DE19840771A1 - A thermostable in vitro polymerase complex for template-dependent elongation of nucleic acids in amplification or reverse transcription methods - Google Patents

Abstract

A thermostable in vitro complex for template-dependent elongation of nucleic acids comprises a thermostable sliding clamp protein, which is connected with an elongation protein that shows thermostable polymerase activity is new. Independent claims are also included for the following: (1) a thermostable accessory in vitro complex characterized in that it contains a sliding clamp protein and a coupling protein, as defined above; (2) a recombinant DNA sequence, which encodes a thermostable in vitro complex or a thermostable accessory complex; (3) a vector that contains recombinant DNA encoding a sliding clamp protein and a coupling protein and/or an elongation protein; (4) a host cell transformed with one or more vectors of (3); (5) a method for the production of a thermostable in vitro complex or thermostable in vitro accessory complex as above; (6) a method for template-dependent elongation of nucleic acids; (7) a method of marking nucleic acids through generating a single break in the phosphodiester bond of the nucleic acid chain and substituting a nucleotide at the break site with a marked nucleotide with the help of a polymerase, where the polymerase is a thermostable in vitro complex as above; and (8) a reagent kit for elongation and/or amplification and/or reverse-transcription and/or sequencing and/or marking of nucleic acids.

Description

The invention relates to a thermostable in vitro complex for Template-dependent elongation of nucleic acids, a thermostable prokaryotic accessory in vitro complex and coding for it DNA sequences and vectors. The invention further relates to Use of the complexes according to the invention in processes for Template-dependent elongation of nucleic acids, such as PCR reactions or DNA sequencing, in which template-dependent DNA in vitro Strand synthesis takes place. Finally, the invention relates to Reagent kits for carrying out the method according to the invention.

DNA polymerases belong to a group of enzymes that single-stranded DNA as a template for the synthesis of a complementary one Use DNA strands. These enzymes play an important role in the Nucleic acid metabolism, including the processes of DNA replication, Repair and recombination. DNA polymerases were found in all cellular Organisms identified, from bacterial to human cells, in many viruses as well as in bacteriophages (Kornberg, A. & Baker, T. A. (1991) DNA replication WH Freeman, New York, NY). You usually take it Archaebacteria and the eubacteria together to form the group of Prokaryotes, the organisms without a real cell nucleus, and presents them with the Eukaryotes, the organisms with a real cell nucleus. Together are often many polymerases from a wide variety of organisms Similarities in the amino acid sequence as well as similarities in the structure (Wang, J., Sattar, A.K.M.A .; Wang, C.C., Karam, J.D., Konigsberg, W.H. & Steitz, T.A. (1997) Crystal Structure of pol a familily replication DNA polymerase from bacteriophage RB69.Cell 89, 1087-1099). Organisms like humans have a variety of DNA-dependent polymerases  which are not all responsible for DNA replication, however some also do DNA repair. Replicative DNA polymerases usually consist of protein complexes with several subunits, which replicate the chromosomes of cellular organisms and viruses. A general property of these replicating polymerases is in general high processivity, that is, their ability to handle thousands of Polymerize nucleotides without dissociating from the DNA template (Kornberg, A. & Baker, T.A. (1991) DNA Replication. WH Freeman, New York, NY).

Until recently, the only well-understood, high-process replication mechanisms in cells were those used by cellular replicases (a protein complex with polymerase activity) and the replication apparatus of the bacteriophage T4 or T7. The mechanism underlying the mechanism includes a protein with a ring-like structure, a "glide clip" that encases the DNA and binds the catalytic polymerase unit - the "elongation protein" - to the DNA (Stukenberg, PT, Studwell-Vaughan, PS &O'Donell, M. (1991) Mechanism of the β-clamp of DNA polymerase III holoenzyme. J. Biol. Chem. 266, 11328-11334; Kuriyan, J. &O'Donnel, M. (1993) Sliding clamps of DNA polymerases. J Mol. Biol. 234, 915-925). The glide clip is often linked to the elongation protein via one or more other proteins, the “coupling proteins”. The three-dimensional structure of various gliding clip proteins has already been determined:

• - that of the eukaryotic proliferating cell nuclear antigen (PCNA) (Krishna, T.S.R., Kong, X.-P., Gary, S., Burgers, P. M. & Kuriyan, J. (1994) Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 79, 1233-1243; Gulbis, J.M. Kelman, Z., Hurwitz, J., O'Donnel, M. & Kuriyan, J. (1996) Structure of the C-terminal region of p21WAF1 / CIP1 complexed with human PCNA. Cell 87, 297-306),  
• - that of the β subunit of polymerase III of the eubacterium Escherichia coli), (Kong, X.-P., Onrust, R., O'Donnell, M. & Kuriyan, J. (1992) Three dimensional structure of the β subunit of Escherichia coli DNA polymerase III holoenzymes; a sliding DNA clamp. Cell 69, 425-437)
• - and that of the bacteriophage T4 Gen45 protein (Kelman, Zvi, Hurwitz, J. O'Donnel, Mike (1998) Structure, 6, 121-125).

The overall structure of these slide clamps is very similar; the pictures of the Overall protein structure of PCNA, the β subunit and the gp45 rings are superimposed on one another (Kelman, Z. & O'Donnell, M. (1995) Structural and functional similarities of prokaryotic and eukaryotic sliding clamps. Nucleid Acids Res. 23, 3613-3620). Every ring has comparable ones Dimensions and a central opening large enough to accommodate duplex DNA, i.e. a DNA double strand consisting of the two complementary DNA strands.

The glide clip cannot wrap itself around the DNA in vivo position; it has to be fixed around the DNA by a clip loader. This clamp loader is a complex protein found in prokaryotes and Eukaryotes consists of a large number of subunits and that of Eubacterium Escherichia coli γ complex or in humans Replication factor C (RF-C) is called (Kelman, Z. & O'Donnell, M. (1994) DNA replication - enzymology and mechanisms. Curr. Opin. Ghent. Dev. 4, 185-195). The glide clip loader recognizes the 3 'end of the Single-strand duplexes (primer template) and positions the glide clip in the presence of ATP around the DNA. The glide clip that holds the DNA then encloses, interacts with polymerases, thus ensuring one fast and processive DNA synthesis.

In the case of the bacteriophage T7, the same goal is a processive DNA Synthesis achieved using a structurally different protein complex. The  Phage expresses its own catalytic polymerase, the T7 polymerase, the product of gene 5, which contains a protein from the host Escherichia coli that binds to thioredoxin and one that replicase highly processive DNA replication enabled (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 also form here, however, this bracket does not have the same structure as e.g. B. in the case of the eukaryotic PCNA.

It is often necessary - as in the case of human polymerase δ - that Proteins (coupling proteins) the connection between the catalytic active part of the polymerase and the processivity factor (sliding clamp) create. In humans, this is the small subunit of δ polymerase (Zhang, S.-J., Zeng, X.-R., Zhang, P., Toomey, N.L., Chuang, R.Y., Chang, L.-S., and Lee, M.Y.W.T. (1994). A conserved region in the amino terminus of DNA polymerase δ is involved in proliferating cell nuclear antigen binding. J. Biol. Chem. 270, 7988-7992). In the case of T7 polymerase, however, binds the processivity factor directly the catalytic unit of the polymerase.

DNA polymerases are characterized by two properties, among others characterized their elongation rate, i.e. the number of nucleotides they can incorporate into a growing strand of DNA per second and their dissociation constant. If the polymerase after each Incorporation of one of the nucleotides into the growing chain again dissociated from the strand (i.e. one elongation step occurs per Binding event), then the processivity has the value 1 and Polymerase is not processive. If the polymerase for repeated Nucleic acid incorporation remains attached to the strand, then the replication mode is called processive and can have a value of reach several thousand (see also: Methods in Enzymology  Volume 262, DNA Replication, Edited by J.L. Campbell, Academic press 1995, pp. 270-280).

For most in vitro applications such as PCR or Sequencing processes, processivity is a desirable property, which, however, are the thermostable ones previously used in these reactions Have only a small amount of enzymes, whereas the temperature sensitive T7 polymerase associated with thioredoxin Processivity of several thousand nucleotides. In comparison - the thermostable DNA polymerase from Thermus thermophilus or aquaticus have only 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).

The U.S. Patents 4,683,195, 4,800,195 and 4,683,202 describe the Application of such thermostable DNA polymerases in the polymerase Chain reaction (PCR). In the PCR, using primers, Template, nucleotides, a DNA polymerase of a corresponding buffer and appropriate reaction conditions, DNA is newly synthesized. Here will the double-stranded target sequence mostly melted thermally, two Oligonucleotides are hybridized and the complementary sequence is used the incorporation of nucleotides by the polymerase on the template synthesized. The extension product of each primer serves as a template for the next cycle. This PCR is preferably used a thermostable polymerase, which cyclic, thermal Melting of the DNA strands survives. So is often Taq DNA Polymerase used (U.S. Patent 4,965,188). The processivity of the Taq However, as stated above, DNA polymerase is relatively low in comparison to the T7 polymerase.

DNA polymerases are also used in DNA sequence determination (Sanger et al., Proc. Natl. Acad. Sci., USA 74: 5463-5467 (1997)). Frequently  becomes a T7 DNA polymerase during Sanger sequencing used (Tabor, S. and Richardson, C.C. Proc. Natl. Acad. Sci., USA 86: 4076-4080 (1989)). Later the cycle sequencing process developed (Murray, V. (1989) Nucleic Acids Res. 17, 8889), which no requires single-stranded template and initiation of the sequence reaction allowed with relatively small amounts of template. The here for Coming polymerases can e.g. B. the above-mentioned Taq Polymerase (U.S. Patent 5,075,216) or the polymerase of Thermotoga neapolitana (WO 96/10640) or other thermostable Polymerases. Newer methods couple exponential amplification and the sequencing of a DNA fragment in one step so that it it is possible to sequence genomic DNA directly. One of the procedures the so-called DEXAS process (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 196 53 439.9 and DE 196 53 494.1) are used a polymerase with reduced discrimination ability Dideoxynucleotides (ddNTPs) vs. Deoxynucleotides (dNTPs) as well as a reaction buffer, two primers that preferentially not equimolar are present, and the above nucleotides, then in several cycles to obtain a complete, sequence-specific DNA ladder of a fragment which is spanned by the primers. A further development of this The method consists in the use of a polymerase mixture, whereby one of the two polymerase discriminates between ddNTPs and dNTPs, while the second has a reduced ability to discriminate (Nucleic Acids Res 1997 May 15; 25 (10): 2032-2034 Direct DNA sequence determination from total genomic DNA. Kilger C, Pääbo S).

DNA polymerases are also used in reverse transcription from RNA to DNA. Here RNA serves as a template and the polymerase synthesizes a complementary strand of DNA. Is used  here z. B. the thermostable DNA polymerase from the organism Thermus thermusphilus (Tth) (U.S. Patent 5,322,770).

It may also be desirable for the polymerase to be proof-read. Has activity, that is 3'-5 'exonuclease activity. This Property is particularly desirable if that too synthesizing product with a low error rate at the Nucleotide incorporation is to be produced.

The above enzymes are commonly used in PCR reactions are largely not part of the actual ones Replication enzymes, but mostly enzymes, one of which assumes that they are involved in DNA repair, which is why Processivity is relatively low.

It was therefore an object of the present invention to provide several of the aforementioned properties of polymerases, especially high Processivity and thermal stability for in vitro use Unite reactions.

This object was achieved according to the invention by providing a thermostable in vitro complex for template-dependent elongation of Nucleic acids comprising a thermostable glide clip protein which with a thermostable polymerase activity Elongation protein is linked. This complex can be Reactions such as B. in PCR reactions, used and has high processivity. It is also an advantage if the complex is a has a low error rate in nucleotide incorporation, i.e. one has increased fidelity. This complex can thus be used for the elongation of the Amplification and sequencing of nucleic acids can be used. This complex can preferably be used in standard PCR reactions.  

To determine the usability of such a complex in standard PCR To enable reactions, simple handling must be guaranteed his. So the replication apparatus of the Simian virus 40 was already in composed in vitro (Wage, S. & Stillman, B. (1994) Nature, Vol. 369, 207-221), but for standard PCR reactions this is Replication apparatus is not suitable because, among other things, it is not thermally stable is. It is therefore the subject of the invention to provide a thermostable in vitro To provide complex with polymerase activity, which in standard PCR Reactions can be used and has a high processivity.

In particular, the invention relates to a thermostable prokaryotic in vitro complex for elongation of nucleic acids thermostable glide clip protein, which is the complementary Completely or partially encloses nucleic acid strands, and a thermostable, Protein comprising polymerase activity, said protein or this protein complex is coupled to the glide clip protein.

In the context of the present invention, the term includes Elongation protein exhibiting polymerase activity too Protein complexes or subunits exhibiting polymerase activity such complexes that carry the polymerase activity.

Thermostable in the sense of the present invention means that the accessory complex with high processivity in growing nucleotides Nucleic acid strands incorporated at both low and high Temperatures that occur in the PCR or other reaction, such as e.g. B. DNA sequencing.

The PCR consists e.g. B. usually from the steps of denaturation (70 ° C to 98 ° C), annealing (40 ° C to 78 ° C) and DNA Strand synthesis (60 ° C to 76 ° C). So this complex must at least between approx. 60 ° C and approx. 70 ° C, in particular between 60 ° C and 76 ° C  and particularly preferably be functional between 40 ° C and 98 ° C. There must be no irreversible during the entire reaction Signs of denaturation of the complex or individual components occur which prevent or inhibit the elongation reaction.

The coupling between slide clamp protein and polymerase activity having elongation protein can by covalent, but also by non-covalent binding. In a preferred embodiment are glide clip protein and elongation protein via a coupling protein connected.

In a further preferred embodiment of the present invention originate in the thermostable in vitro complex according to the invention associated proteins from archaebacteria. However, it is part of the present invention also possible that the associated proteins from Eubacteria. The present invention also relates to an inventive thermostable in vitro complex, in which the associated proteins partly from archaebacteria and partly from Eubacteria.

For the purposes of the present invention, the term prokaryotic includes Protein both proteins from archaebacteria and proteins from eubacteria. It is known that the replication apparatus in Archaea corresponds to that of eukaryotic replication is similar, although the Genome organization in eukaryotes and archaea is completely different and the cellular structure of the eubacteria resembles that of the archaea. (Edgell, D.R. and Doolittle, W.F. (1997). Archaea and the origin (s) of DNA replication proteins. Cell 89, 995-998).

Furthermore, a preferred embodiment of the present Invention a thermostable prokaryotic in vitro complex in which a Proteinase-having protein complex is present, which consists of a  Coupling protein and one or more polymerase activity pointing elongation protein. A is also preferred inventive thermostable prokaryotic in vitro complex, at which the glide clip protein has an annular structure which completely or partially encloses the complementary nucleic acid strands.

The slide bracket

The following explanations are intended to serve the function and the possible manifestations of the glide clip protein better understand.

The glide clip protein fulfills the function of attaching the polymerase activity to the Bind DNA. Either the glide clip protein itself encloses that DNA in whole or in part or by association with the polymerase activity having protein or on the polymerase activity pointing protein complex or its subunit becomes a bracket educated. In any case, this bracketing makes processivity significantly increased, at least one and a half times.

This means that the in vitro complex according to the invention has at least one one and a half times the processivity compared to the elongation protein alone, or in comparison to a polymerase activity Protein complex without slide clip or a subunit thereof.

Homologs of the "proliferating cell Nuclear Antigen "protein complex from the human genome, or homologue of the likewise circular "β-clamp" protein complex from E. coli, which come from thermostable organisms and are therefore thermostable are, or come from non-thermostable organisms, and subsequently were 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. Veltman, Gerard Venema, and Vincent G.H. Eijsink Engineering an enzyme to resist boiling PNAS 1998, 95: 2056-2060). The slide clamp can be used several components. Those in the human genome identified glide clip consists of three PCNA protein components (SEQ ID NO: 11) (homotrimers), which identified in the E. coli genome Slide clamp consists of two components (SEQ ID NO: 35) (Homodimer).

As a slide clamp in the sense of the present invention in particular to understand any protein that has the functional property has the polymerase processivity increase or the decrease in Error rate is used. For this purpose, the slide clip can be an annular one have three-dimensional structure or by coupling to another Protein form a ring-shaped three-dimensional structure, through which they are in the It is able to completely or partially enclose single and double-stranded DNA.

A slide clamp in the sense of the present invention is to be understood in particular as a protein of this type

• 1. to the human (eukaryotes) PCNA amino acid sequence (SEQ ID NO: 11) with a length of at least 100 amino acids a sequence alignment has at least 20% sequence identity has or that
• 2. the bacterial β-clamp sequence from E. coli (Eubacteria) (SEQ ID NO: 35) of at least 100 amino acids in length Sequence alignment has at least 20% sequence identity has or that
• 3. to the amino acid sequence of the PCNA homolog from Archaeoglobus Fulgidus (Archaea) (SEQ ID NO: 12) on a length of at least 100 amino acids in a sequence alignment one at least Has 20% sequence identity.

The slide clip according to the invention can be one or more of the have the aforementioned features.

The sequence identities listed in Fig. 1 were determined with the BLAST algorithm according to Altschul, SF, Gish, W., Miller, W., Myers, EW, and Lipman, DJ, J. Mol. Biol. 215, 403-410 (1990) determined and are explained below.

Proteins that include one or both of the following consensus sequences and that differ from one of these sequences at no more than four positions are also to be understood as sliding clamps in the sense of the present invention ( FIG. 4):

Region 1 (SEQ ID NO: 39)

[G A V L I M P F W] -D-X-X-X- [G A V L I M P F W] -X-X- [G A V L I M P F W] -X- [G A V L I M P F W] -X- [G A V L I M P F W] -X-X-X-X-F-X-X-Y-X-X-D and or

Region 2 (SEQ ID NO: 40)

[G A V L I M P F W] -X (3) -L-A-P- [K R H D E] - [G A V L I M P F W] -E.

The 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 summarized:
G, A, V, L, I, M, P, 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)
In addition, X in the 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. 12. Thus, in the sense of the present invention, a slide clamp is to be understood in particular as any protein that has a score of more than 20 with the Hidden Markov model generated in this way ( FIG. 12). 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).

A hidden Markov model was generated from the multiple alignment of E. coli β-clamp homologs shown in Fig. 13. In the sense of the present invention, a slide clamp is therefore to be understood in particular as any protein which has a score of more than 25 with the Hidden Markov model generated in this way ( FIG. 13).

The slide bracket can be constructed from several components that are firmly bound together by a characteristic bond, so that a stable ring-shaped molecular complex is formed, which is not without Can dissociate more from DNA. This does not make it a fixed one covalent binding to the DNA enables free movement but not hampered. The processivity increasing Slide clip proteins also have characteristic local ones Molecular properties in the region of the interaction region with DNA, which facilitate the free movement and through in this region embedded water molecules can be supported.

A preferred embodiment of the present invention is furthermore in particular a thermostable prokaryotic in vitro complex, where the glide clip protein is one of the following: AF0335 Archaeoglobus Fulgidus, MJ0247 from Methanococcus Jannaschii, PHLA008  from Pyrococcus horikoschii, MTH1312 from Methanobacterium Thermoautotrophicus and AE000761_7 from Aquifex Aeolicus.

In particular, thermostable prokaryotic in vitro complexes from The subject matter of this application comprises, wherein the gliding clip protein is a Amino acid sequence according to SEQ ID No. 11, 12, 13, 14, 15 and 36 (Aquifex Aeolicuc).

The sliding clamp loader

Furthermore, it is to be understood as a preferred embodiment if too this complex according to the invention additionally a sliding clamp loader heard who the components of the slide bracket around the uninterrupted DNA strand around, or this again removed when the reaction is stopped. This sliding clamp loader is preferably associated with the in vitro complex according to the invention.

In humans, the sliding clamp loader consists of five subunits, 4 small (slide clamp loader 1) and a large subunit (Sliding clamp loader 2). According to the invention serve as sliding clamp loaders for example one or more prokaryotic homologues of im Humans identified the "Replication Factor C" protein complex (Slide clip loader 1: SEQ ID NO: 1, 32, 33, 34 and slide clip loader 2 SEQ ID NO: 6).

As a sliding clamp loader 1 in the sense of the present invention in particular to understand such a protein that is related to human (Eukaryotes) amino acid sequence (SEQ ID NO: 1, 32, 33, 34) on one Length of at least 100 amino acids with a sequence alignment one has at least 20% sequence identity.

As a sliding clamp loader 2 in the sense of the present invention in particular to understand such a protein that leads to the  human (eukaryotes) amino acid sequence (SEQ ID NO: 6) on one Length of at least 150 amino acids with a sequence alignment one has at least 20% sequence identity.

For example, the homologs from archaebacteria listed in Fig. 1 are suitable as clip loaders (SEQ ID NO: 3, 4, 5; homologs to slide clip loader 1 and SEQ ID NO: 8, 9, 10, homologues to slide clip loader 2). Therefore, a thermostable prokaryotic in vitro complex is particularly preferred, an RFC-homologous protein or protein complex being additionally present.

For the purposes of the present invention, sliding clamp loader 1 should therefore be understood to mean, for example, any protein which contains the following consensus sequences and does not deviate from this sequence at more than four positions ( FIG. 6):

SEQ ID NO: 41

C-N-Y-X-S- [K R H D E] -I-I-X- [G A V L I M P F W] - [G A V L I M P F W] -Q-S-R-C-X-X-F-R-F- X-P- [G A V L I M P F W].

For the purposes of the present invention, sliding clamp loader 2 is therefore also to be understood, for example, to be any protein which contains the following consensus sequences and does not deviate from this sequence at more than four positions ( FIG. 7):

SEQ ID NO: 42

K-X-X-L-L-X-G-P-P-G-X-G-K-T- [S T N Q Y C] -X- [G A V L I M P F W] -X-X- [G A V L I M P F W].

A hidden Markov model was also generated from the multiple alignment of human glide clip loader 1 homologs shown in Fig. 14. Sliding clamp loader 1 in the sense of the present invention is therefore to be understood as any protein which has a score of more than 25 with the Hidden Markov model generated in this way ( FIG. 14).

A hidden Markov model was also generated from the multiple alignment of human glide clip loader 2 homologs shown in Fig. 15. Sliding clamp loader 2 in the sense of the present invention is therefore to be understood as any protein that has a score of more than 15 with the Hidden Markov model generated in this way ( FIG. 15).

It is also preferred if the Eubacterium Escherichia coli γ-complex homologous protein is present as a sliding clamp loader.

Also preferred is a thermostable in vitro according to the invention Complex for elongation of nucleic acids, in which in addition to the Slide clamp loader may also have ATP, preferably also associated.

The coupling protein

The coupling protein has the function of connecting the elongation protein and the glide clip protein. Coupling protein in the sense of the present invention is to be understood in particular to be any protein which has the function described above. The homologs listed in Fig. 1 for the human sequence of the coupling subunit (DPD2_HUMAN, [SEQ ID NO: 16]) from archaebacteria are suitable as coupling proteins (SEQ ID NO: 17, 18, 19, 20, 21).

As a coupling protein in the sense of the present invention is in particular to understand such a protein that is related to human (eukaryotes) Amino acid sequence (SEQ ID NO: 16) of at least 150 in length Amino acids in a sequence alignment are at least 18% Has sequence identity.  

Coupling protein for the purposes of the present invention is therefore to be understood as any protein which contains the following consensus sequence and does not deviate from this sequence at more than four positions ( FIG. 5):

SEQ ID NO: 43

[FL] - [G A V L I M P F W] -X-X- [G A V L I M P F W] -X-G-X (13) - [G A V L I M P F W] -X- [YR] - [G A V L I M P F W] -X- [G A V L I M P F W] -A-G- [DN] - [G A V L I M P F W] - [G A V L I M P F W] - [DS].

A hidden Markov model was also generated from the multiple alignment of homologues to the human coupling subunit shown in Fig. 16. As a coupling subunit in the sense of the present invention, any protein is to be understood which has a score of more than 10 with the Hidden Markov model generated in this way ( Fig. 16).

Elongation protein exhibiting polymerase activity or Enzyme complex or subunit thereof having polymerase activity

Some elongation proteins require the presence of one Coupling protein (coupling subunit) to at all To have polymerase activity. But it is also conceivable that Elongation protein binds directly to the glide clip. Other Elongation proteins require the presence of a coupling protein for the bond to the slide bracket.

The elongation protein has 5'-3 'polymerase activity or reverse transcriptase activity. Suitable elongation proteins are, for example, the homologues from archaebacteria listed in Fig. 1 for human elongation protein (SEQ ID NO: 22) (SEQ ID NO: 23, 24, 25, 26).

The elongation protein preferably binds to the coupling protein, which in turn is coupled to the glide clip protein.

In particular, as an elongation protein in the sense of the present invention to understand such a protein that is related to human (eukaryotes) Amino acid sequence (SEQ ID NO: 22) of at least 200 in length Amino acids in a sequence alignment are at least 20% Has 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 four positions ( FIG. 8):

SEQ ID NO: 44

D- [G A V L I M P F W] - [G A V L I M P F W] -X-X-Y-N-X-X-X-F-D-X-P-Y- [G A V L I M P F W] - X-X-R-A.

A hidden Markov model was also generated from the multiple alignment of homologues to the human elongation protein (SEQ ID NO: 22) shown in Fig. 17. Elongation protein in the sense of the present invention is therefore to be understood in particular as any protein that has a score 20 more with the Hidden Markov model generated in this way ( FIG. 17).

In particular, as an elongation protein in the sense of the present invention also understand such a protein that is related to the archa-bacterial Amino acid sequence (SEQ ID NO: 27) of at least 400 in length Amino acids in a sequence alignment are at least 25% Has sequence identity. For example, the ones from Proteins derived from archaebacteria with SEQ ID NO: 28, 29, 30 or 31  

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 four positions ( FIG. 9):

SEQ ID NO: 45

A- [G A V L I M P F W] -R-T-A- [G A V L I M P F W] -A- [G A V L I M P F W] - [G A V L I M P F W] -T-E- G- [G A V L I M P F W] -V-X-A-P- [G A V L I M P F W] -E-G-I-A-X-V- [K R H D E] -I.

A hidden Markov model was also generated from the multiple alignment of homologues to the archebacterial elongation protein (SEQ ID NO: 27) shown in Fig. 18. Elongation protein in the sense of the present invention is therefore to be understood in particular as any protein that has a score of more than 35 with the Hidden Markov model generated in this way ( FIG. 18).

The elongation protein can also be of eubacterial origin.

Thus, as an elongation protein in the sense of the present invention also to understand such a protein that leads to the eubacterial Amino acid sequence (SEQ ID NO: 37) of at least 300 in length Amino acids in a sequence alignment are at least 25% Has sequence identity.

Elongation protein for the purposes 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):

SEQ ID NO: 46

[G A V L I M P F W] -P-V-G- [G A V L I M P F W] -G-R-G-S-X- [G A V L I M P F W] -G-S- [G A V L I M P F W] -V-A-X-A- [G A V L I M P F W] -X-I-T-D- [G A V L I M P F W] -D-P-  [G A V L I M P F W] -X-X-X- [G A V L I M P F W] -L-F-E-R-F-L-N-P-E-R- [G A V L I M P F W] -S- M-P-D.

A hidden Markov model was also generated from the multiple alignment of homologs to the eubacterial elongation protein (SEQ ID NO: 37) shown in Fig. 19. Elongation protein in the sense of the present invention is therefore to be understood as any protein which has a score 20 more with the Hidden Markov model generated in this way ( FIG. 19).

So far, some DNA polymerases have been used as elongation proteins Coupling protein and without slide clamp for standard PCR reactions used so. B. DNA polymerase I from Pyrococcus furiosus (United States Patent No. 5,545,552) or Pyrococcus species (European Patent Application No: 0 547 359 A1). These enzymes are characterized by the Property of being thermostable and often a 3'-5 'exonuclease To have activity ('proof-reading' activity). Just recently a Heterodimer with polymerase activity discovered in Pyrococcus furiosus (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.)

It is preferred in the context of the present invention that the Procaryotic in vitro complex according to the invention for the elongation of Nucleic acids consist of proteins that come from Archaea. This means, preference is given to using a slide clamp protein Archaebacteria. Furthermore, it is preferred that the elongation protein or a protein complex having polymerase activity, which consists of elongation protein and coupling protein Archaea bacteria.  

Examples of such proteins suitable for the complex according to the invention are shown in FIGS. 1 and 2.

Of course it is possible through deletions or mutations or through that Adding amino acids to optimize the properties of these proteins. These modified proteins are also the subject of the present Invention as long as they are the in vitro complex according to the invention Form elongation of nucleic acids and those specified in more detail above Perform functions.

Fig. 3 is used to illustrate the complex according to the invention, which shows an example of the replication apparatus in a possible variant, the sliding chamber binding to the elongation protein via a coupling subunit.

Furthermore, it is preferred that in addition to the invention prokaryotic accessory in vitro complex two primers present are. Primers are usually oligonucleotides attached to the two bind complementary DNA strands of the target sequence, wherein they in opposite orientation, their 3 'ends facing each other, towards include amplifying section. They serve as the starting point for the Amplification and usually provide a free 3'-OH end for the Polymerase available for incorporation of a nucleotide.

While using the protein complex for amplification, The complex according to the invention lies in elongation and sequencing preferably in a suitable buffer. Suitable buffers are those used for PCR, sequencing, nucleic acid labeling and others in vitro nucleic acid elongation reactions using polymerase Find. Suitable buffers are described for example in Methods in Molecular Biology Vol. 15 Humana Press Totowa, New Jersey, 1993, edited by Bruce A. White.  

While using the protein complex for elongation, Amplification, reverse transcription and / or sequencing in addition to the complex according to the invention, a mixture of Nucleotides. Deoxynucleotides can be made from dGTP, dATP, dTTP and dCTP are selected, but are not limited to these. In addition can also be derivatives of deoxynucleotides according to the invention are used which are defined as such deoxynucleotides which are described in are able to grow through a thermostable DNA polymerase DNA molecules to be incorporated that are synthesized in the reaction become. Such derivatives include thionucleotides, 7-deaza-2'-dGTP, 7-deaza-2'-dATP as well as deoxyinosine triphosphate, which is also a substitute Deoxynucleotide can be used for dATP, dGTP, dTTP or dCTP one, but are not limited to these. Likewise, marked Deoxynucleotides can be used. All known or / and to the Markings suitable for the purpose of the invention can be used available.

Dideoxynucleotides can be made from ddGTP, ddATP, ddTTP and ddCTP are selected, but are not limited to these. In addition can also derivatives of dideoxynucleotides according to the invention are used which are defined as those dideoxynucleotides which are described in are able to grow through a thermostable DNA polymerase DNA molecules to be incorporated that are synthesized in the reaction become. Such derivatives can contain radioactive dideoxynucleotides (ddATP, ddGTP, ddTTP and ddCTP) or dideoxynucleotides (ddATP, ddGTP, ddTTP and ddCTP), which with z. B. FITC, Cy5, Cy5.5, Cy7 and Texas red or others are marked, include, but are not limited to, these. Within the scope of a sequencing according to the invention, labeled ones can also Deoxynucleotides are used together with unlabeled dideoxynucleotides become.  

Ribonucleotides can be selected from GTP, ATP, TTP and CTP, however, are not limited to these. In addition, derivatives of Ribonucleotides according to the invention are used as such Ribonucleotides are defined that are capable of being thermostable DNA polymerase to be incorporated into growing DNA molecules that be synthesized in the reaction. Such derivatives can be radioactive Ribonucleotides (ATP, GTP, TTP and CTP) or Ribonucleotides (ATP, GTP, TTP and CTP), which with z. B. FITC, Cy5, Cy5.5, Cy7 and Texas red or others are marked, include, but are not limited to, these.

While using the protein complex for amplification, Elongation and sequencing can prove beneficial if a pyrophosphatase is present in the reaction.

Another object of the present invention is a thermostable, accessory in vitro complex, which is a glide clip protein and a Coupling protein comprises, both proteins being as defined above.

To a certain extent, the accessory in vitro complex according to the invention is as a precursor to the thermostable invention described above in vitro complex for template-dependent elongation of nucleic acids understand.

The accessory complex only needs an elongation protein can be combined to give this a significantly increased processivity to lend. The accessory in vitro complex can therefore also be combined can be used with known thermostable polymerases, where also disadvantages of these known proteins with regard in particular to Processivity can be reduced.

Identification of the genes, cloning of the genes, expression of these and purification of the proteins of the in vitro complexes according to the invention:
As a rule, the complexes, 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. In accordance with the present invention, it can happen that genes, in particular from the archaebacteria, intein (Proc Natl Acad Sci USA 1992 Jun 15; 89 (12): 5577-5581, Intervening sequences in an Archaea DNA polymerase gene, Perler FB, Comb DG, Jack WE, Moran LS, Qiang B, Kucera RB, Benner J, Slatko BE, Nwankwo DO, Hempstead SK, et al), which can first be removed.

The identification of further suitable ones for the complex according to the invention Proteins can e.g. B. done by homology searches in databases, which Include genomes from prokaryotes. Some programs are for this suitable, for example the program BLASTP and FASTA (old school, 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. W.R. Pearson & D.J. Lipman PNAS (1988) 85: 2444-2448).

Identification can also be done by using DNA probes used to in z. B. whole genomic banks from prokaryotes to screen for the corresponding genes. The necessary for this experimental methods can be found in Maniatis et al. (Molecular cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, N.Y. (1989)).

The provision of the purified nucleic acid of the genes of the complexes according to the invention can, for. B. about their isolation from a genomic bank of the relevant organism happen or through synthetic DNA production, in each case combined with if desired  amplification by means of PCR with the aid of primers which are suitable for the desired gene segment are specific. Usual procedures are in Maniatis et al. (Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y. (1989)).

The genes of the proteins of the in vitro complexes according to the invention can be found in a variety of methods are cloned and thus by means of a Expression vector for protein expression in a host organism Will be provided. Common processes are in Maniatis et al. (Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y. (1989)). The genes of the complexes can z. B. first into a 'high-copy' vector, e.g. B. pUC18, pBst or pBR322 be cloned and only then into a prokaryotic expression vector, e.g. B. pTrc99 can be cloned, or directly into a prokaryotic Expression vector can be cloned. Among vectors are here Understanding nucleic acids that are capable of another Nucleic acid molecule in or between different organisms, or to transport genetic backgrounds. They usually have that Ability of autonomous replication and / or expression (Expression vectors) of the operatively linked nucleic acid molecule. "Operatively linked" means that the transported nucleic acid molecule is connected to the vector so that it is under the transcription and Translation control of expression control sequences of the vector is available and can be expressed in a host cell. Bacterial Expression systems, the preferred use of those, as well as a Selection of vector systems is e.g. B. in 'Gene Expression Technology', (Meth. Enzymol., Vol 185, Goeddel, Ed., Academic Press, N.Y. (1990)) described. Suitable vectors for the present invention should allow different levels of expression of the proteins in that they possess some or all of the following properties: (1) promoters, or Transcription initiation sites, either right next to the start of the Protein or as a fusion protein, (2) operators that are used  can 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.

Expression vectors with eukaryotic cells, preferably with Vertebrate cells are compatible can also be used. Some known vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1 / pML2d (International Biotechnologies, Inc.), and pTDT1 (ATCC 31255). The A retroviral expression vector is also possible.

Further subjects of the present invention are therefore DNA Sequences which are suitable for the thremostable in vitro Code complex or accessory in vitro complexes, as well corresponding vectors, preferably expression vectors.

The vectors according to the invention contain at least the gene for the Slipclip protein and preferably at least one gene for one Coupling or / and elongation protein, as defined in each case above.

It is preferred in the context of the invention if the vector next to the DNA sequences already contained therein are still suitable Restriction interfaces and, if necessary, polylinkers for the insertion of further DNA Contains sequences. It is particularly preferred if the spatial Arrangement of already contained DNA sequence and additional Insertion site after expression leads to the formation of a fusion protein.

It is also preferred if the vector promoter according to the invention or / and contains operator areas, it being particularly preferred if such promoter and / or operator areas can be induced or repressed are. This makes control of expression in host cells significant simplified and can be designed particularly efficiently.  

Such promoter / operator areas can be in an expression vector also occur several times, so that a possibly independent expression multiple DNA sequences using only one expression vector is made possible.

In the context of the invention, a particularly preferred vector contains DNA Sequences coding for all components of an in vitro complex or accessory in vitro complex.

Another object of the present invention is a host cell, containing one or more vector (s) according to the invention, wherein in expression of this host cell under suitable conditions to proteins can be done. Suitable conditions include, for example Presence of an inductor or a derepressor.

For transformation, phage infection and cell culture exist Standard protocols in Maniatis et al. (Molecular Cloning: A laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.). From the multitude of E. coli strains which are suitable for transformation are the preferred JM101 (ATCC No. 33876), XL1 (Stratagene), RRI (ATCC No. 31343) and BL21 (Pharmacia). Protein expression can e.g. B. also happen with the help of the E. coli strain INVαF '(Invitrogen).

The transformants are made according to the appropriate growth conditions of the host strain. So most E. coli strains are e.g. B. in LB medium cultured at 30 ° C to 42 ° C up to logarithmic or stationary growth phase. The proteins can be transformed from a Culture can be purified, either from a cell pellet, according to Centrifugation, or can be done from the culture solution. If the Proteins from the cell pellet are purified into cells in one suitable buffer resuspended and by means of sonification, enzymatic Treatment or freezing and thawing broken up. If the Cleaning from the culture suspension takes place, that means alone or over  a fusion protein, the supernatant is known from the cells by means of Process such as centrifugation separated.

The separation and purification of the proteins of the invention Complexes either from the supernatant of the culture solution or from the Cell extract can be made by known separation or purification methods happen. These methods are e.g. B. those related to solubilities, such as Salt precipitation, and solvent precipitation, methods that the use different molecular weights, like dialysis, Ultrafiltration, gel filtration, and SDS polyacrylamide gel electrophoresis, Methods that take advantage of different charges Ion exchange chromatography, methods that differ Make use of hydrophobicity, such as reverse-phase HPLC (High Performance Liquid Chromatography), methods that were determined Leverage affinities, such as affinity chromatography, and methods who take advantage of differences in the isoelectric point, such as isoelectric focusing. It is also conceivable that cell extracts, either from the organism, which is the gene of the accessory Complex can be made, which is the inventive Task accomplished, or from the recombinant host organism, e.g. B. E. coli. With these extractions one could possibly do others Avoid cleaning steps.

The methods described above can be in a variety of Combinations are used around the proteins of the in vitro complex to provide.

The thermostable prokaryotic accessory in vitro according to the invention Complex can be used to elongate nucleic acids, e.g. B. for polymerase chain reaction, DNA sequencing, for labeling Nucleic acids and other reactions involving the nucleic acid in vitro Involve synthesis. Reverse application is also possible  Transcription, either the complex according to the invention itself reverse transcriptase activity, or in addition a suitable one Enzyme is added that has reverse transcriptase activity.

Another object of the present invention is therefore a method for template-dependent elongation of nucleic acids, the If necessary, nucleic acid denatured with at least one primer below Hybridization conditions are provided, the primer being complementary to a flanking region of a desired nucleic acid sequence of the Is template strands and with the help of a polymerase in the presence of Nucleotides a primer elongation takes place, being a polymerase thermostable in vitro complex according to the invention is used.

Process for template-dependent elongation of nucleic acids, at which the elongation is based on a primer to the Template nucleic acid was hybridized and a free 3'-OH end for the elongation is available to the person skilled in the art. For Amplification is particularly a polymerase chain reaction carried out. This is usually characterized by a double-stranded DNA Sequence emanated from which a certain target area is amplified shall be. Here two primers are used, which to the Target sequence flanking areas on a partial strand of the DNA Double strands are complementary. To hybridize the primers however, the DNA duplex first denatured, especially thermally melted. After hybridization of the primers, a Elongation using the polymerase, then denatured again and so that the newly formed DNA strands are separated from the template strands, whereupon for another elongation cycle in addition to the original Template strands also the nucleic acid strands formed in the first step are available as templates, each again with primers are hybridized and renewed elongation takes place. This  The procedure is carried out cyclically under each thermal Denaturation as intermediate steps.

For the reverse transcription of RNA into DNA preferred according to the invention also becomes a thermostable in vitro complex according to the invention used, the elongation protein of which is a reverse transcriptase Has activity. This reverse transcriptase activity may be the only one Polymerase activity of the elongation protein, but can also be to an existing 5'-3 'DNA polymerase activity.

Another preferred method according to the invention relates to Sequencing of nucleic acids from a primer that is a region adjacent to the nucleic acid to be sequenced is complementary, whereby again a template-dependent elongation or reverse transcription when sequencing an RNA Use of deoxynucleotides and dideoxynucleotides according to the Method carried out by Sanger. As deoxynucleotides or Dideoxynucleotides are used in this preferred embodiment the respective derivatives described above are also considered suitable. In particular, it is for the method according to the invention for elongating Nucleic acids preferred that the nucleic acids formed are labeled. For this it is possible to use labeled primers and / or labeled deoxynucleotides or / and labeled dideoxynucleotides or / and labeled ribonucleotides or corresponding derivatives, such as those already mentioned above, for example are described.

Another object of the present invention is a Method for labeling nucleic acids by inserting individual ones Breaks in phosphodiester bonds in the nucleic acid chain and replacement of one Nucleotides at the break points by means of a labeled nucleotide Polymerase, a thermostable in vitro complex is used.  

Such a method, commonly called nick translation, enables one easy labeling of nucleic acids. All of the above labeled ribonucleotides or deoxyribonucleotides or derivatives thereof are suitable for this as long as the polymerase accepts them as a substrate.

This marking according to the invention can, for. B. also in the frame or subsequent to a PCR reaction, for which the invention Use of a thermostable in vitro complex is particularly favorable.

The present invention also relates to a kit for elongation or / and amplification or / and reverse transcription or / and sequencing of nucleic acids, which kit can be present in one or more containers

• a) a thermostable in vitro complex according to the invention or
• b) a thermostable, accessory in vitro complex and possibly separately having a polymerase activity Elongation protein and, if necessary, primers, buffer substances, Nucleotides, ATP, other cofactors and / or pyrophosphate.

In particular, the present invention relates to a kit for Elongation, amplification, reverse transcription, labeling or Sequencing of nucleic acids, with additional deoxynucleotides or their derivatives are included.

A preferred reagent kit according to the invention therefore contains Amplification of nucleic acids in addition to substances a) or b), which have 5'-3 'polymerase activity, also deoxynucleotides or / and derivatives thereof. If necessary, here too Ribonucleotides or derivatives thereof are used, namely, if a polymerase is used, which also ribonucleotides as Substrate accepted.  

Another preferred object of the present invention is a Kit for sequencing nucleic acids, in addition to Deoxynucleotides or their derivatives, dideoxynucleotides or their Derivatives for chain termination are included.

Furthermore, the subject of the present invention is in particular a Kit for the reverse transcription of nucleic acids, either the complex according to the invention itself has reverse transcriptase activity, or in addition, a suitable enzyme is present, the reverse Has transcriptase activity, with deoxynucleotides or their Derivatives are contained in the reaction mixture.

In a further particularly preferred embodiment, the kit contains Primers or / and deoxynucleotides or / and dideoxynucleotides or / and Ribonucleotides and / or their respective derivatives in labeled form.

In particular for the sequencing of nucleic acids, it is necessary to use a Insert marker. Suitable markings are already above described in exemplary form and are also considered preferred Embodiments within the scope of the reagent kits according to the invention.

In the context of the present invention, it is also possible that the Reagent kit for labeling nucleic acids as part of a so-called nick translation is used. In this case, the Reagent kit components a) or b) and labeled nucleotides, where Buffer substances, ATP or other cofactors and / or pyrophosphate can also be present. However, primers are in the frame a nick translation is usually not required.

The present invention furthermore relates to the use a thermostable glide clip protein in in vitro processes for  Elongation, amplification, labeling or sequencing or reversing Transcription of nucleic acids.

The kit is preferred if a thermostable protein is used as the glide clip protein Homologous to the PCNA protein (SEQ ID NO: 12, 13, 14, 15), the PCNA homologues from Archeaoglobus Fulgidus or the β-clamp protein (SEQ ID NO: 35, 36). In particular, a kit is preferred which additionally contains a coupling protein.

Furthermore, a kit is preferred which additionally contains another protein containing, the further protein being available as a clamp loader stands, also comes from the group of prokaryotes and is thermostable is.

In particular, a kit is preferred which contains a suitable buffer, as described above. It is also preferred that the kit according to the invention additionally a pyrophosphatase, ATP or / and contains other cofactors.

The following examples are to be used in conjunction with the illustrations Explain the invention in more detail:

Illustrations

In the following, sequence names are often used, among which the protein or nucleic acid sequences in the gene bank and the EMBL Database.

Fig. 1

Protein sequences, paired alignments and multiple alignments Archaebacteria and the corresponding human genes of the Replication apparatus.

The annotation: ¹ means% identity to the corresponding human gene, calculated from the pairwise alignment (see Appendix) with BLASTP 2.0.4 [Feb-24-1998] and the annotation ² % identity to the corresponding gene from Archaeoglobus Fulgidus from the pairwise alignment (see Appendix) with BLASTP 2.0.4 [Feb-24-1998] and the annotation ³ % identity to the corresponding human gene, calculated from the pairwise alignment with FASTA 3.1t02 [March, 1998] ^§ means. The procedures are described in more detail in:
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 and WR Pearson & DJ Lipman PNAS (1988) 85: 2444-2448. In the case of glide clip loader I, glide clip loader II, glide clip, coupling subunit and elongation protein I, the illustration shows the sequence names from the databases and also their SEQ ID number, the values in the brackets representing percent identity per number of amino acids. In the case of elongation protein II, the values relate to percent sequence identity to the Archaeaoglobus fulgidus sequence.

Fig. 2

Protein sequences, paired alignments and multiple alignments from eubacteria of the replication apparatus. Annotation ¹ means% identity to the corresponding gene from E. coli, calculated from the pairwise alignment (see Appendix) with BLASTP 2.0.4 [Feb-24-1998] ^§ . The procedure is described in: 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.

Fig. 3

Sketch of the replication apparatus in a possible variant, the Sliding chamber via a coupling subunit to the elongation protein binds.

Fig. 4

Fig. 4 shows alignments of two conserved regions of the glide clip protein, as well as the consensus sequences derived from them. The following genes are shown: PCNA human (from SEQ ID NO: 11), the corresponding sequence from Archaeoglobus fulgidus (from SEQ ID NO: 12), from Methanococcus janashii (from SEQ ID NO: 13), from Pyrococcus horikoschii (from SEQ ID NO: 14) and from Methanococcus thermoautothrophicus (from SEQ ID NO: 15).

Fig. 5

Fig. 5 shows an alignment of a conserved region of the coupling subunit, as well as the consensus sequences derived from it. The following genes are shown: PfuORF2, DPD2_HUMAN, AF1790 and MJ0702. The SEQ ID numbers can be found in Fig. 1.

Fig. 6

Fig. 6 shows an alignment of a conserved region of the coupling subunit, as well as the consensus sequences derived from it. The following genes are shown: AC11_HUMAN, AF2060, MTH0241, PHBN012 and MJ1422. The SEQ ID numbers can be found in Fig. 1.

Fig. 7

Fig. 7 shows an alignment of a conserved region of the sliding clamp loader 2, as well as the consensus sequences derived from it. The following genes are shown: AC15_HUMAN, MJ0884, AF1195, MTH0240 and MTH0240. The SEQ ID numbers can be found in Fig. 1.

Fig. 8

Fig. 8 shows an alignment of a conserved region of the elonation protein 1, as well as the consensus sequences derived from it. The following genes are shown: DPOD_HUMAN, MJ0885, MTH1208, PHBT047 and DPOL_ARCFU. The SEQ ID numbers can be found in Fig. 1.

Fig. 9

Fig. 9 shows an alignment of a conserved region of the elongation protein 2, as well as the consensus sequences derived from it. The following genes are shown: AF1722, MJ1630, PfuORF3, MTH1536 and PHBN021. The SEQ ID numbers can be found in Fig. 1.

Fig. 10

Fig. 10 shows an alignment of a conserved region of the elongation protein from eubacteria, as well as the consensus sequences derived from it. The following genes are shown: 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 typhimurium).

Fig. 11

Fig. 11 shows an alignment of a conserved region of the gliding clip from eubacteria, as well as the consensus sequences derived from it. The following genes are shown: AAPOL3B, DP3B_ECOLI, S.TYPHIM, DP3B_PROMI, DP3B_PSEPU and DP3B_STRCO (AAPOL3B: Aqufex Aeolicus section 93: DP3B_ECOLI: DNA Pol III, beta chain, Escherichia coli S.TYPHIM: DNA Polmon P3B_PROMI: DNA Pol III, beta chain, Probeus mirabilis DP3B_PSEPU: DNA Pol III, beta chain, Pseudomonas putida DP3B_STRCO: DNA Pol III, beta chain, Strptomyces coelicolor).

Fig. 12

Multiple alignment of glide clip protein sequences for generation of the hidden Markov model.

Fig. 13

Multiple alignment of the eubacterial glide clip protein sequences for Generation of the Hidden Markov Model (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, Probeus mirabilis DP3B_PSEPU: DNA Pol III, beta chain, Pseudomonas putida DP3B_STRCO: DNA Pol III, beta chain, Strptomyces coelicolor).

Fig. 14

Multiple alignment of the glide clip loader 1 protein sequences Generation of the hidden Markov model.

Fig. 15

Multiple alignment of the slide clamp loader 2 protein sequences Generation of the hidden Markov model.

Fig. 16

Multiple alignment of the protein sequences of the coupling subunits to generate the hidden Markov model.

Fig. 17

Multiple alignment of the sequences of the elongation proteins 1 for Generation of the hidden Markov model.

Fig. 18

Multiple alignment of the sequences of the elongation proteins 2 for Generation of the hidden Markov model.

Fig. 19

Multiple alignment of the sequences of the eubacterial elongation proteins to generate the hidden Markov model. The following genes are shown:
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_SALTY: DNA Pol subunit, Salmonella typhimurium).

example

The DNA is purified from the archaeoglobus fulgidus organism (DSM No. 4304) using known methods. Organisms are raised by the DSM (German Collection for Microorganisms). In order to clone the corresponding genes (glide clip loader I / II, glide clip, elongation proteins I / II, coupling subunit) into the expression vector pTrc99, primers are developed for each gene, which span the complete open reading frame, as well as start and stop codon. Here, restriction ends are added to the primers, which facilitate directed cloning into the expression vector. Using about 200 ng of total genomic DNA, PCR reactions are carried out with the appropriate annealing temperatures (about 35 cycles) and the resulting products are purified. After cleaning, the products are treated with restriction enzymes and cleaned 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 ready for ligation with the amplificates of the accessory genes from the above PCR. The ligation is set up and, after incubation, an aliquot is transformed into the E. coli strain INValphaF '(Invtrogen). 3 positive colonies are picked from each gene, plasmid DNA is prepared and the inserts are checked for completeness and correctness by means of DNA sequencing. Correct clones are selected and again used for separation on agar plates (ampicillin). Colonies are picked and overnight cultures are set up. An aliquot (500 µl) of the overnight culture is placed in a one to five liter culture of LB (ampicillin: 80 mg / l). The cultures grow up to an OD ₆₆₀ of 0.8 at 37 ° C. Now IPTG is added for induction (125 mg / l). These cultures now grow another 11 hours. The cultures are centrifuged and the pellets are taken up in a buffer (buffer A: 50 mM Tris-HCl 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). After incubation (15 min.) An equal volume of buffer B is added (B: 10 mM Tris-HCl pH 7.9, 50 mM KCl, 1 mM EDTA, 1 mM PMSF, 0.5% Tween 20, 0.5% Nonidet P40) and the lysis given for incubation at 75 ° C for one hour. After centrifugation, the supernatant is removed and the overexpressed proteins are precipitated using (NH ₄ ) ₂ SO ₄ . After centrifugation, the pellets are collected and the proteins are resuspended with buffer A. The resuspended proteins are dialyzed against storage buffer (50 mM Tris-HCl pH 7.9, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 50% glycerol) and then stored at -70 ° C.

In order to test the activity of the proteins, reactions are composed as follows: Aliquots of the proteins are combined in different configurations and molarities, slide clip loader I / II with slide clip, coupling subunit and elongation protein I, or slide clip loader I / II with slide clip, with and without a coupling subunit and elongation protein II, or glide clip, and elongation protein I or II and finally only elongation protein I or II; the storage buffer above serves as a buffer. DNA polymerization activity is measured by incorporating (methyl- ³ H) TTP in trichloric acid-insoluble material (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 (5′-FITC marked). Dilution series of the template-primer mixture generated in this way are in a reaction consisting of nucleotides (about 200 µM to 1 mM), reaction buffer (final concentration: 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5-5 mM MgCl 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 °. An aliquot is loaded onto an automated sequencer for analysis (e.g. Alf, Pharmacie Biotech) The above protein mixtures also serve to measure fidelity and exonuclease activity, 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) are used, and the protein mixtures are also used in PCR (Methods in Molecular Biology Vol. 15 Humana Press Totowa, New Jersey, 1993, edited by Bruce A White).

SEQUENCE LOG

Claims (43)

1. Thermostable in vitro complex for template-dependent Elongation of nucleic acids, comprising a thermostable Slipclip protein, which with a thermostable Elongation protein having polymerase activity is. 2. Thermostable complex according to claim 1, characterized, that the glide clip protein and the elongation protein over one Coupling protein are linked. 3. Thermostable complex according to claim 1 or 2, characterized, that the glide clip protein and the elongation protein from Archaebacteria originate. 4. Thermostable complex according to claim 1, 2 or 3, characterized, that the glide clip protein has an annular structure, all or part of the template nucleic acid strands encloses. 5. Thermostable complex according to one of claims 1 to 4, characterized in that the gliding clip protein has one or both of the following amino acid consensus sequences:
SEQ ID NO: 39
[GAVLIMPFW] -DXXX- [GAVLIMPFW] -XX- [GAVLIMPFW] -X- [GAVLIMPFW] -X- [GAVLIMPFW] -XXXXFXXYXXD and / or
SEQ ID NO: 40
[GAVLIMPFW] -X (3) -LAP- [KRHDE] - [GAVLIMPFW] -E. 6. Thermostable complex according to one of claims 1 to 5, characterized, that the glide clip protein to human (eukaryotes) PCNA amino acid sequence (SEQ ID NO: 11) in a length of at least 100 amino acids in a sequence alignment has at least 20% sequence identity and / or that Slide clamp protein for the bacterial β-clamp sequence from E. coli (Eubacteria) (SEQ ID NO: 35) over a length of at least 100 Amino acids in a sequence alignment are at least 20 Has% sequence identity and / or the glide clip protein to the amino acid sequence of the PCNA homolog Archaeoglobus Fulgidus (Archaea) (SEQ ID NO: 12) on one Length of at least 100 amino acids in one Sequence alignment has at least 20% sequence identity having. 7. Thermostable complex according to one of the preceding claims, characterized in that the glide clip protein with a Hidden Markow model generated from an alignment from Fig. 12 results in a score of at least 20 and / or wherein the glide clip protein with one from an alignment from Fig. 13 generated hidden markow model gives a score of at least 25. 8. Thermostable complex according to one of the preceding Expectations, characterized,  that the glide clip protein is selected from AF0335 Archaeoglobus Fulgidus, MJ0247 from Methanococcus jannaschii, PHLA008 from Pyrococcus horikoschii, MTH1312 Methanobacterium thermoautotrophicus and AE000761_7 Aquifex aeolicus. 9. Thermostable complex according to one of the preceding Expectations, characterized, that the elongation protein has 5'-3 'polymerase activity or has reverse transcriptase activity. 10. Thermostable complex according to claim 9, characterized in that the elongation protein contains at least one of the following consensus sequences and does not deviate from this sequence at more than four positions:
SEQ ID NO: 44
D- [GAVLIMPFW] - [GAVLIMPFW] -XXYNXXXFDXPY- [GAVKUNOFW] -XXRA
SEQ ID NO: 45
A- [GAVLIMPFW] -RTA- [FAVLIMPFW] -A- [GAVLIMPFW] - [GAVLIMPFW] -TEG- [GAVLIMPFW] -VXAP- [GAVLIMPFW] -EGI- AXV- [KRHDE] -I
SEQ ID NO: 46
[GAVLIMPFW] -PVG- [GAVLIMPFW] -GRGSX- [GAVLIMPFW] -G- S- [GAVKUNOFW] -VAXA- [GAVLIMPFW] -XITD- [GAVKUNOFW] - DP- [GAVLIMPFW] -XXIMFER- [GAVLIMPFW] [GAVLIMPFW] -SMPD. 11. Thermostable complex according to claim 9, characterized,  that the elongation protein is one of the human (Eukaryotes) amino acid sequence (SEQ ID NO: 22) on one Length of at least 200 amino acids in one Sequence alignment has at least 20% sequence identity and / or to the archaebacterial amino acid sequence (SEQ ID NO: 27) with a length of at least 400 amino acids a sequence alignment is at least 25% Has sequence identity and / or eubacterial Amino acid sequence (SEQ ID NO: 37) in a length of at least 300 amino acids in a sequence alignment has at least 25% sequence identity. 12. The thermostable complex according to claim 9, characterized in that the elongation protein with a hidden markow model generated from an alignment from FIG. 17 gives a score of at least 20 and / or wherein the elongation protein with a hidden from an alignment from FIG. 18 Markow model gives a score of at least 35 and / or the elongation protein with a hidden Markow model generated from an alignment from Fig. 19 gives a score of at least 20. 13. Thermostable complex according to one of the preceding Expectations, characterized, that the elongation protein is selected from AF0497 or AF1722 from Archaoglobus fulgidus, MJ0885 or MJ1630 Methanococcus jannaschii, PHBT047 or PHBN021 Pyrococcus horikoschii, MTH1208 or MTH1536 Methanobacterium thermoautotrophicus and PFUORF3 Pyrococcus furiosus.   14. Thermostable complex according to one of the preceding claims, characterized in that the coupling protein contains the following consensus sequence and does not deviate from this sequence at more than four positions:
(SEQ ID NO: 43)
[FL] - [GAVLIMPFW] -XX- [GAVLIMPFW] -XGX (13) - [GAVLIMPFW] - X- [YR] - [GAVLIMPFW] -X- [GAVLIMPFW] -AG- [DN] - [GAVLIMPFW] - [ GAVLIMPFW] - [DS]. 15. Thermostable complex according to one of the preceding Expectations, characterized, that the coupling protein to human (eukaryotes) Amino acid sequence (SEQ ID NO: 16) of a length of at least 150 amino acids in a sequence alignment has at least 18% sequence identity. 16. Thermostable complex according to one of the preceding claims, characterized in that the coupling protein with a Hidden Markow model generated from an alignment from Fig. 16 results in a score of at least 10. 17. Thermostable complex according to one of the preceding Expectations, characterized, that the coupling protein is selected from AF1790 Archaeoglobus fulgidus, MJ0702 from Methanococcus jannaschii, PHBN023 from Pyrococcus horikoschii, MTH1405  Methanobacterium thermoautothrophicum and PFUORF2 Pyrococcus furiosus. 18. Thermostable complex according to one of the preceding Expectations, characterized, that the complex is associated with a protein which is called Slide clamp loader acts. 19. Thermostable complex according to one of the preceding Expectations, characterized, that the complex is associated with ATP or another cofactor is present. 20. Thermostable accessory in vitro complex, characterized, that it contains a slide clamp protein and a coupling protein, as defined in one of the preceding claims are. 21. recombinant DNA sequence, characterized, that they are for a thermostable in vitro complex according to a of claims 1 to 19 or for a thermostable accessory complex encoded according to claim 20. 22.Vector, characterized, that he's one for a glide clip protein and a coupling protein or / and a recombinant DNA encoding an elongation protein Contains sequence.   23. The vector according to claim 22, characterized, that he also has suitable restriction interfaces for insertion contains further DNA sequences in such an arrangement that is a fusion protein from glide clip protein and the Expression product of the other DNA sequences results. 24. Vector according to claim 22 or 23, characterized, that it is suitable for controlling the expression of the DNA sequence (s) Contains promoter and / or operator areas. 25. The vector according to claim 24, characterized, that he has multiple promoter and / or operator areas for contains separate expression of multiple DNA sequences. 26. Vector according to one of claims 22 to 25, characterized, that he repressible and inducible promoter or / and Contains operator areas. 27. Vector according to one of claims 22 to 26, characterized, that it contains a DNA sequence according to claim 21. 28. host cell, characterized, that they match one or more vectors according to one of the Claims 22 to 27 is transformed.   29. Process for the preparation of a thermostable in vitro complex according to one of claims 1 to 19 or a thermostable, accessory in vitro complex according to claim 20, characterized, that according to a corresponding recombinant DNA sequence Claim 21 or one or more corresponding vectors introduces into a host cell according to one of claims 22 to 27, causes the expression of the proteins and this from the Culture medium or isolated after cell disruption and if necessary coupled with other complex components. 30. Use of a thermostable, accessory in vitro Complex according to claim 20 for the production of a thermostable in vitro complex according to one of claims 1 to 19, characterized, that the accessory complex with one Coupling elongation protein having polymerase activity. 31. Process for template-dependent elongation of nucleic acids, the nucleic acid denaturing, if necessary, with at least a primer is provided under hybridization conditions, the primer complementary to a flanking area a desired nucleic acid sequence of the template strand and with the help of a polymerase in the presence of nucleotides a primer elongation takes place, characterized, that according to polymerase a thermostable in vitro complex one of claims 1 to 19 is used. 32. The method according to claim 31, characterized,  that to amplify DNA sequences two desired nucleic acid sequence flanking primers and Deoxynucleotides or / and derivatives thereof or / and ribonucleotides or / and derivatives thereof are used. 33. The method according to claim 32, characterized, that you do a polymerase chain reaction. 34. The method according to claim 31, characterized, that to reverse transcribe RNA into DNA thermostable in vitro complex according to one of claims 1 to 19 uses, whose elongation protein reverse transcriptase Has activity. 35. The method according to any one of claims 31 to 34, characterized, that for sequencing nucleic acids from a primer to one of the nucleic acids to be sequenced neighboring area is complementary, a template-dependent Elongation or reverse transcription using Deoxynucleotides and Dideoxynucleotides or their respective Derivatives carried out according to the Sanger method. 36. The method according to any one of claims 31 to 35, characterized, that when you elongate the nucleic acids, you have labels inserts. 37. The method according to claim 36, characterized,  that labeled primers and / or labeled deoxynucleotides or / and their derivatives or / and labeled dideoxynucleotides or / and their derivatives or / and labeled ribonucleotides or / and their derivatives. 38. Method for labeling nucleic acids by generation single breaks in phosphodiester bonds of Nucleic acid chain and replacement of a nucleotide at the break points by a labeled nucleotide using a polymerase, characterized, that as a polymerase a thermostable in vitro complex after one of claims 1 to 19 is used. 39. Reagent kit for elongation or / and amplification or / and reverse transcription or / and sequencing or / and labeling of nucleic acids, contained in one or more separate containers

• a) a thermostable in vitro complex according to one of claims 1 to 19 or
• b) a thermostable accessory in vitro complex according to claim 20 and, if appropriate, an elongation protein having polymerase activity separately therefrom,

and optionally primers, buffer substances, nucleotides, ATP, one or more other cofactors and / or pyrophosphate. 40. Kit according to claim 39, characterized, that he used to amplify nucleic acids in addition to the substances a) or b) which have 5'-3 'polymerase activity, Contains deoxynucleotides and / or derivatives thereof. 41. Kit according to claim 39,  characterized, that it contains substances a) or b) for reverse transcription, which have reverse transcriptase activity, and Deoxynucleotides or / and derivatives thereof. 42. Kit according to one of claims 39 to 41, characterized, that he used for sequencing alongside deoxynucleotides or Ribonucleotides and / or their derivatives, dideoxynucleotides or / and their derivatives. 43. Kit according to one of claims 39 to 42, characterized, that he has primers and / or deoxynucleotides or / and Contains dideoxynucleotides in labeled form.