WO2006030455A1 - Polymerase d'adn presentant une activite de deplacement de brins - Google Patents

Polymerase d'adn presentant une activite de deplacement de brins Download PDF

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WO2006030455A1
WO2006030455A1 PCT/IS2005/000022 IS2005000022W WO2006030455A1 WO 2006030455 A1 WO2006030455 A1 WO 2006030455A1 IS 2005000022 W IS2005000022 W IS 2005000022W WO 2006030455 A1 WO2006030455 A1 WO 2006030455A1
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polypeptide
dna
seq
sequence
activity
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PCT/IS2005/000022
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WO2006030455A8 (fr
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Sigridur Hjorleifsdottir
Sveinn Ernstson
Thorarinn Blondal
Arnthor Aevarsson
Gudmundur Oli Hreggvidsson
Jakob Kristjansson
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Prokaria Ehf.
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Priority to US11/662,879 priority Critical patent/US20080311626A1/en
Publication of WO2006030455A1 publication Critical patent/WO2006030455A1/fr
Publication of WO2006030455A8 publication Critical patent/WO2006030455A8/fr

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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

  • DNA polymerases The fundamental activities of DNA polymerases are affected by the properties of the enzyme which are greatly variable depending on the source of the enzyme. Characterized DNA polymerases thus display a wide spectrum in terms of their abilities and properties such as optimal working temperature, thermostability, processivity and fidelity (for a review see Brautigam and Steitz 1998b; Steitz 1999).
  • thermostable enzymes foremost thermostable DNA polymerases, has revolutionized the field of recombinant DNA technology and such enzymes are of great importance in the research industry today.
  • DNA polymerases are being used for a variety of biological applications including sequencing and amplification of nucleic acids such as by the polymerase chain reaction (PCR) requiring thermal cycling or through isothermal amplification.
  • PCR polymerase chain reaction
  • a large number of DNA polymerases have been identified and described and shown to have varying suitability for different applications.
  • Many DNA polymerases have also been modified in different ways such as through truncations and site-directed mutagenesis to alter their properties including alterations to abolish basic activities such as the 3'-5' exonuclease activity.
  • DNA polymerases have been described from a number of Thermus species including DNA polymerase I from Thermus aquaticus (Taq DNA polymerase) which is widely used in PCR amplification due to the thermostability of the enzyme (Saiki et al. 1988).
  • Genotyping techniques are for example used for determination and screening of single nucleotide polymorphism (SNP) requiring a certain amount of genomic DNA from different individuals.
  • SNP single nucleotide polymorphism
  • the amount of available DNA is often limited.
  • Whole genome amplification is thus becoming essential for generating sufficient amount of DNA for analysis and to renew genetic material from an original sample.
  • thermocycling protocols For example, the strand displacement methods generally produce larger fragments with higher yield and less sequence bias than PCR-based methods.
  • the PCR-based methods utilizing thermocycling protocols typically give product of short length with incomplete coverage and biased representation of the genome by favoring amplification of certain regions in the genome (Lasken and Egholm 2003; Paez et al. 2004; Lü et al. 2003).
  • MDA Multiple Displacement Amplification offers several advantages. Large amounts of material can be produced even from very small amounts of starting material.
  • the material produced consists of relatively long DNA products, averaging 12 kb, with unbiased coverage of the starting material as mentioned above.
  • MDA can be carried out using crude samples, such as clinical samples consisting of cell or blood lysates. Yields of DNA can be independent of the amount of starting material and thus avoids the need for determination the concentration of DNA and adjustment of the concentration prior to subsequent analysis.
  • MDA offers the possibility of alternative and simplified sampling of genetic material as less material is needed to start with and this can for example simplify the collection of samples from human patients in clinical settings. Furthermore, MDA lends itself relatively easily to automation (Lasken and Egholm 2003).
  • Phi29 DNA polymerase has very tight binding to the DNA substrate giving very high processivity and ability to generate very long DNA products up to more than 100 kb.
  • the essential feature of the enzyme is the ability to synthesize a new DNA strand and at the same time displace previously made DNA strands from the template strand. This is thought to proceed through a mechanism producing hyperbranched product from the starting material as DNA strands are being displaced and becoming new starting points for synthesis of new strands.
  • Phi29 DNA polymerase originates from a mesophilic bacteriophage and the enzyme is normally used at about 3O 0 C in an isothermal reaction. Therefore, avoiding thermocycling and the ability of phi29 DNA polymerase to synthesize through difficult regions in the template material results in even representation of the starting genetic material (Dean et al. 2001, Blanco and Salas 1996, Blanco et al. 1989).
  • the underlying ability for amplification without thermal cycling is based on strand-displacement properties of these polymerases where assumingly the DNA polymerase is able to displace annealed non-template strand and synthesize a new strand whereas conventional DNA polymerases such as Thermus aquaticus DNA polymerase would normally be hindered by the presence of a non-template strand annealed to the template strand.
  • Phi29 DNA polymerase is apparently not a very efficient enzyme compared to conventional DNA polymerase such as Taq DNA polymerase in terms of speed and thus the yield of material produced after a certain time.
  • thermostable DNA polymerases which preferably have DNA strand displacement activity and can be used in a rapid and efficient strand displacement amplification reactions.
  • the DNA polymerases provided by the invention are much more efficient and have other distinctive advantageous properties such as the ability to work at higher temperatures. Enzymes of the type provided by the invention may proof to be valuable tools in various applications in recombinant DNA technology and other molecular biology procedures.
  • the present invention relates to isolated polypeptides having strand-displacement
  • DNA polymerase activity and active derivatives or fragments thereof i.e. fragments and derivatives retaining the DNA polymerase activity of the parent polypeptide from which they are derived
  • the invention encompasses the polypeptides having the amino acid sequences shown as SEQ ID IMO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and polypeptides having strand displacement DNA polymerase activity with substantially similar amino acid sequences to said sequences as well as active derivatives or fragments thereof.
  • the invention provides in one aspect an isolated thermostable polypeptide belonging to the DNA polymerase A family as further defined herein and in more detail in herein referenced articles, which polypeptide is encoded by a gene sequence obtainable from a Thermus sp. and has a non-truncated molecular weight in the range of about 58-68 kDa (kiloDaltons), such as in the range of about 61-65 kDa, including about 61, 62, 63, 64, or 65 kDa.
  • the polypeptides preferably have DNA polymerase strand-diplacement activity..
  • the invention relates to isolated thermostable polypeptides having strand displacement DNA polymerase activity, which are obtainable from strains identified as Thermus antranikianii (strain 2120) and Thermus brockianus (strain 140). Also provided is an isolated polypeptide encoded by a gene isolated from a complex environmental biomass sample. Isolated polypeptides provided by the invention can replace DNA polymerases, such as Phi29 DNA polymerase, in applications that utilize strand displacement activities of a DNA polymerase, in particular in applications that require and/or benefit from elevated temperatures (above about 5O 0 C). The polypeptides of the present invention may also be used in other applications, in particular applications that require elevated temperatures (above about 50 0 C).
  • the invention pertains to methods using DNA polymerases of the invention for DNA synthesis by addition of deoxynucleotides to the 3 'end of a polynucleotide chain, using a complementary nucleic acid strand as a template and displacement intervening strands of nucleic acids hybridized to the template strand.
  • the invention thus pertains to amplification of genetic material such as amplification of genomic DNA.
  • kits for practicing the subject methods are also provided by the invention.
  • the subject methods will be discussed first in greater detail followed by a description of the kits for practicing the subject methods.
  • thermostable polypeptide having DNA polymerase strand displacement activity of the present invention is suitably selected from the group consisting of: a thermostable polypeptides DNA polymerase strand displacement activity obtained from a Thermus species; a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; a polypeptide encoded by a nucleic acid comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; a polypeptide having at least 40% sequence identity with the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; or an active fragment or derivative thereof.
  • thermostable polypeptides having DNA polymerase strand displacement activity described herein have advantageous properties in comparison to prior art strand displacement DNA polymerases, such as very efficient strand displacement activity combined with thermostability and proof-reading activity.
  • the methods of the invention are performed at temperatures in the range of about 5O 0 C up to about 95°C.
  • Figure 1 shows a phylogenic tree of the amino acid sequences of DNA polymerase PoI-H (SEQ ID NO: 4), DNA polymerase Pol-3 (SEQ ID NO: 5), and DNA polymerase Pol-62 (SEQ ID NO: 6), together with selected prior art DNA polymerase I sequences. All the sequences are of DNA polymerases of family A except phi29 DNA polymerase which belongs to family B and is used here as an outgroup.
  • accession numbers of the public sequences are as follows: Thermus fla vus P30313; Thermus filiformis 052225.; Thermus thermophilus P52028.; Thermus aquaticus P19821; Geobacillus stearothermophilus AAB62092; Thermotoga maritima NP229419; Thermomicrobium roseum AAO85272; Desulfitobacterium hafniense ZP_00097788 ; Aquifex pyrophilus AAO15360; Aquifex aeolicus NP_214348; Bacteriophage phi29 X53370.
  • Figure 3 shows activity of PoI-Il and DyNAzyme (DZ) as a function of temperature.
  • Figure 4 shows activity of PoI-Il and DyNAzyme (DZ) as % incorporation of labelled nucleotides (3H-dTTP) over a time period of 120 minutes, measured at 55°C.
  • Figure 5 shows activity of PoI-Il and Pol-3 at 55 0 C for different time periods.
  • DyNAzyme (DZ) is a control sample.
  • Total CPM was 11032.
  • Figure 9 shows the relative activity of PoI-Il DNA polymerase at different temperatures with and without 0.5 M L-Proline.
  • Figure 10 shows heat inactivation (thermostability) of PoI-Il with and without L-Proline as stabilization agent. After 15 min incubation at 94°C the Proline reaction mixture had between 2-3 fold activity (assayed at 55 0 C) compared to the untreated mixture.
  • Figure 11 shows the effects of doubling the amount of template DNA and labelled nucleotide (dNTP) in the reaction mixture for PoI-Il and DyNAzyme (DZ). Total CPM was 15.798 for lx3H and 33.625 for 2x 3H.
  • Figure 13 illustrates purification of PoI-Il on HiTrap Chelatin HP column. Lanes 8,9 and 10 show final fractions. Lanes contain 1: ladder, 2: pol-3 (10 min 65°C), 3: poi-11 (10 min 65 0 C), 4: AlO, 5: A12, 6: B2, 7: B5, 8: Cl, 9: C6, 10: Dl.
  • Figure 18 Amplification from hexamer amplified human genomic DNA. PCR results from human DNA template amplified with pol 11. Marker gene: Beta-actin. 1 ul of 20 ul reactions in Figure 17 used as template in lanes 1 - 8. Lanes 1 - 8 same reactions as in Figure 17. Lanes 9 - 12 and 14 - 17 are PCR reactions that received untreated human DNA as template, 5; 2.5; 1.25; 0.6; 1; 0.5; 0.25 and 0.125 ng respectively. Lane 13 contains a size marker (1 kb ladder from New England Biolabs).
  • Figure 19 shows PCR products from amplified human genomic DNA.
  • Figure 20 The activity for Pol-11 on activated DNA using 0.06 micrograms protein over time with 0.1 mg/ml DNA (diamonds) or 0.6mg/ml DNA (squares).
  • Figure 21 Activity of 0.02 microgram and 0.1 micrograms of Pol-11 and Phi29 DNA polymerases respectively, Specific activity after 10 minutes corresponds to about 360.000 units per mg for Pol-11 and 10.800 units per mg for Phi 29 DNA polymerase. Y-axis shows percent of total incorporation.
  • Figure 22 shows the amino acid sequence alignment of selected DNA polymerase sequences.
  • Taq is DNA polymerase I from Thermus aquaticus (accession number ITAQ)
  • Bst is DNA polymerase from Bacillus stearothermophilus (accession number 2BDP_A)
  • Eco is DNA polymerase I from Echerichia coli (accession number P00582)
  • Aea is DNA polymerase I from Aquifex aeolicus (accession number O67779)
  • PoI-Il is SEQ ID NO: 4
  • Pol-3 is SEQ ID NO: 5
  • Pol-62 is SEQ ID NO: 6.
  • sequence motifs in the 3'-5 exonuclease domain are indicated (Exo I, Exo ii and Exo III) as well as sequence motifs in the polymerase domain (Motif A, Motif B and Motif C).
  • sequence alignment was created using automatic alignment with program ClustalX (ref) followed by some manual adjustments, mainly in the exonuclease domain, using additional information of described sequence motifs and structure information.
  • the sequences of PoI- 11, Pol-3 and Pol-62 are most similar to the Aquifex sequence although the similarity is limited.
  • the Aquifex sequence has similar sequence identity to all three sequences of the invention, for example 33% with respect to the PoI-Il sequence, calculated as percentage of identical matches between the two sequences over the aligned region including any gaps in the length.
  • nucleic acid encompasses the terms “oligonucleotide” and “polynucleotide” and means single-stranded or double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA) and ribonucleotides (RNA).
  • the nucleic acid can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof, linked by intemucleotide phosphodiester bond linkages, and associated counter-ions, e.g., H + , NH 4 + , trialkylammonium, Mg 2+ , Na + and the like.
  • the nucleic acid may also be a peptide nucleic acid (PNA) formed by conjugating bases to an amino acid backbone.
  • PNA peptide nucleic acid
  • isolated means that the material is removed from its original environment (e. g. the natural environment where the material is naturally occurring).
  • a polynucleotide or polypeptide while present in a living source organism is not isolated, but the same polynucleotide or polypeptide, which is separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could for example be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that the vector or composition is not part of the natural environment.
  • isolated refers to a preparation of the polypeptide outside its natural source and preferably substantially free of contaminants.
  • Thermostable is defined herein as having the ability to withstand high temperatures such as above about 60°C for at least 30 minutes while retaining substantial enzymatic activity, at preferred temperatures above 50 0 C, such as between 5O 0 C and 100 0 C, at preferred temperatures of about 5O 0 C to about 75°C and at even more preferred temperatures of about 55°C to about 70 0 C.
  • Thermophilic bacteria also referred to as “thermophiles”, are defined as bacteria having optimum growth temperature above 5O 0 C.
  • Thermophilic bacteriophages or “thermostable bacteriophages” are defined as bacteriophages having thermophilic bacteria as hosts.
  • Thermophilic isolate refers to a bacterial isolate which has been isolated from a high temperature environment and grown and maintained in a laboratory as a pure culture.
  • RNA can be replicated, for example, by RNA directed RNA polymerase, or by reverse transcribing the RNA and then performing a PCR. In the latter case, the amplified copy of the RNA is a DNA with the correlating or homologous sequence.
  • PCR polymerase chain reaction
  • PCR involves repeatedly performing a "cycle” of three steps: 1) “melting”, in which the temperature is adjusted such that the DNA dissociates to single strands, 2) “annealing”, in which the temperature is adjusted such that oligonucleotide primers are permitted to anneal to their complementary nucleotide sequence to form a duplex at one end of the polynucleotide segment to be amplified; and 3) "extension” or “synthesis”, which can occur at the same or slightly higher and more optimum temperature than annealing, and during which oligonucleotides that have formed a duplex are elongated with a thermostable DNA polymerase.
  • the cycle is then repeated until the desired amount of amplified polynucleotide is obtained.
  • Bacterial cells normally carry several DNA polymerases, including DNA polymerases
  • DNA polymerase I from several Thermus species, foremost Thermus aquaticus ⁇ Taq) DNA polymerase I, has been of paramount importance for recombinant DNA technology including the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • DNA polymerase I and consists of a catalytic domain and a 3 '-5 ' proofreading exonuclease domain but is lacking a 5'-3' exonuclease domain.
  • the DNA polymerases of the invention were surprisingly discovered from Thermus species which previously have been extensively studied, including as a source of DNA polymerases. Since the first description of the genus Thermus (Brock and Freeze, 1969) seven other species have been validly described (Oshima and Imahori, 1974; Hudson et al., 1987), (Kristjansson et al., 1994; Williams et al., 1995; Williams et al., 1996; Chung et a)., 2000). The most widely used DNA polymerase to date, Taq DNA polymerase I, is from Thermus aquaticus and similar DNA polymerases from other Thermus species have been characterized and are available commercially.
  • Our results reveal not only one sequence of the DNA polymerases of the present invention but three non-identical but closely related sequences that can be used to define this new protein family through analysis of structural features and phylogenetic relationships to other known DNA polymerases.
  • Certain structural features of the DNA polymerases of the invention set them apart from other known DNA polymerase originating from Thermus strains as well as from all other known DNA polymerases.
  • the DNA polymerases of the present invention belong to family A DNA polymerases as can be seen through sequence comparisons to public database sequences such as using BLAST algorithm.
  • thermophilus strains but only in certain other strains including strains for the species T. scotoductus, T. brockianus, T. oshimai and T. atranikianii.
  • the recently published genomic sequence of Thermus thermophilus HB27 does not contain a gene for a similar DNA polymerase. This fact, together with the unexpectedly high similarity of the obtained DNA polymerase sequences, suggest the presence of DNA polymerases encoded by mobile extra-chromosomal genetic elements with uneven distribution among Thermus strains in nature.
  • Meiothermus strains were used as well as environmental complex biomass samples as described in Example 1. Degenerate PCR methods were used to amplify gene fragments corresponding to polymerase genes as described in Example 2. A number of strains gave amplified gene fragments corresponding to a novel type of DNA polymerase distantly related to Taq DNA polymerase I and other known corresponding polymerases of that type in other Thermus species. Surprisingly, the novel DNA polymerases disclosed here were found to be apparently closest related to Aquifex and Desulfitobacterium DNA polymerases when compared to known DNA polymerases described in the prior art. Still, the similarity to these DNA polymerase is limited (seq.
  • gene fragments of the invention may be obtainable from bacterial strains other than strains belonging to the genus Thermus and may even be of non-bacterial origin such as from bacteriophage genomes.
  • PoI-Il a clone expressing the gene designated as PoI-Il (pAt>17b) expressed in pJOE3075 vector without His-tail and in E.coli cells BL21-RIL. Later, another PoI-Il clone (pAt>18b with vector pJOE3075 in E. coli BL21RIL without DE3) was used as source of the enzyme carrying a His-tail.
  • the gene for PoI-Il originated from T. antranikianii strain 2120. Later in the comparisons another clone with a different gene was used as well.
  • Pol-3 This was designated Pol-3 and was cloned and expressed in p3OE3075 vector with His-tail and in E.coli BL21+DE3.
  • the gene for Pol-3 originated from T. brockianus strain 140.
  • the nucleic acid sequences of the selected DNA polymerase genes are shown as SEQ ID NO: 1 (PoI-Il), SEQ ID NO: 2 (Pol-3) and SEQ ID NO: 3 (Pol-62).
  • Example 6 describes how complete genes of the new type of DNA polymerases were cloned into expression vectors, the genes expressed and the corresponding clones tested for activity.
  • the DNA polymerase PoI-Il polypeptide was chosen as a suitable candidate for the detailed characterization of the type of enzymes disclosed by the invention.
  • Example 7 describes experiments aimed at finding optimal reaction temperature and pH as well as the effects of varying the concentration of some salts.
  • Polymerase PoI-Il had a temperature optimum around 50-55 0 C and a pH optimum pH of approximately 8,5. Heat stability and activity at different temperatures of polymerase PoI-Il was studied as described in Example 8 and the effects of varying the template DNA and nucleotides were observed as described in Example 9.
  • DNA polymerase PoI-Il was expressed and purified (Example 10) for further characterization. PoI-Il polymerase was found to have substantial activity at temperatures up to 90 0 C.
  • the invention thus pertains to DNA polymerases having strand displacement activity at elevated temperatures such as above 5O 0 C, such as up to 100 0 C, such as between 50 and 8O 0 C.
  • the resistance of the polypeptides to heat inactivation may permit their use in applications employing elevated temperatures including denaturing conditions such as in the use of PCR.
  • the invention pertains also to the use of these polypeptides with stabilizing agents such as L-proline.
  • the DNA material used for amplification of the gene was obtained from an environmental sample containing heterogeneous genetic material from the plurality of microbial species found in the ecosystem at the sampling site.
  • the gene obtained from the complex biomass sample may therefore have originated from an organism not belonging to the genus Thermus.
  • the biomass gene product was designated Pol-62 and was found to be very active with strand displacing activity similar to PoI-H and Pol-3 (data not shown).
  • DNA polymerases of the type disclosed by the invention can be obtained from environmental DNA without prior isolation of microbial strains such as Thermus strains.
  • PoI-Il can be successfully used to amplify genomic DNA from minute amounts of starting material, such as human genomic DNA, as demonstrated in Example 17.
  • Genomic material amplified by PoI-Il such as human DNA, can be used for specific amplification of a genomic marker such as a gene.
  • Example 18 demonstrates the amplification of the human B-actin gene from human genomic DNA after whole genome amplification using PoI-Il. It is possible to clone by PCR a normal Beta-actin gene from material amplified by PoI-Il containing less original template than applicable for PCR. Another experiment was done (Example 19) to verify amplification of human DNA from starting material in amounts less than sufficient for normal PCR amplification using specific primers.
  • Vent DNA pol is a commercial archaeal polymerase
  • the Bst DNA polymerase is lacking a functional 3' exonuclease domain (Aliotta et al. 1996) which in contrast is functional in the polypeptides of the invention.
  • This is a very distinctive difference and the consequent lack of proof reading activity in Bst DNA polymerase is a disadvantage for its general use in amplification reaction due to high error rate. This may include for example single base pair errors but due to the nature of the strand displacement reaction, there seems to be also a great risk of other errors in absence of proof reading activity such as chimer formation due to unspecific priming events.
  • Phi29 DNA polymerase seems to be the most commonly used DNA polymerase for strand displacement applications and is considered to be the best suited enzyme for these applications (Technical reference sheet, New England Biolabs), i.e. Phi29 DNA polymerase is considered the current industry standard.
  • the polypeptides of the invention are however distinctively different from Phi29 DNA polymerase, they belong to a different family and are thermostable with optimal activity at temperatures above about 5O 0 C whereas Phi29 DNA polymerase has optimal activity around 30 0 C. The range of applications which can be employed is therefore different for the different types of enzymes.
  • the fingers regions of the polymerase domain have been shown to undergo conformation changes related to binding and hydrolysis of incoming nucleoside triphosphate and thus play important part in fidelity of synthesis as well as translocation of the polymerase along the template and displacement of downstream non-template nucleic acid strand.
  • a correct Watson-Crick basepair between template and incoming nucleotide at the polymerase active site will facilitate the conformational change of the fingers domain, which is essential for catalysis of the reaction, whereas a non-Watson-Crick basepair will hinder the conformational change thereby stalling the reaction.
  • the residue in the polypeptides of the invention which is closest to the first base pair of the downstream duplex, is not a phenylalanine residue by rather a negatively charged glutamate residue (Glu406).
  • This particular amino acid residue could act repelling to the negatively charged sugar-phosphate backbone of the displaced strand and thus facilitate breaking of the hydrogen bonds of the base pair to be disrupted during each cycle. From the observations discussed above, it seems likeiy that a particular region in the polypeptides of the invention is important and plays a direct part in the displacement mechanism and may be crucial for the high efficiency of strand displacement seen in these enzymes.
  • this region refers to the regions of residues Glu406 to Leu422 in PoI-Il polypeptide and corresponding regions in Pol-3 and Pol-62 (see Figure 22), partly overlapping motif B in the fingers domain.
  • This region is well conserved in all three polypeptides and consists of the amino acid sequence 406EGLRRYALTAYGVKLTL422 in PoI- 11 and Pol-3 (one substitution in Pol-62 which has Pro at position 422).
  • this region of the sequence is partly forming an insert in a sequence alignment compared to Taq DNA polymerase, Bst DNA polymerase I and DNA polymerase I from E.
  • the nucleic acid molecules of the invention can be DNA, or can also be RNA, for example, mRJMA.
  • DNA molecules can be double-stranded or single-stranded; single stranded RNA or DNA can be the coding, or sense, strand or the non-coding, or antisense, strand.
  • the nucleic acid molecule comprises at least about 100 nucleotides, more preferably at least about 150 nucleotides, and even more preferably at least about 200 nucleotides.
  • the nucleic acid of the invention comprises a sequence which encodes at least a fragment of the amino acid sequence of a polypeptide of the invention; alternatively, the nucleotide sequence can include at least a fragment of a coding sequence along with additional non-coding sequences such as non-coding 3' and 5' sequences (including regulatory sequences, for example).
  • isolated nucleotide sequences include recombinant DNA molecules in heterologous organisms, as well as partially or substantially purified DNA molecules in solution.
  • isolated RNA transcripts of the DNA molecules of the present invention are also encompassed by "isolated" nucleotide sequences.
  • isolated nucleotide sequences are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences, for gene mapping or for detecting expression of the gene, such as by Northern blot analysis.
  • the present invention also pertains to nucleotide sequences which are not necessarily found in nature but which encode the polypeptides of the invention.
  • DNA molecules which comprise a sequence which is different from the naturally occurring nucleotide sequence but which, due to the degeneracy of the genetic code, encode polypeptides of the present invention are also subject of this invention.
  • the invention also encompasses variations of the nucleotide sequences of the invention, such as those encoding active fragments or active derivatives of the polypeptides as described below. Such variations can be naturally occurring, or non-naturally occurring, such as those induced by various mutagens and mutagenic processes.
  • fragments of the isolated nucleic acid molecules described herein also relates to fragments of the isolated nucleic acid molecules described herein.
  • fragment is intended to encompass a portion of a nucleotide sequence described herein which is from at least about 15 contiguous nucleotides to at least about 50 contiguous nucleotides or longer in length; such fragments are useful as probes and also as primers.
  • Particularly preferred primers and probes selectively hybridize to the nucleic acid molecule encoding the polypeptides described herein.
  • fragments which encode polypeptides that retain enzyme activity, as described below, are particularly useful.
  • nucleic acid molecules of the invention can include, for example, labeling, methylation, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), charged linkages (e.g., phosphorothioates, phosphorodithioates), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids). Also included are synthetic molecules that mimic nucleic acid molecules in the ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates
  • charged linkages e.g., phosphorothioates, phosphorodithioates
  • Such molecules include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule (polypeptide nucleic acids, as described in Nielsen, et a/., 1991)).
  • the invention also encompasses nucleic acid molecules which hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g., nucleic acid molecules which specifically hybridize to a nucleotide sequence encoding polypeptides described herein, and, optionally, have an activity of the polypeptide).
  • Hybridization probes are oligonucleotides which bind in a base- specific manner to a complementary strand of nucleic acid.
  • hybridization conditions By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize (e.g., selectively) with the most similar sequences in the sample can be determined. Exemplary conditions are described in Krause, M. H. and S.A. Aaronson, Methods in Enzymology, 200:546-556 (1991). Also, in, Ausubel, et al., " ' 'Current Protocols in Molecular Biology,” John Wiley & Sons (2001), which describes the determination of washing conditions for moderate or low stringency conditions. Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids.
  • each degree C by which the final wash temperature is reduced (holding SSC concentration constant) allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize.
  • doubling the concentration of SSC results in an increase in T m of 17°C.
  • the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought.
  • a low stringency wash can comprise washing in a solution containing 0.2XSSC/0.1% SDS for 10 minutes at room temperature;
  • a moderate stringency wash can comprise washing in a pre-warmed solution (42°C) solution containing 0.2XSSC/0.1% SDS for 15 min at 42°C;
  • a high stringency wash can comprise washing in prewarmed (68°C) solution containing 0.1 x SSC/0.1%SDS for 15 min at 68°C.
  • washes can be performed repeatedly or sequentially to obtain a desired result as known in the art.
  • Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used.
  • Hybridizable nucleic acid molecules are useful as probes and primers, e.g., for diagnostic applications. Such hybridizable nucleotide sequences are useful as probes and primers for diagnostic applications.
  • ⁇ primer refers to a single- stranded oligonucleotide which acts as a point of initiation of template-directed DNA synthesis under appropriate conditions (e.g., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature.
  • the appropriate length of a primer depends on the intended use of the primer, but typically ranges from 15 to 30 nucleotides.
  • Primer generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
  • a primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the 3' end of the sequence to be amplified.
  • the invention also pertains to nucleotide sequences which have a substantial identity with the nucleotide sequences described herein; particularly preferred are nucleotide sequences which have at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, even more preferably at least about 50%, yet more preferably at least about 70%, still more preferably at least about 80%, and even more preferably at least about 90% identity, and still more preferably 95% identity, with nucleotide sequences described herein. Particularly preferred in this instance are nucleotide sequences encoding polypeptides having DNA polymerase strand displacement activity as described herein.
  • the sequences are aligned for optimal comparison purposes ⁇ e.g., gaps can be introduced in the sequence of a first nucleotide sequence).
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity or similarity scores between two sequences can be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin, et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993).
  • BLAST programs e.g. BLASTN for nucleotide sequences or BLASTP for protein sequences
  • BLASTN for nucleotide sequences
  • BLASTP for protein sequences
  • Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res, 25:3389-3402 (1997).
  • the default parameters of the respective programs ⁇ e.g., BLASTN
  • W determines how many continuous nucleotides must be identical for the program to identify two sequences as containing regions of identity. Alignment of sequences and calculation of sequence identity may also be done using for example the Needleman and Wunsch global alignment algorithm (Needleman and Wunsch 1970) useful for both protein and DNA alignments and discussed further below.
  • the invention also provides expression vectors containing a nucleic acid sequence encoding a polypeptide described herein (or an active derivative or fragment thereof), operably linked to at least one regulatory sequence.
  • Many expression vectors are commercially available, and other suitable vectors can be readily prepared by the skilled artisan.
  • "Operably linked” is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleic acid sequence. Regulatory sequences are art-recognized and are selected to produce the polypeptide or active derivative or fragment thereof. Accordingly, the term “regulatory sequence” includes promoters, enhancers, and other expression control elements which are described in
  • the native regulatory sequences or regulatory sequences native to organism can be employed.
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of polypeptide desired to be expressed.
  • the polypeptides of the present invention can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in an appropriate host cell (see, for example, Broach, et al., Experimental Manipulation of Gene Expression, ed. M. Inouye (Academic Press, 1983) p. 83; Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. Sambrook et al. (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17).
  • expression constructs will contain one or more selectable markers, including, but not limited to, the gene that encodes dihydrofolate reductase and the genes that confer resistance to neomycin, tetracycline, ampicillin, chloramphenicol, kanamycin and streptomycin resistance.
  • selectable markers including, but not limited to, the gene that encodes dihydrofolate reductase and the genes that confer resistance to neomycin, tetracycline, ampicillin, chloramphenicol, kanamycin and streptomycin resistance.
  • prokaryotic and eukaryotic host cells transformed by the described expression vectors are also provided by this invention.
  • cells which can be transformed with the vectors of the present invention include, but are not limited to, bacterial cells such as Thermus scotoductus, Thermus thermophilus, E. coll ⁇ e.g., E.
  • the isolated nucleic acid molecules and vectors of the invention are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other species), as well as for detecting the presence of a DNA construct comprising a nucleic acid sequence of the invention in a culture of host cells.
  • This invention in addition to the isolated nucleic acid molecules encoding an DNA polymerases of the invention, disclosed in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, also pertains to substantially similar sequences.
  • Isolated nucleic acid sequences are substantially similar if: (i) they are capable of hybridizing under stringent conditions as described to any of the nucleic acids shown as SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 or (ii) they encode DNA sequences which are degenerate to any of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • SEQ ID NO: 1 is a particularly useful probe.
  • Other particular useful probes for this purpose are hybridizable fragments to the sequences of SEQ ID NO: 1 (i.e., comprising at least 15 contiguous nucleotides).
  • probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe.
  • Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. The probes are thus useful to isolate complementary copies of DNA from other animal sources or to screen such sources for related sequences.
  • polypeptides having DNA polymerase strand displacement activity are defined as polypeptides having DNA polymerase strand displacement activity which catalyze DNA synthesis by addition of deoxynucleotides to the 3 'end of a polynucleotide chain, using a complementary nucleic acid strand as a template and being able to displace an intervening strand of nucleic acid hybridized to the template strand.
  • Strand displacement thus refers to the dissociation of a nucleic acid strand from its nucleic acid template in a 5' to 3' direction due to template-directed nucleic acid synthesis by the DNA polymerase.
  • DNA polymerase strand displacement activity is suitable assayed by measuring the incorporation of labeled nucleotide such as described for example by Dean et al. (2002).
  • the present invention relates to isolated polypeptides having substantial DNA polymerase strand displacement activity at elevated temperatures, such as above 55°C, and active derivatives or fragments thereof.
  • the invention encompasses the polypeptides having the amino acid sequences shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 and polypeptides having strand displacement activity with substantially similar amino acid sequences to the sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or derivatives or fragments thereof.
  • the polymerases of the invention are more thermostable, i.e.
  • the polymerases of the invention retain a significant portion of their activity at higher temperatures such as temperatures above about 70 0 C or higher such as temperatures above about 75 0 C or 8O 0 C and preferably above about 9O 0 C, e.g. at temperatures in the range of about 55-95°C such as in the range of 75-95°C.
  • the polymerases of the invention retain at least 10% and more preferably at least 15% or at least 20% of their optimal activity at any of the above mentioned temperatures or temperature ranges, when assayed at such temperature.
  • the polymerase has at least about 10% of optimum activity when assayed at a temperature of 90 0 C.
  • the polymerases of the invention also have significant temperature stability, i.e.
  • the polymerase of the invention is a member of family A DNA polymerases as described further hereinabove and in great detail in Steitz T.A. (1999). Additionally, the polymerases of the invention preferably naturally lack a 5'-exonuclease domain, e.g. when isolated from natural sources or after cloning and overexpression of the polymerase of the invention from a suitable host cell.
  • Said sequence is in some embodiments N/Q-F-G-x-x-Y-G-x-x-x-D/E-x-x-R/K- R/K-Y-x-x-x-Y-G-x-K/R-I/L/V-S/T in said region, aligning to residues 396-421 of PoI-Il.
  • the polymerase of the present invention is an isolated polypeptide having DNA polymerase activity encoded by a gene obtained from bacteria of the genus Thermus, said full-length gene encoding a polypeptide having an estimated molecular weight of 61-65 kDa and belonging to family A DNA polymerases.
  • the DNA polymerases of the present invention contain family A DNA polymerase sequence motifs such as "motif A” in the polymerase domain or the "exo I” motif in the 3' exonuclease domain as seen in Figure 22.
  • the invention relates to isolated polypeptides having DNA polymerase activity and a polypeptide sequence such that when said sequence is included in alignment, together with the sequences of Figure 1 using the alignment algorithm in the program ClustalX, and with a subsequent contruction of phyiogenetic tree, using the Neighbour Joining method, the said sequence will belong to the same branch as the sequences of the present invention.
  • the invention provides a novel sub-family sequence-based phyiogenetic branch which is defined by a phyiogenetic tree being prepared as described above, wherein said branch corresponds to internal branch p stemming from node P in the phyiogenetic tree shown in Fig. 1
  • Rhodothermus species e.g. Rhodothermus marinus.
  • the special properties of the isolated polypeptides of the invention as compared to counterparts in the prior art are beneficial for use in various methods. It is an object of the invention to provide methods using the isolated polypeptides of the invention.
  • the methods include methods wherein the isolated polypeptides are used to catalyze DNA synthesis by addition of deoxynucleotides to the 3 1 end of a polynucleotide chain, using a complementary polynucleotide strands as a template.
  • genetic material such as genomic DNA, is amplified using the disclosed isolated polypeptides in a reaction wherein the genetic material is amplified through strand displacement reaction.
  • the GeneBank accession numbers of the polymerase sequences used had the following accession numbers Thermus aquaticus AAA27507, Thermus thermophilus P52028, Thermus flavus P30313, Thermus filiformis AAC46079, Aquifex aeoliticus NP214348. Some strains revealed a novel type of DNA polymerase, which did not show close relation to Taq DNA polymerase I. Some of the strains gave both the expected Taq like polymerase and also the novel type of polymerase gene showing closest sequence identify of 30-35% to Aquifex DNA polymerase (BLAST alignment).
  • PoI-Il does not loose all activity after incubation for 15 minutes up to 94°C. Residual activity is substantially higher after incubation in the presence of L-proline at temperatures above 8O 0 C.
  • Example 12 Incorporation of tritium dTTP into nicked ("activated") calf thymus DNA Incorporation of tritium dTTP by DNA polymerase pol 11 and Thermus eggertssonii
  • reaction components for several reactions were mixed (except enzyme) and dispensed (50 ul) into 1,5 ml Eppendorf tubes. Reactions were preheated at 55°C in a water bath. Enzyme solution was added to reaction(s) and incubated at 55 0 C for 0 - 90min. Three reactions were performed for each time point. Control reactions were without enzyme.
  • Example 13 Strand displacement of PoI-Il and primer requirement using a single strand template.
  • Reaction components were mixed except enzyme solution and heated to 94°C for 4 min. Reaction performed at 55°C overnight (14 hours).
  • a similar reaction was performed using Phi29 instead of PoI-Il.
  • Phi29 reaction a 1Ox buffer for Phi29 was used instead of 1Ox Teg buffer and the reaction was performed at 29°C overnight (14 hours).
  • Example 15 Exonuclease activity of PoI-Il Tritium incorporation by PoI-Il.
  • the enzyme was added last with 1 ul of enzyme diluted into 4 ul of H 2 O.
  • the amount of pol 11 polymerase was 1,14 ug/ul.
  • Samples (10 ul) were drawn from each reaction and dispensed on DE81 filters (Whatman). The filters were dried at 75° for 10' and washed twice in a 100 mM phosphate buffer (pH 7,5).
  • the filters were placed in scintillation vials containing 5 ml of scintillation fluid (Packard Ampligold) and measured in a scintillation counter. The measured incorporation is shown in Figure 16.
  • Example 16 Specific activity of PoI-Il Two experiments were done to measure specific activity of PoI-Il and compare its activity to Phi29 DNA polymerase.
  • Experiment A Specific activity determination of PoI-Il DNA polymerase using salmon sperm activated DNA.
  • Timer intervals were 0/5/10/15/30/60/120 minutes in 0.6 mg/ml DNA and 0/5/10/15/30/60/120/180/240/420 and 720 minutes in 0.1 mg/ml
  • Experiment B Comparison of the activities of PoI-Il and Phi29 DNA polymerases using activated DNA without primers.
  • the specific activity of PoI-Il in the two experiments corresponds to 360.000 units per mg protein, measured from the rate of incorporation during the first 10 minutes.
  • the specific activity of Phi29 DNA polymerase is 10.800 units per mg.
  • Example 17 Amplification using (thiol) hexamers.
  • Hexamers can be utilized to amplify human genomic DNA. Extension for 5 minutes for 5 cycles seems to generate quantities of material on par with longer (20 minute) extension cycles. This indicates that the PoI-Il enzyme works fast enough to allow shorter rather than longer extension times to generate long transcripts (in the form of high molecular weight DNA). Prolonged incubations with hexamers containing a thiol group backbone yield more high molecular weight material than identical incubations containing normal hexamers. The thiol based hexamers seem to be either more suitable for extension or less prone to exonuclease activity.
  • Example 18 Amplification of Beta-actin gene from human genomic DNA.
  • the template used in this experiment was human genomic DNA amplified using PoI-Il as described in Example 17.
  • Example 18 Amplification of human genomic DNA and PCR using specific primers.
  • Salmon genomic DNA was amplified using the following protocol.
  • Amplified DNA is genotyped correctly for the 5 markers.
  • the positive control sample (al-Ssalll) contains sufficient amount of DNA for successful genotyping.
  • the negative control sample (d4_5ngUnampl) using 5ng of starting DNA material without further amplification fails for all markers.
  • Quality control values (GQ) above 0.25 are considered to indicate reliable results. The results indicate that amounts of DNA, that are to limited for analysis of this type, can be amplified to sufficient amounts that can be successfully used for genotyping and assumingly is correctly amplified.
  • sequences of the polypeptides PoI-Il, PoI-3 and Pol-62 were aligned with various other DNA polymerase sequences from public databases using ClustalX software (Thompson et al. 1997). Representative sequences in family A together with sequences of the polypeptides of the invention were selected for final alignment as shown in Figure 22. Known sequence motifs of family A DNA polymerases were identified in the sequences by visual inspection.
  • Coordinates of selected crystal structures were analyzed with molecular graphics.
  • the structures were of Taq DNA polymerase (Protein data bank (PDB) ID: ITAQ), E.coli DNA polymerase Klenow fragment (PDB ID 1D8Y), Bacillus stearothermophilus DNA polymerase fragment (PDB ID 2BDP) and bacteriophage T7 RIMA polymerase in complex with nucleic acids (PDB ID IMSW).
  • the amino acid sequence alignment was partly based on manual adjustment based on the structural superposition of the coordinates of E. coli, Taq and Bst DNA polymerases and published alignments (Blanco et al. 1991, Aliotta et al. 1996; Korolev et al. 1995).
  • the structure of E. coli DNA polymerase was superimposed on the structure of the RNA polymerase using the helices in the fingers domain as reference (Helices O and P) using the program O (Jones et al. 1991).
  • Hjorleifsdottir, S. Diversity of thermostable DNA enzymes from Icelandic hot springs, Dept. of Biotechnology. Lund University, Sweden, Lund, 2002.
  • Hjorleifsdottir, S., et al. Thermostabilities of DIMA ligases and DIMA polymerases from four genera of thermophilic eubacteria. Biotechnol Lett 19 (1997) 147-150.
  • Pratt, L.A. and Kolter, R. Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. MoI Microbiol 30 (1998) 285-93. Rice, P. Longden, I. and Bleasby, A. (2000) Trends Genetics 16:276-277.
  • Skirnisdottir, S., et al. Influence of sulfide and temperature on species composition and community structure of hot spring microbial mats. Appl Environ Microbiol 66 (2000) 2835- 41.

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Abstract

L'invention porte sur de nouveaux polymérases d'ADN à déplacement de brins pouvant être utilisés dans des réactions d'amplification de déplacement de brins rapides et efficaces. Les polymérases sont nettement plus thermostables que les polymérases de la technique antérieure et présentent une activité élevée à des températures élevées. L'invention porte aussi sur des gènes codant les polymérases et sur des vecteurs contenant ces gènes. Des polymérases représentatifs de l'invention peuvent être obtenus à partir de souches bactériennes des espèces Thermus antranikianii et Thermus brockianus mais aussi à partir d'échantillons prélevés dans l'environnement par isolation des espèces sources.
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WO2009106795A1 (fr) 2008-02-28 2009-09-03 Genesys Ltd Enzyme
EP2206790A1 (fr) * 2009-01-13 2010-07-14 Fujifilm Corporation Procédé pour la réduction de la dispersion dans la réaction d'amplification d'acide nucléique
WO2014161712A1 (fr) * 2013-04-05 2014-10-09 Bioron Gmbh Nouvelles adn-polymérases
US10407722B2 (en) 2014-06-06 2019-09-10 Cornell University Method for identification and enumeration of nucleic acid sequence, expression, copy, or DNA methylation changes, using combined nuclease, ligase, polymerase, and sequencing reactions
WO2020185312A2 (fr) 2019-01-25 2020-09-17 President And Fellows Of Harvard College Compositions et procédé de synthèse d'acides nucléiques
WO2021119402A1 (fr) 2019-12-12 2021-06-17 President And Fellows Of Harvard College Compositions et méthodes pour le codage à barres biomoléculaire dirigé par la lumière
US20220136048A1 (en) * 2020-10-30 2022-05-05 Singular Genomics Systems, Inc. Methods and compositions for reducing nucleotide impurities
WO2022082200A3 (fr) * 2020-10-16 2022-06-23 Geaenzymes Co. Saturation biochimique de molécules et son utilisation
CN114761548A (zh) * 2019-10-01 2022-07-15 特罗姆瑟大学-挪威北极圈大学 海洋dna聚合酶i
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Cited By (19)

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WO2008043765A1 (fr) * 2006-10-09 2008-04-17 Qiagen Gmbh Adn-polymérases de thermus eggertssonii
US8183023B2 (en) 2006-10-09 2012-05-22 Qiagen Gmbh Thermus egertssonii DNA polymerases
WO2009106795A1 (fr) 2008-02-28 2009-09-03 Genesys Ltd Enzyme
JP2011512812A (ja) * 2008-02-28 2011-04-28 ジェネシス リミティド 酵素
US8986968B2 (en) 2008-02-28 2015-03-24 Genesys Biotech Ltd. Thermostable DNA polymerase
EP2206790A1 (fr) * 2009-01-13 2010-07-14 Fujifilm Corporation Procédé pour la réduction de la dispersion dans la réaction d'amplification d'acide nucléique
WO2014161712A1 (fr) * 2013-04-05 2014-10-09 Bioron Gmbh Nouvelles adn-polymérases
US9896671B2 (en) 2013-04-05 2018-02-20 Bioron Gmbh DNA polymerases
US10407722B2 (en) 2014-06-06 2019-09-10 Cornell University Method for identification and enumeration of nucleic acid sequence, expression, copy, or DNA methylation changes, using combined nuclease, ligase, polymerase, and sequencing reactions
EP3567122A1 (fr) 2014-06-06 2019-11-13 Cornell University Procédé d'identification et d'énumération de changements en matière de séquence d'acide nucléique, expression, copie ou méthylation d'adn en utilisant des réactions associant nucléase, ligase, polymérase et séquençage
EP3889271A1 (fr) 2014-06-06 2021-10-06 Cornell University Procédé d'identification et d'énumération de changements en matière de séquence d'acide nucléique, expression, copie ou méthylation d'adn en utilisant des réactions associant nucléase, ligase, polymérase et séquençage
US11486002B2 (en) 2014-06-06 2022-11-01 Cornell University Method for identification and enumeration of nucleic acid sequence, expression, copy, or DNA methylation changes, using combined nuclease, ligase, polymerase, and sequencing reactions
EP4170044A1 (fr) 2014-06-06 2023-04-26 Cornell University Procédé d'identification et de numération de modifications de méthylation de séquences d'acides nucléiques, expression, copie ou adn
US11390857B2 (en) 2017-12-15 2022-07-19 Universitetet I Tromsø—Norges Artiske Universitet DNA polymerases
WO2020185312A2 (fr) 2019-01-25 2020-09-17 President And Fellows Of Harvard College Compositions et procédé de synthèse d'acides nucléiques
CN114761548A (zh) * 2019-10-01 2022-07-15 特罗姆瑟大学-挪威北极圈大学 海洋dna聚合酶i
WO2021119402A1 (fr) 2019-12-12 2021-06-17 President And Fellows Of Harvard College Compositions et méthodes pour le codage à barres biomoléculaire dirigé par la lumière
WO2022082200A3 (fr) * 2020-10-16 2022-06-23 Geaenzymes Co. Saturation biochimique de molécules et son utilisation
US20220136048A1 (en) * 2020-10-30 2022-05-05 Singular Genomics Systems, Inc. Methods and compositions for reducing nucleotide impurities

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