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WO2000008164A2 - Complexe in vitro thermostable a activite de polymerase - Google Patents

Complexe in vitro thermostable a activite de polymerase Download PDF

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Publication number
WO2000008164A2
WO2000008164A2 PCT/DE1999/002480 DE9902480W WO0008164A2 WO 2000008164 A2 WO2000008164 A2 WO 2000008164A2 DE 9902480 W DE9902480 W DE 9902480W WO 0008164 A2 WO0008164 A2 WO 0008164A2
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Prior art keywords
protein
thermostable
fro
gavlimpfw
elongation
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PCT/DE1999/002480
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German (de)
English (en)
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WO2000008164A3 (fr
Inventor
Christian Kilger
Ingo Kober
Hartmut Voss
Gerd Moeckel
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Lion Bioscience Ag
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Priority claimed from DE19840771A external-priority patent/DE19840771A1/de
Application filed by Lion Bioscience Ag filed Critical Lion Bioscience Ag
Priority to EP99942776A priority Critical patent/EP1100923A2/fr
Priority to AU56171/99A priority patent/AU5617199A/en
Priority to CA002338185A priority patent/CA2338185A1/fr
Priority to DE19981494T priority patent/DE19981494D2/de
Priority to JP2000563788A priority patent/JP2002522042A/ja
Publication of WO2000008164A2 publication Critical patent/WO2000008164A2/fr
Publication of WO2000008164A3 publication Critical patent/WO2000008164A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • 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)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to a thermostable in vitro complex for template-dependent elongation of nucleic acids, a thermostable in vitro complex and DNA sequences and vectors coding therefor.
  • the invention further relates to the use of the complexes according to the invention in methods for template-dependent elongation of nucleic acids, such as PCR reactions, reverse transcription, DNA labeling or DNA sequencing, in which template-dependent DNA strand synthesis takes place in vitro.
  • the invention also relates to kits or reagent kits for carrying out the method according to the invention.
  • DNA polymerases belong to a group of enzymes that use single-stranded DNA as a template for the synthesis of a complementary DNA strand. These enzymes play an important role in nucleic acid metabolism, including the processes of DNA replication, repair and recombination. DNA polymerases have been identified in all cellular organisms, from bacterial to human cells, in many viruses and in bacteriophages (Kornberg, A. & Baker, TA (1991) DNA Replication WH Freeman, New York, NY).
  • the archaebacteria and the eubacteria are combined to form the group of prokaryotes, the organisms without a real cell nucleus, and the eukaryotes, the organisms with a real cell nucleus, are compared.
  • Common to many polymerases from a wide variety of organisms are often similarities in the amino acid sequence and similarities in structure (Wang, J., Sattar, AKMA; Wang, CC, Karam, JD, Königsberg, WH & Steitz, TA (1997) Crystal Structure of pol ⁇ familily replication DNA polymerase from bacteriophage RB69.Cell 89, 1087-1099).
  • Organisms like humans have a large number of DNA-dependent polymerases, but not all of them are responsible for DNA replication, but some also carry out DNA repair.
  • replicative DNA polymerases mostly consist of protein complexes with multiple units that replicate the chromosomes of cellular organisms and viruses.
  • a general property of these replicating polymerases is generally high processivity, that is, their ability to polymerize thousands of nucleotides without dissociating from the DNA template (Kornberg, A. & Baker, TA (1991) DNA Replication. WH Freeman, New York, NY).
  • the replication apparatus includes a variety of components. These include, inter alia, a) proteins having polymerase activity, b) proteins which are involved in the formation of a clamp structure, the clamp structure having the task, inter alia, of binding polymerase activity to its template, stabilizing the binding and thus the dissociation constant to be changed accordingly, c) proteins which load the clip onto the template, d) proteins which stabilize the template and optionally e) proteins which carry the polymerase to the template.
  • the proteins mentioned under b) form structures that are either open or closed, for example ring-shaped or semi-ring-shaped structures. Such structures can be formed by one or more species of proteins. It is possible that one of said protein species has polymerase activity.
  • the proteins responsible for the formation of these structures, if they have no polymerase activity, are hereinafter referred to as “glide clip proteins” or “clip proteins”. 5
  • the glide clip is often linked to an elongation protein via one or more other proteins, in other words coupled to the elongation protein.
  • a coupling protein is hereinafter referred to as a coupling protein or coupling subunit, where the coupling can optionally take place via a plurality of coupling proteins.
  • Elongation protein is to be understood herein to mean a protein or complex which has polymerase activity and which has at least one or more of the following properties: use of RNA as a template for the synthesis of DNA and / or RNA, use of DNA as a template for the synthesis of DNA and / or RNA, synthesis of RNA, synthesis of DNA, synthesis of nucleic acids from DNA and RNA, exonuclease activity in 5'-3 'direction and exonuclease activity in 3'-5' direction, strand displacement activity, thermostability and processivity or non-processivity.
  • PCNA eukaryotic proliferating cell nuclear antigen
  • such a protein complex consists of a large number of subunits, which is called Escherichia coli ⁇ complex in the Eubacterium and replication factor C (RF-C) in humans (Kelman, Z. &O'Donnell, M. (1994) DNA replication - enzymology and mechanisms. Curr. Opin. Gent. Dev. 4, 185-195).
  • the protein complex recognizes the 3 'end of the primer of the "primer-template duplex" and positions the glide clip around the DNA in the presence of ATP.
  • the phage expresses its own catalytic polymerase, the T7 polymerase, which is the gene product of the gene 5, which binds to a protein from the host Escherichia coli, the thioredoxin, and which enables highly processing DNA replication as a replicase (Proc. Natl. Acad. Sci. USA 1992 Oct 15; 89 (20): 9774-9778 Genetic analysis of the interaction between bacteriophage T7 DNA polymerase and Escherichia coli thioredoxin, Himawan JS, Richardson CC). Brackets are also formed here, but this bracket does not have the same structure as e.g. in the case of eukaryotic PCNA.
  • DNA polymerases are characterized, among other things, by two properties, their elongation rate, that is, the number of nucleotides that they can incorporate into a growing DNA strand per second, and their dissociation constant. If, after each incorporation step, the polymerase disassociates one of the nucleotides into the growing chain again (i.e. one elongation step occurs per binding event), then the processivity has the value 1 and the polymerase is not processive. This synthesis is called distributive.
  • the replication mode is called processive and can reach a value of several thousand (see also: Methods in Enzymology Volume 262, DNA Replication, Edited by JL Campbell, Academic press 1995, pp. 270-280)
  • thermostable enzymes previously used in these reactions have only a small amount
  • temperature-sensitive T7 polymerase associated with thioredoxin has a processivity of several thousand nucleotides .
  • thermostable DNA polymerase from Thermus thermophilus or Thermus aquaticus only have a processivity of about 50 nucleotides (Biochim Biophys Acta 1995 Nov 7; 1264 (2): 243-248 Inactivation of the 5'-3 'exonuclease of Thermus aquaticus DNA polymerase, Merkens LS, Bryan SK, Moses RE).
  • PCR Chain reaction
  • primers also called template
  • nucleotides nucleotides
  • a DNA polymerase of a corresponding buffer and suitable reaction conditions. This is usually based on a double-stranded DNA sequence from which a specific target area is to be amplified.
  • two primers are used, which are complementary to flanking regions of the target sequence on a partial strand of the DNA double strand.
  • the DNA double strands are first denatured, in particular thermally melted. After hybridization of the primers, an elongation takes place using the polymerase.
  • the PCR preferably uses a thermostable polymerase that survives the cyclic, thermal melting of the DNA strands. So Taq DNA polymerase is often used (US Patent 4,965,188). The processivity of the Taq DNA polymerase, however, is, as stated above, relatively low in comparison to the T7 polymerase.
  • DNA polymerases are also used in DNA sequence determination (Sanger et al., Proc. Natl. Acad. Sei., USA 74: 5463-5467 (1997)).
  • a T7 DNA polymerase is often used in Sanger sequencing (Tabor, S. and Richardson, CC. Proc. Natl. Acad. Sei., USA 86: 4076-4080 (1989)).
  • the cycle sequencing method was later developed (Murray, V. (1989) Nucleic Acids Res. 17, 8889), which does not require a single-stranded template and allows the initiation of the sequence reaction with relatively small amounts of template.
  • the polymerases used here can be, for example, the above-mentioned tag polymerase (US Patent 5,075, 216) or the polymerase from Thermotoga neapolitana (WO 96/10640) or other thermostable polymerases.
  • Newer methods combine the exponential amplification and the sequencing of a DNA fragment in one step, so that it is possible to sequence genomic DNA directly.
  • DEXAS method Nucleic Acids Res 1997 May 15; 25 (10): 2032-2034 Direct DNA sequence determination from total genomic DNA.Kilger C, Pääbo S, Biol. Chem. 1997 Feb; 378 (2 ): 99-105 Direct exponential amplification and sequencing (DEXAS) of genomic DNA.Kilger C, Pääbo S and DE 19653439.9 and DE
  • DNA polymerases are also used in the reverse transcription of RNA into DNA.
  • RNA serves as a template and the polymerase synthesizes a complementary DNA strand.
  • thermostable DNA polymerase from the organism Thermus thermusphilus (Tth) (U.S. Patent 5,322,770).
  • the polymerase may also have proof-reading activity
  • thermostable in v / yro complex comprising a thermostable gliding clip protein and a thermostable elongation protein having polymerase activity.
  • the complex according to the invention can serve for the template-dependent elongation of nucleic acid (s).
  • This complex can be used in in wfro reactions, such as in PCR reactions, and is highly processive. It is also advantageous that the complex has a low error rate in nucleotide incorporation, that is, it has an increased accuracy in base incorporation. This complex can thus be used advantageously in elongation, amplification, reverse transcription, DNA labeling and the sequencing of nucleic acids.
  • the possible uses outlined above each represent particularly preferred embodiments of the invention.
  • the sliding chamber protein is linked to the elongation protein.
  • thermostable in ' fra complex the glide clip protein and the elongation protein are linked via a coupling protein.
  • the coupling between slide clamp protein and elongation protein having polymerase activity can be carried out by covalent but also by non-covalent binding.
  • the gliding clip protein and / or the elongation protein come from archaebacteria.
  • the complex according to the invention is a prokaryotic in w ' fro complex.
  • the prokaryotic complex according to the invention can be an archaebacterial in v / fro complex.
  • the prokaryotic complex according to the invention can be an eubacterial in vitro complex.
  • the complex according to the invention is a eukaryotic in v / fro complex.
  • a prokaryotic in v / fro complex is one in which the gliding clip protein is of prokaryotic origin regardless of the origin of the elongation protein.
  • a eubacterial complex is one in which the glide clip protein is of eubacterial origin, regardless of the origin of the elongation protein.
  • an archaebacterial complex is one in which the glide clip protein is of archaebacterial origin, regardless of the origin of the elongation protein.
  • a eukaryotic complex is still one in which the glide clip protein is of eukaryotic origin, regardless of the origin of the elongation protein.
  • the present invention also relates to those thermostable in wYro complexes in which the proteins which form the complexes originate in part from archaebacteria, eukaryotes and from eubacteria.
  • any permutations of the in ⁇ ro complexes with regard to the protein components and their respective origins are the subject of the present invention. Origin in the above sense is intended to denote the source to which the gene, the gene information or the protein can be attributed.
  • the actual manner of obtaining the gliding clip protein or the elongation protein is independent of this, which can be done, for example, by chemical synthesis, genetic engineering processes or isolation from natural sources.
  • thermostable prokaryotic in w ⁇ ro complex for elongation in particular for template-dependent elongation of nucleic acids, which comprises a thermostable gliding clip protein (FIG. 20) which completely or partially encloses the complementary nucleic acid strands, and a thermostable, polymerase activity (FIG 21 and 22) comprising protein, said protein or protein complex being coupled or associated with the gliding clip protein.
  • polymerase activity-having elongation protein also includes polymerase activity-having protein complexes or subunits of such complexes which carry the polymerase activity.
  • thermostable means that the in vitro complex with high processivity incorporates nucleotides into growing nucleic acid strands, both at low and at high temperatures, which occur in PCR or another reaction, such as e.g. DNA sequencing.
  • the PCR consists e.g. usually from the steps of denaturing (70 ° C to 98 ° C), annealing (40 ° C to 78 ° C) and DNA strand synthesis (60 ° C to 76 ° C). This complex must therefore at least between approx. 60 ° C and approx.
  • Denaturation phenomena of the complex or individual components occur which prevent or inhibit the elongation reaction.
  • the gliding clip protein fulfills the function of binding the elongation protein to the DNA.
  • the glide clip protein itself completely or partially encloses the single-stranded or double-stranded nucleic acid or by association with the protein having polymerase activity or with the protein complex having polymerase activity or a subunit thereof, and thereby forms a bracket around the nucleic acid.
  • the processivity is significantly increased by this bracketing, at least one and a half times (Example 5 and Fig. 23).
  • the in v / yro complex according to the invention has at least one and a half times the processivity in comparison to an elongation protein alone, or in comparison to a protein complex having polymerase activity, or a subunit thereof, without a glide clip (example 5 and FIG. 23).
  • homologs or functional analogs of the "proliferating cell nuclear antigen" protein complex, encoded in the human genome, or homologs of the likewise circular " ⁇ -clamp" protein complex from E. coli, which originate from thermostable organisms and are thermostable, can serve as the sliding chamber , or if they are not thermally stable are made thermostable, or come from non-thermostable organisms and are thermostable or are subsequently made thermostable by changing the amino acid sequence (Eijsink VG, van der Zee JR, van den Burg B, Vriend G, Venema G, FEBS Lett 1991 Apr 22 ; 282 (1): 13-16, Improving the thermostability of the neutral protease of Bacillus stearothermophilus by replacing a buried asparagine by leucine, Bertus Van den Burg, Gert Vriend, Oene R.
  • homologous sequences are to be understood as sequences which are distinguished by the fact that they have sequence similarity to one or more other sequences, to an extent to which a coincidental similarity cannot be assumed. The degree of sequence similarity is expressed in percent and is called homology. The term "sequence identity" is sometimes also used.
  • a homolog is a nucleic acid or amino acid sequence which is a homologous sequence to a reference sequence.
  • the slide clamp can be constructed from several components.
  • the glide clip identified in the human genome consists of three PCNA protein components (SEQ ID NO: 11) (homotrimers), the glide clip identified in the E. co // genome consists of two components (SEQ ID NO: 35) (homodimers).
  • a slide clamp is to be understood here in particular to mean any protein which has the functional property of increasing the polymerase processivity (example 5 and FIG. 23) and / or serves to lower the error rate.
  • the glide clip can have an annular three-dimensional structure or, by coupling to another protein, form an annular three-dimensional structure, by means of which it is able to completely or partially enclose single and double-stranded nucleic acids.
  • a slide clamp in the sense of the present invention is to be understood in particular as a protein of this type
  • SEQ ID NO 11 over a length of at least 100 amino acids with a sequence alignment has an at least 20%, preferably at least 25% and more preferably at least 30% sequence identity or the 2nd to the bacterial ß-clamp sequence from E . coli (eubacteria)
  • Archaeoglobus fulgidus (Archaebacteria) (SEQ ID NO 12) has at least 20%, preferably 25% and more preferably 30% sequence identity over a length of at least 100 amino acids with a sequence alignment.
  • the slide clip according to the invention can have one or more of the aforementioned features.
  • Invention are also to be understood as including proteins that contain one or both of the following consensus sequences (Region 1 and Region 2) no more than four positions from region 1 (SEQ ID No .: 39) or no more than four positions from region 2 (SEQ ID No .: 40) (Fig. 4):
  • amino acids are named according to the standard IUPAC - single letter nomenclature and listed according to the Prosite Pattern description standard.
  • the following amino acid groups are often combined: G, A, V, L, I, MP, F or W (amino acids with non-polar side chains) S, T, N, Q, Y, or C (amino acid with uncharged polar side chains) K , R, H, D or E (amino acid with charged and polar side chains)
  • X in the sequences or sequence listing means any amino acid or insertion or deletion
  • a hidden Markov model was also generated from the multiple alignment of human PCNA homologs shown in FIG.
  • a slide clamp is to be understood in particular to mean any protein which, with the hidden Markov model thus generated (hereinafter referred to as “HMM”), has a “score” of more than 20, preferably 25, most preferably 30 (Fig. 12), where a "score” is the output value of an HMM analysis.
  • the hidden Markov models and the corresponding scores were calculated with the hmmfs program (version 1.8.4, July 1997) from the HMMER package (HMMER Protein and DNA Hidden Markov Models (Version 1.8) by Sean Eddy, Dept. of Genetics, Washington University School of Medicine, St. Louis, USA;).
  • Markov models with a hidden profile are statistical models of the consensus of the primary structure of a sequence family.
  • the profiles use position-specific scores (" scores ") for amino acids (or nucleotides) and position-specific scores to open or expand an insertion or deletion.
  • scores position-specific scores
  • Methods for creating profiles based on multiple alignments have been described by Taylor (1986), Gribskov et al. (1987), Barton (1990) and Heinikoff (1996).
  • HMM provide a completely probabilistic description of profiles, ie Bayes' teaching defines how the entire probability (evaluation) parameter should be set (see Krogh et al. 1994, Eddy 1996 and Eddy 1998).
  • the central idea is that an HMM is a finite model that describes a probability distribution over an unlimited number of possible sequences.
  • the HMM is composed of a number of states that correspond to the pillars of a multiple alignment, as is usually shown. Each state emits symbols (residues) according to the (state-specific) symbol emission probabilities, and the states are interconnected by state transition probabilities. Starting from an initial state, a sequence of states is generated by moving from one state to another according to the state transition probabilities until a final state is reached.
  • Each state then emits symbols according to the emission probability distribution of that state, creating an observable sequence of symbols.
  • the attribute "hidden” is derived from the fact that the underlying state sequence cannot be observed; what is seen is the symbol sequence.
  • An estimate of the transition and emission probabilities (the training of the model) is carried out achieved dynamic programming algorithms implemented in the HMMER package.
  • the HMMER package provides a numerical quantity (the score or output value) that is proportional to this probability, i.e. H. the information content of the sequence, expressed in bits, measured according to the HMM.
  • a slide clamp is to be understood in particular as any protein that has a score of more than 25, preferably 30 and most preferably 35 with the HMM thus generated (FIG. 13).
  • the slide clip can be constructed from several components which are firmly bonded to one another by a characteristic bond, so that a stable ring-shaped molecular complex is formed which cannot readily dissociate from the nucleic acid. This enables a firm, but not covalent, bond to the nucleic acid, which, however, does not hinder the free movement on it.
  • the processivity-enhancing glide clip proteins also have characteristic local molecular properties in the region of the interaction region with DNA, which facilitate the free movement and which can be supported by water molecules stored in this region.
  • a preferred embodiment of the present invention is furthermore in particular a thermostable prokaryotic in w ⁇ ro complex, the gliding clip protein being one of the following: AF 0335 from Archaeoglobus fulgidus (SEQ ID NO .: 12) (FIG. 24), MJ0247 from Methanococcus jannaschii (SEQ ID NO .: 13), PHLA008 from Pyrococcus horikoschii (SEQ ID NO .: 14), MTH1312 from Methanobacterium Thermoautotrophicus (SEQ ID NO .: 15), and AE000761_7 from Aquifex aeolicus (SEQ ID NO .: 36).
  • thermostable in w / o complexes are the subject of this application, the gliding clip protein comprising an amino acid sequence which is selected from the group SEQ ID No. 11, 12, 13, 14, 15 and 36.
  • the complex according to the invention comprises a slide clamp loader.
  • a slide clamp loader is understood here to mean a protein, protein complex or subunit of a protein which comprises a homologue of the "Replication Factor C" protein complex identified in humans.
  • this protein complex consists of five subunits, which are encoded by 5 separate genes in humans.
  • the 4 small subunits, each encoded by a gene form a protein complex in humans.
  • the large subunit protein in humans is encoded by a gene (referred to herein as a sliding clip loader 2).
  • the sequences of the four small subunits are shown as SEQ ID NO .: 1, 32, 33, 34, the sequence of the large subunit is shown as SEQ ID NO .: 6.
  • homologs or functional analogs, individually or in any combination, of each of the sequences SEQ ID NO .: 1, 32, 33, 34 mentioned above can be used as slide clamp loader 1.
  • the homologues can be of prokaryotic, such as eubacterial or archaebacterial, or eukaryotic origin.
  • homologs or functional analogs individually or in any combination, of the sequence SEQ ID NO .: 6 mentioned above can be used as slide clamp loader 2.
  • the homologues can be of prokaryotic, such as eubacterial or archaebacterial, or eukaryotic origin.
  • a slide clamp loader 1 in the sense of the present invention can also be understood to be such a protein which has at least 100 amino acids in a sequence alignment with the human (eukaryotes) amino acid sequence (SEQ ID NO: 1, 32, 33, 34) with a sequence alignment %, preferably at least 25% and more preferably at least 30% sequence identity.
  • a slide clamp loader 2 in the sense of the present invention can also be understood to be such a protein which has at least 20%, preferably at least 20% sequence identity to the human (eukaryotes) amino acid sequence (SEQ ID NO: 6) over a length of at least 150 amino acids with a sequence alignment Has 25% and more preferably at least 30%.
  • Sliding clip loader homologs in the sense of the above definition are, for example, the genes from archaebacteria listed in FIG. 1.
  • the sequences SEQ ID NO correspond to these genes for slide clamp loader 1: 2, 3, 4, and 5 and for the sliding clamp loader 2 the sequences SEQ ID NO: 7, 8, 9, and 10.
  • a slide clamp loader 1 in the sense of the present invention can also be understood to be a protein which comprises a sequence according to the following consensus sequence and does not deviate from this sequence at more than four positions (see also FIG. 6 for alignment):
  • a slide clamp loader 2 in the sense of the present invention can also be understood to be a protein which comprises a sequence according to the following consensus sequence and does not deviate from this sequence at more than four positions (see also FIG. 7 for alignment):
  • An HMM was also generated from the multiple alignment of sequences of slide clamp loader 1 comprising the human sequence and some sequences from archaebacteria homologous to it, shown in FIG. 14.
  • a slide clamp loader 1 in the sense of the present invention is therefore also to be understood as a protein which, with the HMM thus produced, has a score of more than 25, preferably more than 30 and most preferably more than 35 (see also FIG. 14 for alignment).
  • a slide clamp loader 2 in the sense of the present invention is therefore also to be understood as a protein which, with the HMM thus generated, has a score of more than 15, preferably more than 20 and most preferably more than 25 (see also FIG. 15 for alignment).
  • the in w ' fro complex according to the invention comprises a protein homologous to the Eubaktehum Escherichia coli ⁇ complex or parts thereof as a slide clamp loader 1 or slide clamp loader 2.
  • a sliding clip loader in the sense of the present invention can accordingly be a sliding clip loader 1 alone, a sliding clip loader 2 alone, or a combination of one or more sliding clip loaders 1 or sliding clip loader 2, each as defined above.
  • thermostable in / ro complex for elongation of nucleic acids additionally comprises a compound which releases energy during cleavage, such as ATP, GTP, CTP, TTP, dATP, dGTP, dCTP or dTTP.
  • a compound which releases energy during cleavage such as ATP, GTP, CTP, TTP, dATP, dGTP, dCTP or dTTP.
  • a slide clamp loader appears to assemble the components of the slide clamp around the uninterrupted strand of DNA or to remove them again when the reaction is complete.
  • the sliding clamp can have an annular three-dimensional structure or, by coupling to another protein, form an annular three-dimensional structure, by means of which it is able to completely or partially enclose single- or double-stranded nucleic acids.
  • the sliding clamp loader can bring advantages if the template nucleic acid molecule is present in a closed ring.
  • thermostable in w ' fro complexes are the subject of this application, the sliding clip loader 1 comprising an amino acid sequence which is selected from the group SEQ ID No. 2, 3, 4 and 5.
  • thermostable in / ro complexes are the subject of this application, the sliding clip ladder 2 comprising an amino acid sequence which is selected from the group SEQ ID No. 7, 8, 9 and 10.
  • the coupling protein has the function of linking an elongation protein with one or more glide clip proteins or a glide clip protein complex.
  • Coupling protein in the sense of the present invention is therefore to be understood in particular as any protein which has the function described above. Coupling proteins of archaebacterial origin are preferred.
  • Coupling proteins in the sense of the present invention are, for example, homologs or functional analogs, individually or in any combination, of each of the sequences listed in FIG. 1, the homologs to the human sequence of the small subunit of polymerase ⁇ , referred to herein as the coupling subunit (sequence name: DPD2JHUMAN , shown in the sequence listing as SEQ ID NO: 16).
  • a coupling protein can be a protein which comprises a sequence which is selected from the group which comprises the sequences SEQ ID NO: 17, 18, 19, 20 and 21.
  • a coupling protein in the sense of the present invention is to be understood in particular to be a protein that has a length of at least 150 to the human (eukaryotes) amino acid sequence (SEQ ID NO: 16)
  • Amino acids in a sequence alignment an at least 18%, preferably has at least 22% and more preferably at least 26% sequence identity.
  • Coupling protein in the sense of the present invention is to be understood in particular as a protein which has at least 20%, preferably at least 20% sequence identity to the amino acid sequence (SEQ ID NO: 19) of Pyrococcus horikoshii with a sequence alignment of at least 150 amino acids 25% sequence identity and most preferably a sequence identity greater than 30%.
  • Coupling protein for the purposes of the present invention is also to be understood as any protein which comprises the following consensus sequence and does not deviate from this sequence at more than four positions.
  • the generation of the consensus sequence is shown in FIG. 5, which is shown as SEQ ID No. 43 is disclosed herein.
  • a coupling subunit in the sense of the present invention is therefore to be understood as any protein which has a score of more than 10, preferably more than 15, most preferably more than 20 with the HMM thus generated.
  • thermostable in v / fro complexes are the subject of this application, the coupling protein comprising an amino acid sequence which is selected from the group SEQ ID No. 17, 18, 19, 20 and 21.
  • an elongation protein can be used which has the features defined at the outset.
  • forms of elimination proteins can be used, as described below and at least in part are already known in the prior art.
  • Elongation proteins require the presence of a coupling protein to have any polymerase activity at all. It is also known that some elongation proteins can bind directly to glide clip proteins, whereas other elongation proteins require the presence of a coupling protein for binding to the glide clip proteins. Elongation proteins can furthermore combine the two properties mentioned above, that is to say both via a coupling protein and also directly, that is to say without a coupling protein, bind to a stick clamp protein.
  • Preferred elongation proteins for the in w ' / ro complex according to the invention can be selected from the group comprising the organisms carboxydothermus hydrogenoformans, Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus oshimai, Thermus ruber, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species sp.
  • Thermus thermusphilus Therotoga maritima, Therotoga neapolitana, Thermosipho af canus, Anaerocellum thermophilum, Bacillus caldotenax, or Bacillus stearothermophilus.
  • Suitable elongation proteins are, for example, the elongation proteins from archaebacteria homologous or functionally analogous to human elongation protein (SEQ ID NO: 22) (SEQ ID NO: 23, 24, 25, 26).
  • the invention relates to such thermostable in w-ro complexes, the elongation protein comprising an amino acid sequence which is selected from the group SEQ ID No. 23, 24, 25, 26, 27, 28, 29, 30 and 31.
  • An elongation protein in the sense of the present invention is to be understood in particular as a protein that has at least 20%, preferably 25%, sequence identity to the human (eukaryotes) amino acid sequence (SEQ ID NO: 22) over a length of at least 200 amino acids with a sequence alignment. sequence identity, most preferably 30% sequence identity.
  • An elongation protein in the sense of the present invention is also to be understood in particular as a protein which contains the following consensus sequence (SEQ ID No. 44) and does not deviate from this sequence at more than four positions. 8 shows the alignment on which the consensus sequence is based.
  • An HMM was also generated from the multiple alignment of homologs to the human elongation protein (SEQ ID NO: 22) shown in FIG. 17.
  • An elongation protein in the sense of the present invention is therefore also to be understood as a protein which, with the HMM thus generated, has a score of more than 20, preferably more than 25, most preferably more than 30.
  • An elongation protein in the sense of the present invention is thus also to be understood as a protein which, in relation to the archaebacterial amino acid sequence (SEQ ID NO: 27), has a sequence identity of at least 25%, preferably at least 30%, over a length of at least 400 amino acids with a sequence alignment Sequence identity, most preferably has at least 35% sequence identity.
  • the proteins with the SEQ ID NO: 27, 28, 29, 30 or 31 originating from archaebacteria are suitable.
  • Elongation protein in the sense of the present invention is therefore to be understood in particular as any protein which contains the following consensus sequence, referred to herein as SEQ ID No .: 45, and does not deviate from this sequence at more than four positions. (FIG. 9 shows the alignment on which the consensus sequence is based.)
  • HMM was also generated from the multiple alignment of homologs to the archaebacterial elongation protein (SEQ ID NO: 27) shown in FIG. 18.
  • Elongation protein in the sense of the present invention is thus to be understood in particular as any protein that has a score of more than 35, preferably more than 40 and even more preferably more than 45 with the HMM produced in this way (FIG. 18).
  • the elongation protein can also be of eubacterial origin.
  • An elongation protein in the sense of the present invention is therefore also to be understood as a protein that has a length of at least 300 to form the eubacterial amino acid sequence (SEQ ID NO: 37)
  • Amino acids in a sequence alignment have at least 25% preferably has at least 30% and more preferably at least 35% sequence identity.
  • Elongation protein in the sense of the present invention is therefore to be understood in particular as any protein which contains the following consensus sequence and does not deviate from this sequence at more than eight positions (FIG. 10):
  • Elongation protein in the sense of the present invention is therefore to be understood as any protein which has a score of more than 20, preferably more than 25, most preferably more than 30 with the HMM thus generated.
  • thermostable in w ' / ro complexes are the subject of this application, the elongation protein comprising an amino acid sequence which is selected from the group SEQ ID No. 38 includes.
  • DNA polymerases have been used as elongation proteins without coupling protein and without slide clamp for standard PCR reactions, for example DNA polymerase I from Pyrococcus furiosus (US No. 5,545,552) or Pyrococcus species (EP-A-0 547 359). These enzymes are characterized by the property of being thermostable and often one To have 3'-5 'exonuclease activity (' proof-reading 'activity).
  • a heterodimer with polymerase activity in Pyrococcus furiosus has only recently been discovered (Uemori, T., Sato, Y., Kato, I., Doi, H., and Ishino, Y. (1997).
  • a novel DNA polymerase in the hyperthermophilic archaeon , Pyrococcus furiosus gene cloning, expression, and characterization. Genes to Cells 2, 499-512.
  • thermostable in w ' fro complex illustrates an example of an embodiment of the thermostable in w ' fro complex according to the invention, in which case the glide clip binds to the elongation protein via a coupling protein.
  • the reaction mixture according to the invention in v / fro or the reaction mixture according to the invention containing it can furthermore comprise a nucleic acid, the nucleic acid being, for example, the nucleic acid to be elongated, sequenced, amplified or reversely transcribed and / or a primer, the latter Primer is preferably hybridized to a nucleic acid.
  • Primers are usually oligonucleotides which are complementary to a target sequence in order to be able to bind to them, whereby in opposite orientation, with their 3 'ends directed towards one another, includes the nucleic acid section to be elongated, sequenced, amplified or reversely transcribed . They serve as the starting point for the enzyme activity and generally provide a free 3'-OH end for the polymerase to incorporate a nucleotide.
  • the complex according to the invention optionally in a reaction mixture, is preferably in a suitable buffer.
  • Suitable buffers are those which are used for PCR, sequencing, nucleic acid tag and others in w 'rro-nucleic acid elongation reactions by polymerase. Suitable buffers are described, for example, in Methods in Molecular Biology, Vol. 15 Humana Press Totowa, New Jersey, 1993, ed. Bruce A. White.
  • thermostable invention in vi 'TRO for the elongation complex, amplification, reverse transcription and / or sequencing may additionally be present to the inventive complex is a nucleotide or a mixture of nucleotides, or used or to be encompassed.
  • Deoxynucleotides can be selected from, but are not limited to, dGTP, dATP, dTTP and dCTP.
  • derivatives of deoxynucleotides can also be used which are defined as deoxynucleotides which are able to be incorporated into growing nucleic acid molecules by a thermostable polymerase.
  • Such derivatives include the thionucleotides, 7-deaza-2 '-dGTP, 7-deaza-2' - dATP, digoxigenin-dUTP (Boehringer Mannheim) -dATP and deoxyinosine triphosphate, which is also a replacement deoxynucleotide for dATP, dGTP, dTTP or dCTP can be used, but are not limited to these. Labeled deoxynucleotides can also be used.
  • the use of pyrenes and pyrene derivatives is also possible. All known markings and / or markings suitable for the purpose according to the invention can be present or used.
  • Dideoxynucleotides can be selected from, but are not limited to, ddGTP, ddATP, ddTTP and ddCTP. Alternatively, you can
  • Dideoxynucleotides are defined that are capable of being incorporated by polymerase into growing nucleic acid molecules that are synthesized in the reaction.
  • Such derivatives can be radioactive dideoxynucleotides (ddATP, ddGTP, ddTTP and ddCTP) or dideoxynucleotides (ddATP, ddGTP, ddTTP and ddCTP) which are labeled with, for example, FITC, Cy5, Cy5.5, Cy7, Texas red or other dyes, but are not limited to these.
  • Labeled deoxynucleotides can also be used together with unlabeled dideoxynucleotides in the course of sequencing using the thermostable in ⁇ ro complex according to the invention.
  • Ribonucleotides can be selected from, but are not limited to, GTP, ATP, TTP and CTP.
  • derivatives of ribonucleotides defined as those ribonucleotides capable of being incorporated by polymerase into growing nucleic acid molecules that are synthesized in the reaction can also be used in accordance with the invention.
  • Such derivatives can be radioactive ribonucleotides (ATP, GTP, TTP and CTP) or ribonucleotides (ATP, GTP, TTP and CTP) which are labeled with e.g. FITC, Cy5, Cy5.5, Cy7 and Texas red or others are marked, include, but are not limited to, these.
  • the present invention furthermore relates to a reaction mixture which comprises the in w ' ro complex according to the invention. It can be provided that the reaction mixture further comprises one or more elongation proteins which have at least one or more of the above activities. Such an additional elongation protein is advantageously a thermostable polymerase. Such a reaction approach allows increased processivity compared to the use of the known thermostable polymerases.
  • the complexes preferably completely or partially consisting of recombinant proteins, can be provided with the following steps: provision of the nucleic acid fragment which codes for the desired protein, ligation into an expression vector, transformation into a host, expression and purification of the protein ( Fig. 25).
  • provision of the nucleic acid fragment which codes for the desired protein ligation into an expression vector
  • transformation into a host transformation into a host
  • purification of the protein Fig. 25.
  • genes in particular from the archaebacteria, intein (Proc. Natl. Acad. Sci.
  • genes and / or proteins suitable for the complex according to the invention can be identified, for example, by homology searches in databases which include genomes from prokaryotes.
  • Programs that are suitable for this include, but are not limited to, the BLAST, BLASTP, and FASTA programs.
  • BLAST Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs ", Nucleic Acids Res. 25: 3389-3402. WR Pearson & D. Lipman PNAS (1988) 85: 2444-2448).
  • Identification can also be done by using DNA probes to screen for prokaryotes or eukaryotes in genomic banks, for example, for the corresponding genes.
  • the experimental procedures required for this can be found in Maniatis et al. (Molecular Cloning (2 " d edition, 3 Volume set): A laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989)) or in Ausubel et al (Current Protocols in Molecular Biology, John Wiley and Sons (1988)).
  • the provision of the purified nucleic acid of the genes, the proteins which build up the complexes according to the invention can e.g. by isolating them from a genomic library of the relevant organism or by synthetic DNA production, in each case if desired combined with amplification by means of PCR, with the aid of primers which are specific for the desired gene segment.
  • Usual methods are described in Maniatis et al. (Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y. (1989)).
  • genes of the proteins of the ⁇ ra complexes according to the invention can be cloned using a variety of methods and thus made available by means of an expression vector for protein expression in a host organism. Common processes are in Maniatis et al. (Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989)).
  • the genes of the complexes can first be cloned into a high-copy vector, for example pUC18, pBst or pBR322, and only then into a prokaryotic expression vector, for example pTrc99, pQE30, pQE31 or pQE32, or directly into one prokaryotic or viral expression vector can be cloned.
  • vectors are to be understood as nucleic acids which are capable of to transport another nucleic acid molecule in or between different organisms or genetic backgrounds. They usually have the ability to autonomously replicate and / or express (expression vectors) the operatively linked nucleic acid molecule. "Operatively linked” herein means that the transported nucleic acid molecule is linked to the vector such that it is under the transcriptional and translational control of expression control sequences of the vector and can be expressed in a host cell.
  • Bacterial and viral expression systems their preferred use and a selection of vector systems are described, for example, in Gene Expression Technology, (Meth. Enzymol., Vol 185, Goeddel, Ed., Academic Press, NY (1990)).
  • Suitable vectors for the present invention should enable different expression levels of the proteins by having some or all of the following properties: (1) promoters, or transcription initiation sites, either immediately next to the start of the protein or as a fusion protein, (2) operators, can be used to switch gene expression on or off, (3) ribosomal binding sites for improved translation, and (4) termination sites for transcription or translation, which lead to improved stability of the transcript.
  • Expression vectors that are compatible with eukaryotic cells, preferably with vertebrate cells, can also be used.
  • Some known vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1 / pML2d (International Biotechnologies, Inc.), pAcHLT-ABC (Pharmingen) and pTDTI (ATCC 31255). It is also possible to use a retroviral expression vector.
  • the vectors according to the invention contain at least one gene for the glide clip protein or at least one gene for the coupling protein or at least one gene for a glide clip loader 1 and / or 2 or at least one gene for an elongation protein.
  • the vectors simultaneously contain several of the various genes mentioned above, for example the genes for an elongation protein and a glide clip protein.
  • the vector also contains suitable restriction sites and, if appropriate, polylinkers for the insertion of further DNA sequences. It is particularly preferred if the spatial arrangement of the already contained DNA sequence and additional insertion site leads to the formation of a fusion protein after expression.
  • the vector according to the invention contains promoter and / or operator areas, it being particularly preferred if such promoter and / or operator areas are inducible or repressible. Control of expression in host cells is thereby considerably simplified and can be designed particularly efficiently.
  • Such promoter / operator areas can also occur multiple times in an expression vector, so that, if necessary, independent expression of several DNA sequences is made possible using only one expression vector.
  • Another object of the present invention is a host cell containing one or more vector (s) according to the invention, the expression of proteins in this host cell under suitable conditions can be done.
  • suitable conditions include, for example, the presence of a repressor, inductor, or a derepressor.
  • the transformants are cultivated under the appropriate growth conditions of the host strain.
  • Most E. co // strains are e.g. cultivated in LB medium at 30 ° C to 42 ° C until the logarithmic or stationary growth phase.
  • the proteins can be purified from a transformed culture, which can be done either from a cell pellet, after centrifugation, or from the culture fluid. If the proteins are purified from the cell pellet, the cells are resuspended in a suitable buffer and broken up by means of ultrasound treatment, enzymatic treatment or freezing and thawing. If the purification from the culture suspension takes place, either without or with a fusion protein, the supernatant is separated from the cells by means of known methods, such as centrifugation.
  • the separation and purification of the proteins of the complexes according to the invention either from the supernatant of the culture solution or from the cell extract can be carried out by known separation or purification processes.
  • These methods are, for example, those that relate to the solubilities, such as salt precipitation and solvent precipitation, methods that take advantage of the different molecular weights, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis, methods that use the different charges, such as Ion exchange chromatography, methods that take advantage of the different hydrophobicity, such as reverse-phase HPLC (High Performance Liquid Chromatography), methods that take advantage of certain affinities, such as affinity chromatography (examples 6, 7, 24 and 25), and methods that take advantage of differences in the isoelectric point, such as isoelectric focusing.
  • solubilities such as salt precipitation and solvent precipitation
  • methods that take advantage of the different molecular weights such as dialysis, ultrafiltration, gel filtration, and SDS
  • cell extracts can be made either from the organism which carries the gene of the accessory complex which fulfills the task according to the invention, or from the recombinant host organism, for example E. coli. With these extractions one could avoid other cleaning steps.
  • thermostable in v / o complexes according to the invention can be used to elongate nucleic acids, e.g. B. for polymerase chain reaction, (example 3, 4, and Fig. 21; Fig. 22) DNA sequencing, for labeling nucleic acids and other reactions that involve the in vitro synthesis of nucleic acids.
  • Another object of the present invention is therefore a method for template-dependent elongation of nucleic acids, the nucleic acid being denatured, if necessary, provided with at least one primer under hybridization conditions, the primer being sufficiently complementary to a flanking region of a desired nucleic acid sequence of the template strand and with the aid of a polymerase in the presence of nucleotides, a primer elongation takes place, where as Polymerase a thermostable in w ' f / o complex is used.
  • Methods for the template-dependent elongation of nucleic acids in which the elongation takes place starting from a primer which has been hybridized to the template nucleic acid and provides a free 3'-OH end for the elongation, are known to the person skilled in the art.
  • a PCR polymerase chain reaction is carried out for the amplification.
  • thermostable in vitro complex according to the invention in reverse transcription, with either the complex according to the invention itself having reverse transcriptase activity, or else a suitable enzyme having a reverse transcriptase activity being added, regardless of whether the thermostable in ⁇ ra complex itself has reverse transcriptase activity.
  • thermostable in w ' fro complex for the reverse transcription of RNA into DNA preferred according to the invention, a thermostable in w ' fro complex according to the invention is likewise used, the elongation protein of which itself has a reverse transcriptase activity.
  • This reverse transcriptase activity can be the only polymerase activity of the elongation protein, but can also be present in addition to an existing 5'-3 'DNA polymerase activity.
  • a preferred embodiment of the in w ' fro complex according to the invention comprises the elongation protein from the organism Carboxydothermus hydrogenoformans, as disclosed in EP-A 0 834 569.
  • Sequencing Another preferred use of the 'TRO in vi complex of the invention is the sequencing of nucleic acids according to the method of Sanger. Starting from at least one primer which is sufficiently complementary to a part of the nucleic acid to be sequenced, a template-dependent elongation is carried out. In the case of the sequencing of RNA, it is necessary to carry out a reverse transcription. In the context of this preferred embodiment, the respective derivatives described above are also considered suitable as deoxynucleotides or dideoxynucleotides. In particular, it is preferred for the methods according to the invention for elongating nucleic acids that the nucleic acids formed are labeled.
  • Another object of the present invention a method for labeling nucleic acids, for example by insertion of individual fractures in phosphodiester bonds of the nucleic acid chain, and replacement of one nucleotide at the break points by a labeled nucleotide using a polymerase, wherein the polymerase, an inventive thermostable in w 'fro- Complex is used.
  • a preferred method is the method generally referred to as nick translation, which enables simple labeling of nucleic acids. All of the labeled ribonucleotides or deoxyribonucleotides or derivatives thereof already described above are suitable for this as long as the polymerase accepts them as a substrate. Labeling in the above sense is also a label which is carried out as part of a PCR reaction, in which case labeled nucleotides or derivatives thereof are incorporated into the nucleic acid sequence.
  • the present invention also relates to a kit for elongation and / or amplification and / or reverse transcription and / or sequencing of nucleic acids, it being possible for this kit to comprise one or more containers.
  • the kit itself comprises a) a thermostable in w ' fro complex according to the invention or b) a thermostable in w ' fro complex and optionally an elongation protein having polymerase activity, and optionally one or more of the components selected from the group , which includes primers, buffer substances, nucleotides, ATP, other cofactors and pyrophosphatase.
  • thermostable in w ' fro complex in said kit is present in the form of its individual constituents, that is to say as a thermostable gliding clip protein and thermostable elongation protein, separately or combined in one container, and only forms as such at a later point in time .
  • the present invention relates to a kit for elongation, amplification, reverse transcription, labeling or sequencing of nucleic acids, which additionally contains deoxynucleotides or their derivatives.
  • ribonucleotides or derivatives thereof can also be included in the kit according to the invention, namely if an elongation protein is used which accepts ribonucleotides as a substrate.
  • a preferred embodiment of the kit according to the invention is a kit for sequencing nucleic acids, which in addition to deoxynucleotides or their derivatives, dideoxynucleotides or their derivatives are included for chain termination.
  • the present invention particularly relates to a kit for reverse transcription of nucleic acids, either the complex according to the invention itself having reverse transcriptase activity, or additionally a suitable enzyme which has reverse transcriptase activity, with deoxynucleotides or their derivatives being present Reaction mixture can be included.
  • the kit contains primers and / or deoxynucleotides and / or dideoxynucleotides and / or ribonucleotides and / or their respective derivatives in labeled form.
  • Suitable markings have already been described above in exemplary form. Suitable marking agents are included in a preferred embodiment of the kit according to the invention.
  • the kit also referred to herein as the reagent kit
  • the reagent kit contains the components a) or b) and labeled nucleotides, buffer substances, ATP or other cofactors and / or pyrophosphatase may also be present.
  • a kit is preferred which comprises a suitable buffer, as described above. It is also preferred that the kit according to the invention additionally comprises a pyrophosphatase, ATP and / or other cofactors.
  • the present invention furthermore relates to the use of a thermostable gliding clip protein in in vitro methods for elongation, amplification, labeling or sequencing or reverse transcription of nucleic acids.
  • thermostable in w ' fro complex according to the invention can be used for the purposes of sequencing, amplification, reverse transcription and the like with both short and long nucleic acid fragments, with a reduced overall frequency of errors
  • thermostable in w ' fro complex is used for the purposes of sequencing, amplification and reverse transcription of long nucleic acid fragments.
  • thermostable in w ' fro complex is used for the purposes of sequencing, amplification and reverse transcription of those nucleic acid fragments which form the secondary structure.
  • sequence names are often used, under which the protein or nucleic acid sequences are in the gene bank and the EMBL database. It shows:
  • Fig. 2 is a table similar to that of Fig. 1, but limited to
  • Gliding clip protein and elongation protein from E. coli and A. aeolicus Gliding clip protein and elongation protein from E. coli and A. aeolicus
  • thermostable in w ' fro complex shows a schematic representation of the thermostable in w ' fro complex according to the invention
  • Elongation protein 1; 9 shows an alignment of a conserved region of the
  • HMM hidden Markov models
  • 27 shows the results of a Y2H experiment
  • 28 shows the PCR amplification result of the human collagen gene using the thermostable in w ' fro complex according to the invention
  • FIG. 1 lists, in tabular form, protein sequence names of the thermostable in w ' fro complex according to the invention, and values from pairwise alignments and multiple alignments between archaebacteria and between archaebacteria and the corresponding human genes.
  • the annotation " 1" means the percent identity (%) to the corresponding human gene, calculated from the pairwise alignment (see figures) with BLASTP 2.0.4 [Feb-24-1998] and the annotation " 2" percent identity to the corresponding gene from Archaeoglobus fulgidus, calculated from the pairwise alignment (see figures) with BLASTP 2.0.4 [Feb-24-1998] and the annotation " 3" percentage identity to the corresponding human gene, calculated from the pairwise alignment with FASTA 3.1t02 [March, 1998] ⁇ .
  • the methods are described in more detail in: Altschul, Stephen F., Thomas L. Madden, AIejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J.
  • Die 1 shows the sequence names from the databases and also their SEQ ID number in the case of the glide clip loader 1, the glide clip loader 2, the glide clip, the coupling protein and the elongation protein 1, and also their SEQ ID number, the values in the brackets representing percent identity per number of amino acids The values relate to percent sequence identity to the Archaeoglobus fulgidus sequence in the case of elongation protein 2.
  • the sequence names from the databases and also their "SEQ ID No.” are also shown here.
  • FIG. 3 shows a sketch of a possible form of the thermostable in w ' fro complex according to the invention, the sliding chamber binding to the elongation protein via a coupling protein.
  • Fig. 4 shows a sketch of a possible form of the thermostable in w ' fro complex according to the invention, the sliding chamber binding to the elongation protein via a coupling protein.
  • PCNA human from SEQ ID NO: 11
  • Methanococcus janashii from SEQ ID NO: 13
  • Pyrococcus horikoschii from SEQ ID NO: 14
  • Methanococcus thermoautothrophicus from SEQ ID NO: 15
  • FIG. 5 shows an alignment of a conserved region of the coupling protein and the consensus sequences derived therefrom.
  • the following genes are shown: PfuORF2, DPD2JHUMAN, AF1790 and MJ0702.
  • the SEQ ID numbers can be found in FIG. 1.
  • FIG. 6 shows an alignment of a conserved region of the coupling subunit and the consensus sequences derived therefrom.
  • the following genes are shown: AC11_HUMAN, AF2060, MTH0241, PHBN012 and MJ1422.
  • the SEQ ID numbers can be found in FIG. 1.
  • FIG. 7 shows an alignment of a conserved region of the sliding clamp loader 2 and the consensus sequences derived therefrom.
  • the following genes are shown: AC15_HUMAN, MJ0884, AF1195, MTH0240 and MTH0240.
  • the SEQ ID numbers can be found in FIG. 1.
  • FIG. 8 shows an alignment of a conserved region of the elongation protein 1, and the consensus sequences derived therefrom. The following genes are shown: DPODJHUMAN, MJ0885, MTH1208, PHBT047 and DPOL_ARCFU. The SEQ ID numbers can be found in FIG. 1.
  • FIG. 9 shows an alignment of a conserved region of the elongation protein 2, and the consensus sequences derived therefrom.
  • the following genes are shown: AF1722, MJ1630, PfuORF3, MTH1536 and PHBN021.
  • the SEQ ID numbers can be found in FIG. 1.
  • DP3A_ECOLI DNA Pol III, alpha subunit, Escherichia coli, BB0579: DNA Pol III, alpha subunit, Borrelia burgdorferi, DP3A_HELPY: DNA Pol III, alpha subunit, Helicobacter pylori AA50: Aquifex aeolicus, section 50 and DP3A_ALT DNA Pol III, alpha subunit, Salmonella typhimuhum).
  • AAPOL3B AAPOL3B, DP3B_ECOLI, S.TYPHIM, DP3B_PROMI, DP3B_PSEPU and DP3B_STRC0
  • AAP0L3B Aquifex aeolicus Section 93: DP3B_ECOLI: DNA Pol III, beta chain, Escherichia coli S.TYPHIM, DNA Salmonella P3, DNAMM3 PYBM3, DNA Polmonella P3: DNA Polmonella P3 : DNA Polmonella P3 : DNA Pol III, beta chain, Proteus mirabilis
  • DP3B_PSEPU DNA Pol III, beta chain, Pseudomonas putida DP3B_STRCO: DNA Pol III, beta chain, Streptomyces coelicolo ⁇ .
  • Figure 12 shows a multiple alignment of the glide clip protein sequences to generate the HMM.
  • AAPOL3B Aqufex Aeolicus section 93: DP3B_ECOLI: DNA Pol III, beta chain, Escherichia coli S.TYPHIM: DNA Pol III, beta chain, Salmonella typhimurium: P3B_PROMI DNA Pol III, beta chain, Proteus mirabilis
  • DP3B_PSEPU DNA Pol III, beta chain, Pseudomonas putida DP3B_STRCO: DNA Pol III, beta chain, Streptomyces coelicolor).
  • FIG. 14 shows a multiple alignment of the sliding clip loader 1 protein sequences for generating the HMM.
  • FIG. 16 shows a multiple alignment of the protein sequences of the coupling proteins to generate the HMM.
  • DP3A_ECOLI DNA Pol III, alpha subunit, Escherichia coli, BB0579: DNA Pol III, alpha subunit, Borrelia burgdorferi, DP3AJHELPY: DNA Pol III, alpha subunit, Helicobacter pylori AA50: Aquifex aeolicus, section 50 and DP3A_ALT DNA Pol III, alpha subunit, Salmonella typhimurium).
  • Fig. 20 (Example 2)
  • Recombinant Archaeoglobus fulgidus-PCNA (AF 0335) is a trimer under native conditions.
  • 20A shows proteins with His tag (histidine tag) in fractions 15 (lane 1), 17 (lane 2), 19 (lane 3), 21 (lane 4), 23 (lane 5), 25 ( Lane 6) of the chromatography carried out without urea.
  • 20B shows His-tagged proteins in fractions 10 (lane 1), 11 (lane 2), 12 (lane 3), 13 (lane 4), 14 (lane 5), 15 (lane 6), 16 ( Lane 7), 17 (lane 8) of the denaturing chromatography carried out in the presence of urea.
  • Sample 23 shows a comparison of the activity of an elongation protein with and without glide clip protein.
  • Sample 1 represents an enzyme-free approach.
  • Samples 2-12 also each contained 3 ⁇ l of a 1: 1000 dilution of a fraction of recombinant archeoglobe / u / gr / divs DNA polymerase (starting concentration 7.5 ⁇ g / ⁇ l).
  • Samples 3-7 and 8-12 also 0.5; 1 ; 2; 4 and 8 ul of a fraction of recombinant slide clamp protein from Archaeoglobus fulgidus PCNA.
  • Lane 24 shows the result of the purification of a slide clamp protein:
  • Lane 1 shows a molecular weight standard ((BIO RAD Cat No. 161-0317).
  • 500 ⁇ l bacteria were sedimented directly before induction, according to the manufacturer's instructions for running NuPage gels (NOVEX; Fig. 25) treated and applied.
  • Lane 3 shows the same amount of bacteria 16 hours after the elution.
  • Lanes 4 and 5 each show 8 ⁇ l of the two eluates of the Ni-Nta agarose column after dialysis. By cleaning over Ni-NTA Agarose (Qiagen) are obtained as high-purity fractions of the sliding clip protein of the organism Archaeoglobus fulgidus (see lanes 4 and 5).
  • FIG. 25 shows the expression and purification of the Archaeoglobus fulgidus DNA polymerase (see also Example 7):
  • Lane 1 shows a molecular weight standard ((BIO RAD Cat No. 161-0317).
  • 500 ⁇ l bacteria were sedimented directly before induction, treated and applied according to the instructions for running NuPage gels.
  • Lane 3 shows the same amount of bacteria after 16 hours
  • lanes 4 and 5 each show 8 ⁇ l of the two eluates of the Ni-Nta agarose column after dialysis
  • Lane 6 shows 8 ml of a dialyzed coarse extract
  • Lanes 4 and 5 show highly pure fractions of the Archaeoglobus fulgidus DNA polymerase, which are purified by purification over Ni -NTA- agarose are obtained.
  • FIG. 26 shows the results of the use of an in w ' fro complex according to the invention in the PCR.
  • Lane one shows a PCR reaction without using a glide clip
  • lane 2 shows the result of a PCR reaction using a glide clip protein.
  • Figure 27 shows the results of a "yeast two hybrid" experiment, referred to herein as a Y2H experiment, where row A is populated with cells carrying the empty pGAD424 vector (Clontech, Palo Alto, USA) so that the Transcription activation domain is expressed, the row B is populated with cells that carry the pGAD424 vector, of which the Sacharomyces cerevesiae gene CDC48 is expressed as a fusion protein with the transcription activation domain, the row C is populated with cells which carry the pGAD424 vector, of which the sliding clip gene from Archaeoglobus fulgidus is expressed as a fusion protein with the transcription activation domain, the row D is not is populated with cells and row E is populated with cells which carry the pGAD424 vector, from which the elongation protein gene from Archaeoglobus fulgidus is expressed as a fusion protein with the transcription activation domain.
  • Column 1 is populated with cells that carry the empty pGBT9 vector (Clontech, Palo Alto, USA) so that the DNA binding domain is expressed
  • column 2 is populated with cells that carry the pGBT9 vector, of which the Saccharomyces cerevisiae -Gen UFD3 is expressed as a fusion protein with the DNA binding domain
  • column 3 is populated with cells that carry the pGBT9 vector, from which the gliding clip protein from Archaeoglobus fulgidus is expressed as a fusion protein with the DNA binding domain
  • column 4 is populated with cells that carry the pGBT9 vector, from which the coupling protein from Archaeoglobus fulgidus is expressed as a fusion protein with the DNA binding domain
  • column 5 is populated with cells that carry the pGBT9 vector, from which the elongation protein from Archaeoglobus fulgidus as a fusion protein the DNA binding domain is expressed.
  • FIG. 28 shows the PCR amplification result of the human collagen gene in each case using the thermostable in w ' fro complex according to the invention and without it.
  • the expected amplificate has a size of approximately 1 Kb in both cases.
  • Lane 1 shows a molecular weight marker
  • lane 2 shows the result of the amplification using an elongation protein according to the invention without Slide clamp
  • track 3 shows the result of the amplification using the thermostable in w ' fro complex according to the invention.
  • the DNA is purified from the archaeoglobus fulgidus organism (DSM No. 4304) using known methods. The organisms were grown by the DSM (German Collection for Microorganisms).
  • DSM German Collection for Microorganisms.
  • primers are developed for each gene, which span the complete open reading frame including the stop codon.
  • the primer sequences also contain the start codon only for cloning in pTRC99.
  • the corresponding primers for cloning in pQE30 contain the nucleotides immediately following the start codon as the first gene-specific sequences.
  • restriction ends are added to the primers, which facilitate directed cloning into the expression vector.
  • PCR reactions are carried out with the appropriate annealing temperatures (approximately 35 cycles) and the resulting products are purified. After cleaning, the products are treated with restriction enzymes and purified on an agarose gel in order to be ready for ligation.
  • the expression vector is linearized, purified and diluted by means of restriction enzymes in such a way that it is used for ligation with the amplificates of the genes of the in vitro Complex from the above PCR is ready. The ligation is set up and, after incubation, an aliquot is transformed into one of the E.
  • IPTG is added for induction (125 mg / l). These cultures now grow another 4-21 hours.
  • the cultures are centrifuged and, starting from the vector pQE30, recombinant proteins expressed according to protocol 8 and 11 are extracted and purified from "The QIAexpressionist" (third edition, QIAGEN).
  • the pellets are taken up in a buffer (buffer A: 50 mM Tris -HCI pH 7.9, 50 mM dextrose, 1 mM EDTA. After centrifugation, the cells are taken up again, but buffer A now additionally contains lysozyme (4 mg / ml).
  • buffer B 10 mM Tris-HCl (pH 7.9), 50 mM KCI, 1 mM EDTA, 1 mM PMSF, 0.5% Tween 20, 0.5% Nonidet P40
  • B 10 mM Tris-HCl (pH 7.9), 50 mM KCI, 1 mM EDTA, 1 mM PMSF, 0.5% Tween 20, 0.5% Nonidet P40
  • the supernatant is removed by centrifugation and the overexpressed proteins are precipitated using (NH) 2 S0 4. After centrifugation, the pellets are collected and the proteins are resuspended with buffer A.
  • the resuspended proteins are washed against storage buffer (50 mM Tris HCI (pH 7.9), 50 mM KCI, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 50% glycerol) dialyzed and then stored at + 4 ° C to -70 ° C.
  • storage buffer 50 mM Tris HCI (pH 7.9), 50 mM KCI, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 50% glycerol
  • DNA polymerization activity is measured by incorporating (methyl- 3 H) TTP in trichloric acid-insoluble material or by incorporating digoxigenin-dUTP in unlabeled double-stranded DNA regions with free internal 3 ' ends (Ishino, Y., Iwasaki, H., Fukui, H., Mineno, J., Kato, I., & Schinigawa, H. (1992) Biochimie 74, 131-136). To determine the processivity, the above protein mixtures are used in primer elongation experiments.
  • An M13 single-strand template is introduced into 10 mM Tris-HCl (pH 9.4) and heated (92 ° C.) and cooled (room temperature) together with a universal primer (New England Biolabs) (5′-FITC labeled).
  • Dilution series of the template-primer mixture generated in this way are prepared in a reaction consisting of nucleotides (about 200 ⁇ M to 1 mM), reaction buffer (final concentration: 50 mM KCI, 10 mM Tris-HCl (pH 8.3), 1.5-5 mM MgCI 2 , ATP (0 mM - 200 mM) and protein-stabilizing agents are combined and incubated for 10 minutes at 37 ° C, 52 °, 62 °, 68 °, 74 °, and 78 °.
  • protein mixtures above also serve to measure fidelity and exonuclease activity, whereby the methods described in Kohler et al. (Proc. Natl. Acad. Sci. USA 88: 7958-7962 (1991) or Chase et al. (J. Biol. Chem., 249: 4545-4552 (1972).
  • the protein mixtures in PCR was used (Fig. 26; Methods in Molecular Biology Vol. 15 Humana Press Totowa, New Jersey, 1993, edited by Bruce A. White).
  • FIG. 20A shows His-tagged proteins in fractions 15 (lane 1), 17 (lane 2), 19 (lane 3), 21 (lane 4), 23 (lane 5), 25 (lane 6) or without Urea carried out Chromatography.
  • Example 3 Isolation, provision and use of an elongation protein (Pyrococcus horikoshii DNA polymerase (PH1947)) for the formation of the fro complex according to the invention.
  • an elongation protein Pyrococcus horikoshii DNA polymerase (PH1947)
  • the elongation protein from Pyrococcus horikoshii (Pyrococcus horikoshii DNA polymerase (PH 1947)) was used in PCR reactions and leads to efficient amplification of a specific DNA product.
  • each reaction contained 5 ng template DNA (421 bp Rsa I fragment cloned with adapters in PCR 2.1; (Kaiser C, v.
  • the samples went through 40 cycles of 30 s at 95 ° C; 30 s at 55 ° C and 120 s at 68 ° C. 5 ⁇ l of the batches were then removed and separated on a 1% agarose gel in 1 ⁇ TAE buffer (40 mM Tris acetate; 20 mM sodium acetate; 10 mM EDTA; pH 7.2) at 10 V / cm.
  • the Archaeoglobe fw / g / ' dus DNA polymerase AF 0497 can generate PCR products as efficiently as Taq polymerase.
  • 1 unit Taq polymerase and 1 ⁇ l Archaeoglobus fu / g / dus DNA polymerase AF 0497 crude extract (see 25) were used individually in standard PCR reactions to compare the activity.
  • Each reaction contained 20 ng M13 MP18 ssDNA in addition to the enzyme; 0.2 mM each of dATP, dTTP, dCTP and dGTP; 1.5 ⁇ M DNA each Primer of the following nucleotide sequence:
  • GGATTGACCGTAATGGGATAGGTTACGTT SEQ ID NO .: 47
  • AGCGGATAACAATTTCACACAGGAAACAG SEQ ID NO .: 48
  • the samples went through a different number of cycles consisting of 30 s at 95 ° C; 30 s at 59 ° C and 60 s at 68 ° C.
  • the final components of a 50 ⁇ l volume included the following components of an in w ' fro complex according to the invention: 10 mM Tris / HCl pH 7.5; 50 mM KCI; 2 mM MgCl 2 ; 10 ⁇ g BSA (can also be omitted); 0.5 mM digoxigenin-dUTP (DIG-dUTP, Boehringer Mannheim); 40 nM; 0.5 ⁇ g poly dA / 40 nM OligodT (20mer) hybrid. Sample 1 without elongation protein.
  • Samples 2-12 also contained 3 ⁇ l of a 1: 1000 dilution of a fraction of recombinant Archaeoglobus ftv / g / dus DNA polymerase (elongation protein). Samples 3-7 and 8-12 also 0.5; 1 ; 2; 4 and 8 ⁇ l of a fraction of recombinant slide clamp protein from Archaeoglobus fulgidus.
  • the samples were incubated at 68 ° C. for 30 minutes and then nucleic acids by precipitation with 3 parts by volume of ethanol / 3 M sodium acetate pH 5.2 (30/1). Like. The precipitate was resuspended in 20 ⁇ l 100 mM Tris-HCl (pH 7.9) and 10 ⁇ l each in individual cavities of a 96-well silent screen plate with Nylon 66 Biodyne B 0.45 ⁇ m pore (Nalge Nunc) dripped on and the nucleic acids fixed for 10 minutes at 70 ° C on the membrane. Incorporated digoxigenin-dUTP (Boehringer Mannheim) was detected using the DIG Luminiscent Detection Kit for Nucleic Acids (Boehringer Mannheim).
  • PCNA clearly stimulates the integration of DIG-dUTP by the DNA polymerase (cf. lanes 3-7 with lane 2).
  • the PCNA fraction used has no endogenous polymerase activity (lanes 8-12).
  • Example 6 Amplification; Cloning, Expression and Purification of a
  • Archipelaginus protein from Archaeoglobus fulgidus is Archipelaginus protein from Archaeoglobus fulgidus:
  • Example 7 Provision of an elongation protein Expression and purification of the archaeoglobus fu / g / dus DNA polymerase: pQE 30 plasmid DNA (QIAGEN) with the gene inserted in the correct reading frame for the elongation protein, the Archaeoglobus fulgidus DNA polymerase AF 0497, was followed Instructions from "The QIAexpressionist (third edition; QIAGEN)" transformed into competent E.coli M15 [prep 4]. Transfer to 1 L culture medium and induced protein expression also took place according to the schemes given there. After an induction time of 16 hours, the bacteria were sedimented for 10 minutes at 5000 g.
  • QIAGEN plasmid DNA
  • the QIAexpressionist (third edition; QIAGEN; protocol 8, protocol 11) was used to obtain highly pure fractions. Only the bound proteins were eluted with 2 x 2 ml elution buffer and not as described there with 4 x 0.5 ml elution buffer. To obtain crude extracts from recombinant proteins, the bacterial sediments were alternatively washed with 100 ml of buffer A (50 mM Tris-HCl pH 7.9; 50 mM glucose; 1 mM EDTA) and, after renewed centrifugation, resuspended in 50 ml of buffer A with 4 mg / ml of lysozyme .
  • buffer A 50 mM Tris-HCl pH 7.9; 50 mM glucose; 1 mM EDTA
  • lysis buffer (10 mM Tris-HCl pH 7.9; 50 mM KCI; 1 mM EDTA; 0.5% Tween 20; 0.5% IGPAL) and E. coli proteins were added Incubated for 60 minutes at 75 ° C in a water bath. After subsequent centrifugation for 15 minutes at 27,000 g, the supernatant was precipitated by slowly adding 40 mg of crystalline ammonium sulfate per ml of extract with permanent stirring. Precipitated proteins were sedimented at 27000 g for 10 minutes and the sediment was resuspended in 20 ml of buffer A.
  • Example 8 Use of an In Vitro Complex According to the Invention in PCR
  • each reaction contained a PCR-amplified and unpurified PCNA gene fragment (PCR reaction for specific amplification of the PCNA fragment: 0.5 ⁇ l Archeoglobus fulgidus polymerase; 0.2 mM each dATP, dTTP, dCTP and dGTP; 1.5 ⁇ M each of the specific primers (5 ' -ACG CGC GGA TCC ATA GAC GTC ATA ATG ACC GG- 3 '(SEQ ID No .: 49); 5 ' -TAC GGG GTA CCC GAG CCA AAA TTG GGT AAA G-3 '(SEQ ID No.:50); 50 mM KCI; 2 mM MgCI 2 ; 10 mM Tris- HCI (pH 7.5) and 50
  • a positive control was also amplified by PCR, cloned into the vectors pGBT9 (see also vertical columns in FIG. 27) and pGAD424 (see also horizontal rows in FIG. 27) and by gap repair in the yeast strain PJ69-4a (for pGAD424 ) and PJ69-4alpha (for pGBT9) as hybrid proteins. Diploid cells containing both vectors were generated by pairing according to the grid shown in Fig. 27. The expression of three independent reporters (HIS3, ADE2 and MEL1) was measured. The growth on medium without histidine and adenine is shown in FIG. 27. In this experiment, the cells that carry both vectors and in which the cells also grow Expression products of these two vectors bind to each other. As a result of the binding of the expression products, transcription of the reporter genes is initiated, so that the histidine and adenine auxotrophy are abolished and these cells are able to grow in said medium.
  • Fig. 27 shows a binding between the proteins of the positive control, the elongation protein and the glide clip protein, the glide clip protein and the glide clip protein and the coupling protein and the glide clip protein.
  • thermostable in w ' fro complex according to the invention can be used very well in the amplification of genomic DNA fragments. This results in efficient amplification even when using small amounts of a template or an elongation protein.
  • Example 10 a section of the human collagen gene was amplified, once using the thermostable in w ' fro complex according to the invention and once using an elongation protein alone.
  • nucleotide mix 25 mM starting solution containing each nucleotide A, C, G and T
  • 0.2 ⁇ l of each primer 100 pmol / ⁇ l starting solution “collagen forward” 5′-TAA AGG GTC ACC GTG GCT TC-3 '(SEQ ID NO .: 53), 100 pmol / ⁇ l starting solution "collagen reverse”5'- CGA ACC ACA TTG GCA TCA TC-3' SEQ ID NO .: 54), 0.8 ⁇ l DNA (200 ng / ul human genomic DNA from Boehringer Mannheim), 5 ul 10 x PCR buffer (pH 7.5) (100mM Tris-HCl pH7.5, 500mM KCI, 15mM MgCI 2 ), 1 ⁇ l elongatin protein (AF1722, 7.5 ⁇ g / ⁇ l protein concentration) and 8 ⁇ l slide clamp protein (AF 0335, protein concentration 0.3

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Abstract

Le complexe in vitro thermostable selon la présente invention destiné à l'élongation, dépendante de matrices, d'acide nucléique comporte une protéine agrafe thermostable et une protéine d'élongation thermostable.
PCT/DE1999/002480 1998-08-06 1999-08-06 Complexe in vitro thermostable a activite de polymerase WO2000008164A2 (fr)

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EP99942776A EP1100923A2 (fr) 1998-08-06 1999-08-06 Complexe in vitro thermostable a activite de polymerase
AU56171/99A AU5617199A (en) 1998-08-06 1999-08-06 Thermostable (in vitro) complex with polymerase activity
CA002338185A CA2338185A1 (fr) 1998-08-06 1999-08-06 Complexe in vitro thermostable a activite de polymerase
DE19981494T DE19981494D2 (de) 1998-08-06 1999-08-06 Thermostabiler in Vitro-Komplex mit Polymeraseaktivität
JP2000563788A JP2002522042A (ja) 1998-08-06 1999-08-06 ポリメラーゼ活性を有する熱安定性ｉｎｖｉｔｒｏ複合体

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WO2001011051A3 (fr) * 1999-08-06 2001-06-21 Lion Bioscience Ag Proteines chimeres
WO2000065062A3 (fr) * 1999-04-21 2002-02-14 Centre Nat Rech Scient Sequence genomique et polypeptides de pyrococcus abissy, leurs fragments et leurs utilisations
WO2003014151A3 (fr) * 2001-08-10 2003-09-04 Genset Sa Adn complementaire et proteines humains et leurs utilisations
JP2010000089A (ja) * 2000-05-26 2010-01-07 Bio Rad Lab Inc 改良された核酸修飾酵素
EP3381947A4 (fr) * 2015-11-27 2019-04-10 Kyushu University National University Corporation Mutant d'adn polymérase

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JP6699560B2 (ja) * 2014-11-28 2020-05-27 東洋紡株式会社 Ｐｃｎａ単量体
WO2016084879A1 (fr) * 2014-11-28 2016-06-02 東洋紡株式会社 Dispositif d'amplification d'acide nucléique

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US5583026A (en) * 1994-08-31 1996-12-10 Cornell Research Foundation, Inc. Process for reconstituting the polymerase III* and other subassemblies of E. coli DNA polymerase III holoenzyme from peptide subunits
WO1993015115A1 (fr) * 1992-01-24 1993-08-05 Cornell Research Foundation, Inc. HOLOENZYME ADN POLYMERASE III et E. COLI ET SOUS-UNITES
US5633159A (en) * 1995-03-10 1997-05-27 Becton, Dickinson And Company DNA polymerase III β-subunit from mycobacteriophage DS6A
EP0983365A1 (fr) * 1997-04-08 2000-03-08 The Rockefeller University Enzyme derivee d'organismes thermophiles fonctionnant comme replicase chromosomique, production et emplois de cette enzyme
US6238905B1 (en) * 1997-09-12 2001-05-29 University Technology Corporation Thermophilic polymerase III holoenzyme
AU7105798A (en) * 1998-04-09 1999-11-01 Rockfeller University, The Enzyme derived from thermophilic organisms that functions as a chromosomal replicase, and preparation and uses thereof

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000065062A3 (fr) * 1999-04-21 2002-02-14 Centre Nat Rech Scient Sequence genomique et polypeptides de pyrococcus abissy, leurs fragments et leurs utilisations
WO2001011051A3 (fr) * 1999-08-06 2001-06-21 Lion Bioscience Ag Proteines chimeres
JP2010000089A (ja) * 2000-05-26 2010-01-07 Bio Rad Lab Inc 改良された核酸修飾酵素
WO2003014151A3 (fr) * 2001-08-10 2003-09-04 Genset Sa Adn complementaire et proteines humains et leurs utilisations
EP3381947A4 (fr) * 2015-11-27 2019-04-10 Kyushu University National University Corporation Mutant d'adn polymérase

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