sequence 5' to a target binding region; b) extending the 3' ends of the hybridized material using a DNA polymerase of the present invention, preferably one in which the exonuclease activity has been removed, in the presence of three dNTPs and one dNTP ⁇ S; c) nicking at the hemiphosphorothioate recognition site with a restriction enzyme, preferably; d) extending the 3' end at the nick using a DNA polymerase of the present invention, displacing the downstream complement of the target strand; and e) repeating steps (c) and (d).
  • This SDA method proceeds at a linear amplification rate if one primer is used as above. However, if two primers are used which hybridize to each strand of a double-stranded DNA fragment, then the method proceeds exponentially (Walker, G. T., Little, M.C., Nadeau, J.G. and Shank D.D. (1992) Proc. Natl. Acad. Sci. USA 89:392-396).
  • the present invention also provides a method for determining the nucleotide base sequence of a DNA molecule.
  • the method includes providing a DNA molecule, annealing with a primer molecule able to hybridize to the DNA molecule; and incubating the annealed molecules in a vessel containing at least one, and preferably four deoxynucleotide triphosphate, and a DNA polymerase of the present invention preferably one containing the phenylalanine to tyrosine mutation. Also provided is at least one DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base. The method further includes separating the DNA products of the incubating reaction according to size, whereby at least a part of the nucleotide base sequence of the DNA molecule can be determined. In preferred embodiments, the sequencing is performed at a temperature between
  • the DNA polymerase has less than 1000, 250, 100, 50, 10 or even 2 units of exonuclease activity per mg of polymerase (measured by standard procedure, see below) and is able to utilize primers having only 4, 6 or 10 bases; and the concentration of all four deoxynucleoside triphosphates at the start of the incubating step is sufficient to allow DNA synthesis to continue until terminated by the agent, e.g. a ddNTP.
  • the agent e.g. a ddNTP.
  • more than 2, 5, 10 or even 100 fold excess of a dNTP is provided to the corresponding ddNTP.
  • the invention features a kit or solution for DNA sequencing including a DNA polymerase of the present invention and a reagent necessary for the sequencing such as dITP, deaza dGTP, a chain terminating agent such as a ddNTP, and optionally a pyrophosphatase.
  • a DNA polymerase of the present invention and a reagent necessary for the sequencing such as dITP, deaza dGTP, a chain terminating agent such as a ddNTP, and optionally a pyrophosphatase.
  • the DNA polymerases of the present invention containing the phenylalanine to tyrosine mutation are suitably used in sequencing, preferably in combination with a pyrophosphatase. Accordingly, the present invention provides a composition which comprises a DNA polymerase of the present invention containing the phyenylalanine to tyrosine mutation in combination with a pyrophosphatase, preferably a thermostable pyrophosphatase from Thermoplasma acidophilum.
  • the invention features a method for sequencing a strand of DNA essentially as described above with one or more (preferably 2, 3 or 4) deoxyribonucleoside triphosphates, a DNA polymerase of the present invention, and a first chain terminating agent.
  • the DNA polymerase causes the primer to be elongated to form a first series of first DNA products differing in the length of the elongated primer, each first DNA product having a chain terminating agent at its elongated end, and the number of molecules of each first DNA products being approximately the same for substantially all DNA products differing in length by no more than 20 bases.
  • the method also features providing a second chain terminating agent in the hybridized mixture at a concentration different from the first chain terminating agent, wherein the DNA polymerase causes production of a second series of second DNA products differing in length of the elongated primer, with each second DNA product having the second chain terminating agent at its elongated end.
  • the number of molecules of each second DNA product is approximately the same for substantially all second DNA products differing in length from each other by from 1 to 20 bases, and is distinctly different from the number of molecules of all the first DNA products having a length differing by no more than 20 bases from that of said second DNA products.
  • three or four such chain terminating agents can be used to make different products and the sequence reaction is provided with a magnesium ion, or even a manganese or iron ion (e. g. at a concentration between 0.05 and 100 mM, preferably between 1 and 10mM)i and the DNA products are separated according to molecular weight in four or less lanes of a gel.
  • a magnesium ion or even a manganese or iron ion (e. g. at a concentration between 0.05 and 100 mM, preferably between 1 and 10mM)i and the DNA products are separated according to molecular weight in four or less lanes of a gel.
  • the invention features a method for sequencing a nucleic acid by combining an oligonucleotide primer, a nucleic acid to be sequenced, between one and four deoxyribonucleoside triphosphates, a DNA polymerase of the present invention, and at least two chain terminating agents in different amounts, under conditions favoring extension of the oligonucleotide primer to form nucleic acid fragments complementary to the nucleic acid to be sequenced.
  • the method further includes separating the nucleic acid fragments by size and determining the nucleic acid sequence.
  • the agents are differentiated from each other by intensity of a label in the primer extension products.
  • the present invention provides a method for preparing complementary DNA by combining an oligonucleotide primer, a sample of RNA, a DNA polymerase of the present invention, and between one and four deoxyribonucleoside phosphates, under conditions favoring preparation of the complementary DNA.
  • the DNA polymerases of the present invention which act as reverse transcriptases lack appreciable, and preferably have no RNaseH activity and, as such, are useful in RT/PCR, the generation of hybridization probes and RNA sequencing.
  • the present invention provides a purified reverse transcriptase having a reverse transcriptase activity of greater than 1000 units per milligram.
  • the reverse transcriptase lacks RNaseH activity; the reverse transcriptase is from Spirochaeta thermophila; the reverse transcriptase has an N-terminal deletion or amino acid changes that remove the exonuclease function.
  • the invention features a method for reverse transcription/polymerase chain reaction (RT/PCR) which utilizes a DNA polymerase of the present invention and a DNA polymerase suitable for PCR in the same reaction vessel.
  • RT/PCR reverse transcription/polymerase chain reaction
  • the DNA polymerase of the present invention is from Spirochaeta thermophila
  • the polymerase has one or more amino acids deleted from the amino terminus or amino acid changes to remove the exonuclease activity
  • the DNA polymerase suitable for PCR is Taq DNA polymerase.
  • the present invention features a kit or solution for RT/PCR comprising a DNA polymerase of the present invention and a DNA polymerase suitable for PCR.
  • the DNA polymerase of the present invention is from Spirochaeta thermophila, the polymerase has one or more amino acids deleted from the amino terminus or amino acid changes to remove the exonuclease function, and the DNA polymerase suitable for PCR is Taq DNA polymerase.
  • the invention features polymerases that have exonuclease activity removed by replacing an aspartic acid residue at position 8 with an alanine residue. This is a preferred mutation for it not only removes the exonuclease activity but removes an RNaseH activity, which may effect reverse transcription.
  • the invention features a method for polymerase chain reaction in the presence of a polymerase stabilizing agent, utilizing an enzymatically active DNA polymerase having at least 80% identity in its amino acid sequence to the DNA polymerase of Spirochaeta thermophila and an exonuclease activity removed.
  • the polymerase stabilizing agents include, but are not limited to, glycerol (10 to 50% final concentration), trimethylamine-N-oxide (TMANO; up to 4 M final concentration), and N-methylmorpholine-N-oxide (MMO; up to 3 M final concentration).
  • TMANO trimethylamine-N-oxide
  • MMO N-methylmorpholine-N-oxide
  • glycerol is used at a final concentration of 30%.
  • polymerase stabilizing agent an agent which allows the use of the polymerase in PCR and RT/PCR. These agents reduce the denaturing temperature of the template and stabilize the polymerase. By stabilize is meant make temperature stable. By final concentration is meant the final concentration of the agent in the PCR or RT/PCR solution.
  • the DNA polymerases of the present invention can be used to carry out polymerase chain reaction (PCR) when the reaction is carried out in the presence of a polymerase stabilizing agent, such as glycerol, TMANO, or MMO, and the like.
  • a polymerase stabilizing agent such as glycerol, TMANO, or MMO, and the like.
  • the glycerol concentration is in the range of 10 to 50%, and most preferably it is at a final concentration of 30%.
  • TMANO and MMO can be used at concentrations up to 4 M and 3 M final concentration, respectively. These agents have been shown to stabilize the polymerase reaction.
  • the polymerases of the present invention are also suitable for reverse transcription/polymerase chain reaction (RT/PCR) under these conditions.
  • the invention features a method for reverse transcription/polymerase chain reaction, in the presence of a polymerase stabilizing agent, utilizing an enzymatically active DNA polymerase having at least 80% identity in its amino acid sequence to the DNA polymerase of Spirochaeta thermophila and an exonuclease activity removed.
  • the polymerase stabilizing agents include, but are not limited to, glycerol (10 to 50% final concentration), TMANO (up to 4 M final concentration), and MMO (up to 3 M final concentration).
  • glycerol is used at a final concentration of 30%.
  • Example 1 Cloning of native polymerase and generation of mutant enzymes
  • the Spirochaeta thermophila bacterial strain was obtained from ATCC culture collection (ATCC 700085) as a freeze-dried culture, and was dissolve in 50 ul of sterile water. Genomic DNA amplification was performed as described (GenomiPhiTM, GE Healthcare). The polymerase gene was then amplified by PCR and cloned into PKK vector at the EcoRI and HindIII Site. The primers were designed using the known genomic DNA sequence, with the introduction of restriction enzyme sites. The primer at N-terminus (with EcoRI site) is 5'- GCG CGA ATT CAT GAA ACC GCT CTT CCT CAT AGA TGC CTA CGG - 3' (SEQ ID NO: 16).
  • the primer at the C-terminus is 5' - GCG CAA GCT TGT CGA CCC CCT AGT GGG CCT CTC CCC AG - 3' (SEQ ID NO:17).
  • the PCR product was generated using Taq DNA polymerase and cloned into PKK vector.
  • the full length clone was sequenced to identify the DNA sequence (SEQ ID NO: 1) that encodes the full length DNA polymerase (SEQ ID NO:2).
  • SEQ ID NO: 1 that encodes the full length DNA polymerase
  • the full length enzyme clones were fermented using 2X LB medium. Overnight cultures of the plasmid-containing strains were inoculated into 2X LB medium. The polymerase expression was induced at 1.0 OD by adding ImM IPTG. The cells were harvested after growth for 4 hours at 37 0 C.
  • Cell broth was centrifugation to collect cell pellets.
  • the cell paste containing full length enzymes was resuspended in 4 volume lysis buffer (50 mM Tris-Cl, pH 7.5; 50 mM NaCl; 0.5 mM EDTA; 0.2% NP-40; 0.2% Tween 20, ImM PMSF) and normalized to OD. Lysis was carried out by heat at 75 0 C for 15 min, then lmg/ml lysozyme was added and the cell lysate was centrifuged at 6,000 rpm for 20 min. The clear supernatant was collected and analyzed on SDS-PAGE. A protein of expected size (about 100,000 daltons) can be detected.
  • the supernatant was loaded onto 5ml HiTrap Heparin Sepharose HP.
  • the column was washed with 10 column volumes with Buffer A (5OmM Tris pH 8.5; ImM EDTA; 5OmM NaCl).
  • Buffer A 5OmM Tris pH 8.5; ImM EDTA; 5OmM NaCl
  • the column was further washed with gradient: 0 - 60% Buffer B (5OmM Tris pH 8.3; ImM EDTA; IM NaCl) in 10 column volumes. Elution was continued by stepping up to 100% buffer B for 5 column volumes. 5ml fractions were collected.
  • Peak fractions from HiTrapQ were dialyzed overnight against Buffer A until conductivity is less than lOmS/cm. Sample was loaded onto 5ml HiTrap Q Sepharose HP, washed with 10 column volumes of Buffer A, then with gradient: 0 - 60% Buffer B in 10 column volumes. Elution was continued by stepping up to 100% buffer B for 5 column volumes. 5ml fractions were collected.
  • Fractions containing polymerase activity was pooled and dialyzed against final storage buffer (20 mM Tris-Cl, pH 8.5, 25 mM KCL, 0.1 mM EDTA, 50% glycerol, 0.5% NP-40, 0.5% Tween 20 and ImM DTT).
  • Thermostability of the enzymes is assayed at 95°C, as 50% activity at time determined by DAPI assay.
  • the enzyme is diluted to 20ng/ ⁇ l in enzyme dilution buffer (25mM Tris pH 8.0, 5OmM KCl, 0.5% (v/v) Tween 20, and 0.5% (v/v) NP-40).
  • a 20 ⁇ l reaction is set up with the following final composition (5OmM Tris pH 8.0, 5mM MgCl 2 , 50ng M13 DNA, 50 ⁇ M dNTPs and 20ng enzyme).
  • the reaction is incubated at 95 0 C for 0, 1, 2, 4, 6, 8, 10 and 12 minutes.
  • DAPI assay is performed by taking 5 ⁇ l of the 20 ⁇ l reaction, adding it to 195 ⁇ l
  • DAPI reaction buffer 8OmM Tris pH 9.0, 2.4mM MgCl 2 , 5 ⁇ M FAS42 template, 7.5 ⁇ M DAPI and 250 ⁇ M dNTPs. DAPI plate assay is run at 37°C, and the maximum slopes are plotted.
  • the sequencing premix composition is formulated with DYEnamic ET terminators and dITP/dA, T, C (2500 ⁇ M dITP, 500 ⁇ M dCTP, 500 ⁇ M dATP, and 500 ⁇ M dTTP).
  • the ratio of dNTP:ddNTP is 156.
  • the composition also contains 20 units of TS II enzyme mix or Spirochaeta thermophila polymerase per reaction.
  • composition and water are added to a total volume of 20 ⁇ l. Reaction mixtures are cycled through 95°C, 20 seconds; 50 0 C, 30 seconds; and 60 0 C, 60 seconds, repeated 30 times.
  • the samples are purified and analyzed according to manufacturer's instructions, and run on ABI 3100 capillary sequencing instrument (Applied Biosystems). The resulting electropherograms using TS II enzyme mix and Spirochaeta thermophila polymerase are compared.

Abstract

L'invention concerne une ADN polymérase issue de Spirochaeta thermophila. Cette enzyme se révèle utile pour des procédés qui requièrent une synthèse d'ADN impliquant le déplacement de brins, telle que l'amplification par déplacement de brin (SDA), pour un séquençage de l'ADN, et la réaction en chaîne par polymérase. La présente invention inclut également dans son champ de réalisation divers mutants (délétion et substitution) qui conservent la capacité de répliquer l'ADN sous la forme de la polymérase native de Spirochaeta thermophila.
PCT/US2007/069837 2006-05-30 2007-05-29 Nouvelle adn polymérase issue de spirochaeta thermophila WO2007143436A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104726418A (zh) * 2015-02-10 2015-06-24 江南大学 Spirochaeta thermophila生产耐热超氧化物歧化酶(SOD)的方法
WO2020013058A1 (fr) * 2018-07-13 2020-01-16 タカラバイオ株式会社 Mutant d'adn polymérase adapté à l'amplification d'acide nucléique à partir d'arn

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