Modified thermostable dna polymerase, its preparation and use

 

The invention relates to the field of molecular biology and genetic engineering and can be used in various methods of analysis of nucleic acids associated with the synthesis of complementary DNA sequences. The proposed modified form thermostable DNA polymerase obtained by substitution of the glutamate residue in the consensus sequence SerGlnIleGluLeuArgVal/Ile of natural thermostable enzyme other amino acid residue and selection Malinov, showing an increased ability to incorporate in the reaction of unusual substrates, preferably rntf. Described encoding a modified DNA polymerase nucleotide sequence containing vectors and strains of E. coli, transformed with these vectors and obtain recombinant forms of mutant enzymes. The proposed use of a new thermostable DNA polymerase, including the composition of the kits and compositions, reaction sequencing of nucleic acids allows the use of new approaches in the analysis of the survey results. 9 C. and 11 C.p. f-crystals, 4 PL.

The technical field to which the invention relates

The present invention relates to thermostable Sposoby allocation of such polymerases. The enzymes according to the invention can be used for various purposes, in particular for sequencing nucleic acids. Thus, the invention also relates to improved methods of analysis of nucleic acid sequences.

Background of invention

DNA sequencing typically involves obtaining four populations of fragments of single-stranded DNA with one particular end and one variable end. Variable end usually ends at a specific nucleotide base [guanine (G), adenine (A), thymidine (T) or cytosine (C)]. Each of the four different sets of fragments separated on the basis of their length. For this purpose, use of polyacrylamide gel with high resolution. Each band on such gels corresponds to a specific nucleotide in the DNA sequence, thus showing its position in the sequence.

A commonly used method of DNA sequencing is dideoxy - or chain-termination sequencing method, which includes the enzymatic synthesis of DNA chain (Sanger and others, 1977, the OEWG. Natl. Acad. Sci. 74: 5463). Usually carry four different synthesis reaction, each of which must end at some founded the San. The reaction products are easy to identify since each bar corresponds to only G, a, T or C.

In dideoxy - or chain-termination method, short single-stranded annealed primer corresponding to single-stranded matrix. Primer is lengthened at its 3’end by including deoxynucleotides (dNTP) to enable dideoxynucleotide (ddntp). After you enable ddntp elongation stops. However, to ensure proper DNA replication DNA polymerase have a more pronounced ability to include natural substrates, i.e., dNTP, and do not include analogs of nucleotides, called unnatural nucleotides. In the synthesis of DNA ribonucleotides (MTF) are considered as unnatural nucleotides, because unlike ddntp rntf usually are not natural substrates for DNA polymerase in vivo. In the cell this property helps reduce the inclusion of abnormal bases, such as deoxyinosine (DITF) or rntp, in the growing DNA chain.

The two most frequently used methods for automatic sequencing is sequencing using colored primer and painted terminator. These methods are suitable for use with fluorescently labeled frags, sequencing based on fluorescence, is preferable. In General, sequencing painted with primer fluorescently labeled primer is used in combination with unlabeled dNTP. The process is carried out using the four reactions of synthesis and to obtain a gel of four bands for each subject sequencing matrix (corresponding to the product of the termination, which are specific to each base). After extension of the primer to be sequencing mixtures containing products termination enabled dideoxynucleotide, is usually analyzed using gel electrophoresis for DNA sequencing. After electrophoretic separation of fluorescently labeled products are cut with a laser from the base gel and the fluorescence determined using the appropriate monitor. In automatic systems with the passage of reaction mixtures through the matrix of the gel during electrophoresis detector scans the base of the gel to detect which of the labeled fragments are used (Smith and others, 1986, Nature 321: 674-679). In a modification of this method, each of the four primers have been labelled with a different fluorescent marker. Upon completion of four different reacts each band, whereby the individually identified different fluorescent labels (corresponding to four different products termination, which are specific to each base).

In another embodiment, method used sequencing using colored terminator. In this method, to enable dNTP used DNA polymerase and fluorescently labeled ddntp on the extensible end of the DNA primer (Lee and others, 1992, Nucleic Acid Research 20: 2471). The advantage of this technique is no need to synthesize dye-labeled primers. In addition, reactions using colored terminator are more convenient because all four reactions can be carried out in the same tube. Previously described modified thermostable DNA polymerase having a reduced ability to distinguish ddntp (see publication of the European application EP-A-655506 and application for U.S. patent 08/448223). An example of a modified thermostable DNA polymerase is a mutant form of DNA polymerase from T. aquaticus having a tyrosine residue at position 667 (instead of a phenylalanine residue), i.e. the so-called F667Y-mutant form of DNA polymerase Taq. AmpliTaqFS, which is firebase nucleic acid target, several hundred or several thousand times. AmpliTaqFS is a mutant form of the DNA polymerase from T. aquaticus, with the F667Y mutation and an additional aspartic acid residue at position 46 (instead of a glycine residue; G46D - mutation).

Thus, there is a need for thermostable DNA polymerase used for alternative methods of synthesis of nucleic acids suitable for accurate and efficient from the point of view of analysis of the nucleotide sequence in DNA. There is also a need to develop methods based on fluorescence, which do not require the use of dideoxynucleotides. The present invention is directed to solving these problems.

Summary of the invention

The present invention relates to dependent matrix thermostable DNA polymerase, which include the characteristic amino acid sequence motif SerGlnIleXaaLeuArgXaa (SEQ ID NO: 1), whereby "XAA" at position 4 of the sequence represents any amino acid residue except residue glutamic acid (Glu), and "XAA" at position 7 of this sequence is a valine residue (Val) or an isoleucine residue (Il). When the expression is Annex 4 of this sequence represents any amino acid residue, in addition to the residue of glutamic acid. The ability of thermostable DNA polymerase having the amino acid sequence comprising the sequence of the motif, where X at position 4 of the sequence-motif is not a glutamic acid residue, to limit the incorporation of ribonucleotides in comparison with the previously described thermostable polymerase is reduced. For the growing DNA chain, the ribonucleotides are unnatural nucleotides. Thus, the first subject of the invention are new enzymes that can include unnatural analogues of reasons, such as ribonucleotides, in the growing DNA chain and several orders of magnitude more efficient than previously identified thermostable DNA-synthesizing enzymes. Genes encoding these enzymes also belong to the present invention, as well as recombinant expression vectors and cell host comprising these vectors. Using such transformed host cells can be obtained large quantities of purified thermostable polymerases enzymes.

In accordance with the present izobreteniem was identified region or sequence motif within the amino acid sequence of a thermostable DNA plno include deoxyribonucleotides. Changes in this area, such as replacing one or more amino acids (for example, introduction by siteprovides mutagenesis), produce thermostable enzyme is a polymerase that can synthesize RNA or RNA/DNA Chimera or hybrid circuit on the DNA matrix.

Another subject of the invention are improved methods and compositions for determining the sequence of nucleic acid target, and the need for termination circuit ddntp disappears. With the help presented in this description of the improved methods of the ribonucleotides (MTF) are included in the products of primer extension. Because the enzymes that are the subject of the invention, accurately and efficiently include RTF and dNTP in the sequencing reactions can be used a mixture of both nucleotides. After elongation of the newly synthesized primer oligonucleotide products can be split to enable RTF techniques known in the art, for example by hydrolysis, leading thereby to obtain a population of fragments suitable for fractionation and sequence analysis standard methods, such as gel electrophoresis. research institutes. Thus, the subject invention relates to thermostable DNA polymerases, which are distinguished by the fact that the polymerase includes critical motive SerGlnIleXaaLeuArgXaa (SEQ ID NO: 1), where "XAA" at position 4 can be any amino acid residue except residue glutamic acid (Glu), and "XAA" at position 7 is a residue of valine (Val) or an isoleucine residue (Il).

Another subject of the invention are modified polymerase presented in the present description, which includes ribonucleotides or analogs containing a hydroxyl group or other substituent in the 2’ position, which usually do not have deoxyribonucleotides. These nucleotides can be differently marked that creates alternatives to the conventional use of dideoxynucleotides for the purpose of DNA sequencing.

Mutant thermostable polymerase according to the invention is able to more effectively incorporate unnatural nucleotides, in particular the ribonucleotides, compared with the corresponding wild-type enzymes. In a preferred embodiment of the invention be included unusual nucleotide may be analogous to the termination circuit substrate, such as 2’-hydroxy-3’-d is the Chida, such as RTF.

Another object of the invention avalude mutant thermostable polymerase that is able to more effectively incorporate unnatural nucleotides, in particular the ribonucleotides than the corresponding wild-type enzymes. Thus, the subject invention relates to a recombinant thermostable DNA polymerase, each of which is characterized by the fact that (a) in its native form, the polymerase comprises the amino acid sequence SerGlnIleGluLeuArgXaa (SEQ ID NO: 2), where "XAA" at position 7 of this sequence indicates the residue of valine (Val) or an isoleucine residue (Il); (b) in the amino acid sequence of the recombinant enzyme, preferably in position 4 of the sequence, get a mutation, in which the glutamic acid residue in position 4 represents a residue of a different amino acid, preferably glycine residue; and (C) the ability of the recombinant enzyme to limit the incorporation of ribonucleotides and analogs of ribonucleotides, compared with the native form of this enzyme is reduced.

Another object of the invention is the polymerase that are designed to fragment amplification products and the products of the elongation of the primer, and such is I different strategies to determine the sequence.

The enzymes of the present invention and encode their genes can be used to create compositions that are suitable for the implementation of reactions of DNA sequencing and comprising a mixture of normal nucleotides and at least one ribonucleotide or similar ribonucleotide. In a preferred embodiment of the invention unusual nucleotide is ribonucleotide, and the concentration of ribonucleotide lower concentration corresponding deoxyribonucleotide, i.e., the ratio RTF:dntf is 1:1 or less. The enzymes according to the invention is also suitable for sale as sets, which may include any of the additional elements required for the implementation of the reaction sequencing a nucleic acid, such as, for example, dNTP, rntp, buffers and/or primers.

Detailed description of the invention

The present invention relates to a new and improved modified thermostable DNA polymerase, compositions and kits described in the claims. The enzymes according to the invention more effectively include unusual nucleosidase in comparison with previously known polymerase or appropriate polymerase wild-type, which are these new pepperminty, vectors for the expression of modified enzymes and cells with embedded them in vectors. The enzymes according to the invention is suitable for practical use in the new methods of DNA sequencing, which have several advantages compared to the methods of DNA sequencing, known from the prior art.

Below to facilitate understanding of the present invention defined some concepts.

The concept of "normal", if it is mentioned in connection with the bases of nucleic acids, nucleotidyltransferase or nucleotides, refers to those that occur in natural conditions in the specified polynucleotide (for example, DNA is DTF, DSTF, dCTP and dTTP). In addition, in the reaction of DNA synthesis in vitro, such as sequencing, instead dCTP often use sdgt and DITF (although they are less efficient). In General, they can be summarized by the notion of deoxyribonucleosides (dntf).

The term "expression system" refers to posledovatelnostei DNA containing a desired coding sequence and functionally linked regulatory sequences, so that hosts transformed with these sequences are capable of producing codere the th DNA can also be incorporated into the chromosome of the host.

The term "gene" refers to a DNA sequence that comprises control and coding sequences necessary for the reproducible production of a biologically active polypeptide or precursor. The polypeptide can be encoded full-length sequence of a gene or any having sufficient length part of the coding sequence, in order to preserve enzymatic activity.

The term "cell(s)-owner(eve)" refers to unicellular prokaryotic or eukaryotic organisms such as bacteria, yeast, actinomyces, and to separate the cells from plants or animals of higher units, if they are able to grow in cell culture.

Used in the present description, the term "reaction mixture for DNA sequencing" refers to the reaction mixture, which includes the elements necessary for the reaction DNA sequencing. Thus, the reaction mixture for DNA sequencing are suitable for use in a method of DNA sequencing to determine the sequence of the nucleic acid target, although initially the reaction mixture may be incomplete, so that the user could control the initiation of the reaction sequence. In this case, the reaction may what s the complete reaction mixture for DNA sequencing. Usually the reaction mixture for DNA sequencing should contain a buffer suitable for polymerization, nucleosidases and at least one unnatural nucleotide. The reaction mixture may also contain a primer that can be extended to the target polymerase, polymerase, and a nucleic acid target. Primer, as well as one of the nucleotides, usually includes containing the label, for example, fluorescent fragment that can be detected. Usually reacciona mixture is a mixture that includes four normal nucleotides and at least one unnatural nucleotide. In a preferred embodiment, the polymerase is a thermostable DNA polymerase, and unusual nucleotide is ribonucleotide.

The term "oligonucleotide" as used herein, refers to a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size of the oligonucleotide may depend on many factors, including the basic function or use of the oligonucleotide.

Oligonucleotides can be obtained by any suitable method, including, for example, m phosphocreatine method of Narang and others, 1979, Meth. Enzymol. 68: 90-99; fosfodiesterzy method of Brown and others, 1979, Meth. Enzymol. 68: 109-151; diethylphosphoramidite method of Beaucage and others, 1981, Tetrahedron Lett. 22: 1859-1862; Threepenny method of Matteucci and others, 1981, J. Am. Chem. Soc. 103: 3185-3191; automated methods of synthesis, or by a method using a solid substrate, is described in U.S. patent 4458066.

The term "primer" as used herein, refers to a natural or synthetic oligonucleotide, which can serve as a point of initiation of synthesis when creating conditions under which begins elongation of the primer. The primer preferably represents a single-stranded oligodeoxyribonucleotide. The appropriate length of a primer depends on the purpose of the primer but typically ranges from 15 to 35 nucleotides. Short primer molecules generally require more low temperature for the formation of a sufficiently stable hybrid complexes with the matrix. A primer need not reflect the exact sequence of the matrix, but that was the elongation of the primers should have sufficient complementarity for hybridization with the matrix.

If necessary, the primer can be labeled by incorporating a label that can be detected by spectroscopic, photogenic isP, fluorescent dyes, electronmobility reagents, enzymes (as commonly used in the methods ELISA), Biotin, or haptens and proteins for which we have antisera or monoclonal antibodies.

The term "thermostable polymerase" refers to an enzyme which is stable when heated and which is resistant to heat and retains sufficient activity for posleduuschego implementation of the extension reaction of the primer when exposed to elevated temperatures for the time necessary for the implementation of denaturation of double-stranded nucleic acids. According to this description, thermostable polymerase suitable for use in the reaction with temperature cycles, such as polymerase chain reaction (PCR). For thermostable polymerase under enzymatic activity understand the ability to properly catalyze the joining of nucleotides for the formation of products of the elongation of the primer, which is complementary to the chain nucleic acid as a matrix.

Necessary to denature the nucleic acid conditions of heating can vary, for example, the salt concentration in the buffer, and the composition and length of the nucleic acid to be denatured, but typically aitlines heating mainly depends on the temperature and length of the nucleic acid, and it usually ranges from a few seconds up to four minutes.

The concept of "unusual" or "modified" when it describes the basis of nucleic acids, nucleosides or nucleotide comprises a modification, derivatives or analogues of the usual bases or nucleotides that occur in DNA in vivo. In particular, according to this description, unusual nucleotides modified at the 2’ position of the sugar ribose compared with conventional dNTP. Thus, although the ribonucleotides (i.e., ATP, GTP, TTF, CTS, called in General rntf) and avalude naturally occurring nucleotides RNA, in the context of this description, these ribonucleotides are unusual nucleotides, as they have in the 2’ position of the sugar hydroxyl group, which is absent in dNTP. Analogs of ribonucleotides, containing substituents in the 2’ position, such as 2’-fluoro-, 2’-aminosilane analogues, fall under the scope of the invention. In addition, analogues of ribonucleotides can be modified at position 3’, for example, by replacing the normal hydroxyl group hydrogen group (3’-deoxy), resulting in an analog-terminator ribonucleotide. Such nucleate is their MTF should be seen as unnatural, and, therefore, RTF should be regarded as alien to the base. Thus, in a preferred embodiment of the invention when implementing methods to extend a DNA primer, including methods of DNA sequencing, as products get the nucleic acids, which contain both traditional and unusual nucleotides, but mostly they contain normal nucleotides, which are dNTP.

Unusual base can be fluorescently labeled, for example, fluorescent or rhodamine; afluorescent labeled, for example, Biotin; labeled with isotopes, such as32R33P or35S or unlabeled.

To clarify the essence of the invention in the description of specific examples of thermostable enzymes DNA polymerase according to the invention, however, these references should not be construed as limiting the scope of the invention. In a preferred embodiment, thermostable enzyme according to the invention is used in various methods of sequencing nucleic acids, although new thermostable polymerase, in the present description, can be used for any purpose where such enzymatic activity is necessary or desirable. The enzyme takeouts fact, each of them contains a critical motive SerGlnIleXaaLeuArgXaa (SEQ ID NO: 1), where "XAA" at position 4 of the sequence represents any amino acid residue except residue glutamic acid (Glu), and "XAA" at position 7 of this sequence is a valine residue (Val) or an isoleucine residue (Il). Genes encoding thermostable polymerases that have a glutamic acid residue in position 4 of this motif can be modified according to the present description with obtaining suitable modified polymerases. These modified thermostable polymerases differ in that compared to the native enzymes or enzymes of the wild type, they are modified in the amino acid sequence motif SerGlnIleGluLeuArgXaa (SEQ ID NO: 2), where "XAA" at position 7 of this sequence indicates the residue of valine (Val) or an isoleucine residue (Il); i.e., this motif has been modified by substitution of the glutamic acid residue in position 4 of the residue by another amino acid. The critical motive thermostable DNA polymerase of the present invention below, using standard one-letter code of amino acids (Lehninger, Biochemistry, New York, Worth Publishers Inc., 1970, page glutaminovoi acid (Glu), and "XAA" at position 7 is a residue of valine (Val) or an isoleucine residue (Il).

Coding sequences of genes as well as proteins containing this critical amino acid sequence, where XAA in position 4 is not a residue glutamic acid (Glu), refer to the polymerase, the ability to limit the inclusion of rntp lowered, and included in the scope of the present invention. Inside critical motive may be made additional modifications of residues of other amino acids, preferably amino acid residues selected from the group comprising glutamine (Gln or Q), leucine (Leu or L) or arginine (Arg or R).

The present invention can be used to obtain a thermostable DNA polymerases with improved properties by certain modifications in the sequence of the gene encoding thermostable DNA polymerase. In a preferred embodiment of the invention the sequence of the gene and the encoded enzyme originate from species of the genus Thermus, although eubacteria not related to the genus Thermus are also included in the present invention, as described in more detail below. Similarly, due to the highly conserved nature revealed in the present study, criticism is with Taq polymerase. Such thermostable polymerase is included in the scope of the present invention, provided that the amino acid sequence comprises the motif S Q I X L R V/I, where X denotes any amino acid residue except glutamic acid residue, and this amino acid sequence in General homology (sequence identity) of at least about 39%, preferably at least about 60%, more preferably at least about 80% amino acid sequence of native Taq polymerase. The entire length of this sequence Taq polymerase described in WO 89/06691 and registered under registration number R in the database of patented sequences GENESEQ or under registration number M in the database sequences EMBL under registration number A in the database sequences PIR.

Examples of thermostable DNA polymerases of the present invention are recombinant derivatives of native polymerases from organisms listed in table 1. Table 1 shows the specific sequence of critical motive and the position of the balance of "X" for each of these native polymerases. Since each stable is each enzyme. For the following polymerases amino acid residue at position "X" of the critical motif S Q I X L R V/I is a glutamic acid. The molecular weight of the preferred polymerases of the present invention is from 85000 to 105000 Yes, more preferably from 90000 to 95000 Yes. The amino acid sequence of these polymerases consists of about 750-950 of amino acid residues, preferably from 800-850 amino acid residues. The polymerases of the present invention may consist of approximately 540 or more amino acids and include, at least, the polymerase domain and the portion corresponding to the 3’-5’-ectonucleoside domain (obtained polymerase can possess or not possess 3’-5’-ectonucleoside activity), and possibly part of the 5’-3’-ectonucleoside domain, which is located on the first third of the amino acid sequences of many full-sized thermostable polymerases.

For thermostable DNA polymerases, not shown in table 1, the choice of the corresponding glutamic acid, subject to the modification is simple, if you have defined a critical motif or consensus motif in their amino acid sequence.

Regardless of the exact position inside thermostable Dynasty-motive SerGlnIleGluLeuArgXaa (SEQ ID NO: 2), where "XAA" at position 7 of this sequence indicates the residue of valine (Val) or an isoleucine residue (Il), polymerase domain allows to obtain a thermostable polymerase, capable of effective inclusion of extraneous nucleotides. In a preferred embodiment, the glutamic acid is replaced by an amino acid having an unloaded polar R group, such as glycine, serine, cysteine, threonine, or to an amino acid having a small non-polar R-group, such as, for example, alanine. In the most preferred embodiment, the glutamic acid residue is substituted for a glycine residue (G). Program that allows you to install the amino acid and nucleotide sequence, can be purchased from the firm Genetics Computer Group, 575 Science Drive, Madison, pieces of Wisconsin. Program suitable for a particular, defined in this motif include, for example, "GAP", "BESTFIT" and "PILEUP" to help identify the exact sequence to update the field.

As can be seen from the table 1, there are two necessary forms of conservative sequence motif SerGlnIleGluLeuArgXaa (SEQ ID NO: 2) within the polymerase domain of thermostable DNA polymerases these thermophilic organisms. Sequence-the ILO is EP, Thermus aquaticus, Thermus caldophilus, Thermus thermophilus, Thermus flavus and Thermus filiformis, and Thermus species spsl7 and Z05. Sequence-motif SerGlnIleGluLeuArgVal (SEQ ID NO: 3) is also present in the polymerase domain of other thermostable DNA polymerases derived, for example, from Thermosipho africanus and from various strains of Bacillus, such as Bacillus caldotenax and Bacillus stearothermophilus. Sequence-motif SerGlnIleGluLeuArgIle (SEQ ID NO: 4), for example, is present in native thermostable polymerase, Thermotoga maritima, Thermotoga neapolitana and Anaerocellum thermophilum.

The full sequence of the nucleic acid and amino acid sequence for each of the Taq-, Tth-, Z05-, sps17-, Tma and Taf polymerase is described in U.S. patent 5466591. The sequence of the DNA polymerase from the TSA, Tfl, Tne, Ath, Bca and Bst are described in the following publications: Tja - in database sequences EMBL under registration number U62584 (see also Kwon, 1997, Mol. Cells 7(2): 264-271); Tfl - Akhmetzjanov and Vakhitov, 1992, Nucleic Acids Research 20(21): 5839; Tne - WO 97/09451 and in WO 96/41014; Ath - database EMBL sequences under registration number H (more strain Ath described by Rainey and others, 1993, J. Bacteriol. 175(15): 4772-4779); Bst - Uemori and others, 1993, J. Biochem. 113: 401-410 and database sequences EMBL under registration number U23149 (see also Phang and others, 1995, Gene 163: 65-68). Amino acids iovanna Japanese patent 05/304964 And, published European application EP-A-699760 and Aliotta and others, 1996, Genet. Anal. 12: 185-195; the sequence can also be obtained from the database of sequences EMBL under registration number U33536. The sequence, as described in Gene 163: 65-68 (1995), contains the remainder of the "E" at position 661 critical motive. ICA-polymerase described in Uemori and others, 1993, J. Biochem. 113: 401-410 and database sequences EMBL under registration number D12982. Thermostable DNA polymerase from Thermus filiformis (see FEMS Environ. Lett. 22: 149-153, 1994; also deposited in ATSC number 43280) can be recovered using the methods described in U.S. patent 4889818, and based on the sequence information provided in table 1. Each of the above sequences and publications included in the present description by reference. Homology (sequence identity) between the amino acid sequence of the native form of Taq polymerase, as described in WO 89/06691, and the sequence specified previously Tfl polymerase is more 87,4%. The corresponding homology with Tth polymerase is 87.4 per cent, with TCA polymerase is 86.6%, with Bst polymerase (registration number U23149) is 42,0% with ICA-polymerase is 42,6% G thermostable DNA polymerases. In the case where "X" denotes the residue is glutamic acid, the change of the gene encoding polymerase, allows to obtain the enzyme according to the invention, which easily includes MTF compared to, for example, Taq polymerase, which has a critical motive is not modified. Consequently, the invention relates to a class of enzymes that also includes, for example, thermostable DNA polymerase, and the corresponding gene and expression vectors from Thermus oshimai (Williams and others, 1996, Int. J. Syst. Bacteriol. 46(2): 403-408); Thermus silvanus and Thermus chliarophilus (Tenreiro and others, 1995, Int. J. Syst. Bacteriol. 45(4): 633-639); Thermus scotoductus (Tenreiro and others, 1995, Res. Environ. 146(4): 315-324); Thermus brockianus (Munster, 1986, Gen. Environ. 132: 1677) and Thermus ruber (Contact and others, 1984, Int. J. Syst. Bacteriol. 34: the 498-499; also deposited in ATSC number 35948). In addition, the invention includes, for example, modified forms of thermostable DNA polymerase and the corresponding genes and vectors for the expression of Thermotoga elfii (Ravot and others, 1995, Int. J. Syst. Bacteriol. 45: 312; also deposited under the number DSM 9442) and Thermotoga thermarum (Windberger and others, 1992, Int. J. Syst. Bacteriol. 42: 327; also deposited under the number DSM 5069). Each of the above sequences and publications included in the present description by reference.

In a preferred embodiment, subject to modification of critical twms of the objects of the invention to provide a mutant thermostable DNA polymerase, exhibiting significantly enhanced efficiency for incorporating unnatural nucleotides using matrices. In a particularly preferred example, the sequence of the polymerase includes LeuAspTyrSerGlnIleGlyLeuArgValLeualahisleuser (SEQ ID NO: 6). Such thermostable DNA polymerase is particularly suitable in processes such as DNA sequencing, RNA synthesis on the basis of DNA synthesis in vitro DNA having as substituents rntf.

Obtaining thermostable DNA polymerases with enhanced efficiency for incorporating unnatural bases can be carried out using techniques such as site-directed mutagenesis. See, for example, the publication Sambrook and other, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 1989, second edition, Chapter 15.51, "Oligonucleotide-Mediated Mutagenesis". For example, a mutation consisting in the replacement of "A" to "G" in the second position of the codon encoding residue glutamic acid 615 gene sequence DNA polymerase Thermus aquaticus (Taq) (see SEQ ID NO: 7), leads to a more than 500-fold increase in the efficacy of including extraneous nucleotides according to this description, while maintaining the ability of the enzyme to mediashout PCR in the presence of normal nucleotides, i.e. dntf.

This particular mutation in the DNA polymerase Taq leads to samsonet the ability of the enzyme to incorporate unnatural nucleotides, it should be expected that the replacement of the glutamic acid residue at the remains of any other amino acids, such as, for example, residues of serine, cysteine, threonine, alanine, valine or leucine, will have the same effect. Therefore, the present invention includes other amino acid substitutes that replace E, although the mutant E615G is preferred. Thus, the basis of the invention lies in the fact that the fourth amino acid residue in the motif is presented in SEQ ID NO: 1, is not a residue of glutamic acid.

Site-directed mutagenesis can also be performed by site-specific primaryprevention mutagenesis. This method is currently the standard in the art, and it is carried out by using a synthetic oligonucleotide primer complementary single-stranded DNA phage, which is subject to matirovanie, except for a limited erroneous pairing, representing the desired mutation. In General, as a primer to direct synthesis of the circuit, the complementary plasmid or phage, using synthetic oligonucleotide, and the resulting double-stranded DNA is transformed into bacteria-host, supporting FA is desali probe to identify those plaques who are required a modified sequence of a gene. Alternatively, you can apply methods "recombinant PCR" as described in PCR Protocols, San Diego, Academic Press, edited by Innis and others, 1990, Chapter 22, entitled "Recombinant PCR", Higuchi, pp. 177-183.

As shown in table 1, a glutamic acid in the critical motive Taq polymerase is stored in other thermostable DNA polymerases, however, can be localized in a separate but related situation amino acid sequence. Change conservative glutamic acid in SEQ ID NO: 2 thermostable DNA polymerases of the genus Thermus and DNA-polymerases from related genera Thermotoga, Thermosipho and Anaerocellum should also enhance the ability of the polymerase for the effective inclusion of unusual nucleotides compared to Taq polymerase, containing SEQ ID NO: 2.

The change in the balance of glutamic acid in the critical motive other thermostable DNA polymerases can be implemented using the principles and methods used in site-directed mutagenesis. In GeneBank or database SwissProt/PIR there are several sequences of DNA polymerase from Bacillus stearothermophilus. These sequences have a high degree of kinship, however, are somewhat different ), where "XAA" at position 7 of this sequence indicates the residue of valine (Val) or an isoleucine residue (Ile), albeit in different positions in the sequence.

Based on published information about the available amino acid and nucleotide sequences of thermostable DNA polymerases, are presented in this description, using standard methods of recombination can also be designed chimeric polymerase, which consist of domains originating from different thermostable DNA polymerases. In U.S. patent 5466591 and 5374553 described methods of exchange of different functional segments of thermostable polymerases, such as 5’-3’-ectonucleoside domain, 3’-5’-ectonucleoside domain and the polymerase domain, obtaining new enzymes. Preferred chimeric thermostable polymerase enzymes include 5’-3’-ectonucleoside domain, 3’-5’-ectonucleoside domain and the polymerase domain, and one of the domains is derived from another polymerase, and the polymerase domain contains critical motive SerGlnIleXaaLeuArgXaa (SEQ ID NO: 1), where "XAA" at position 4 of the sequence represents any amino acid residue except residue glutamic acid (Glu), and "XAA" at position 7 of this sequence p is armenti Taq/TMA, the structure of which is shown in table 2. As can be seen from this table, the polymerase domain of these chimeric enzymes Taq/Tma contains a mutation in the above critical motive.

Plasmid PC1 in accordance with the Budapest Treaty was deposited in ATSC July 17, 1996, and received a registration number 98107. Plasmid PC1 contains the gene encoding thermostable DNA polymerase with a mutation in the codon encoding the glutamic acid residue at position 615 amino acid sequence of native Taq polymerase, resulting in a mutant form of Taq polymerase, which has a glycine residue at position 615 (615G-mutant Taq polymerase). This Deposit allows the use of alternative methods for obtaining thermostable DNA polymerases with enhanced efficiency for incorporating unnatural analogues of nucleotides. In the example I shows that the use of flanking restriction sites suitable for sublimirovanny mutations E615G, allows you to create other thermostable enzymes DNA polymerase. Because many thermostable DNA polymerases known the full sequence of the gene based on the sequence information of criticism in the art ways such as splitting restrictase and the replacement fragment or site-specific mutagenesis in vitro.

A modified gene or a gene fragment obtained by site-specific mutagenesis can be obtained from a plasmid or phage by standard methods and Legerova with expression vector for further cultivation and selection of the resulting enzyme. For the practical implementation of the invention are suitable for numerous cloning and expressing vectors, including system suitable for mammals and bacteria, and they are described, for example, Sambrook and other, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, second edition, 1989. For convenience in the present invention illustrates the use of the PL promoter originating from phage lambda (Shimatake and others, 1981, Nature 292: 128). The use of this promoter, in particular, are described in U.S. patents 4711845 and 5079352.

Thermostable DNA polymerase of the present invention are usually isolated from microorganisms, such as E. coli that had been transformed by the expression vector functionally associated with the gene coding for DNA polymerase wild-type or modified thermostable DNA polymerase. An example of a suitable microorganism host is E. coli strain DG116 described by the Lawyer and d(American Type Culture Collection) under the registration number of ATSS 53601. Methods of purification of thermostable polymerases are also described, for example, Lawyer and others, 1993, PCR Methods and Applications 2: 275-287.

To a person skilled in the art it is obvious that the above thermostable DNA polymerase with enhanced efficiency for incorporating unnatural nucleotides, most can easily be obtained using methods based on recombinant DNA technology. The need for one of the enzymes of the present invention or a derivative or homologue of these enzymes assumes the creation of a recombinant form of the enzyme, which usually includes the construction of the expression vector, transformation of host cells with this vector and culturing the transformed host cell under conditions in which such expression will occur. Methods for producing expression vectors, transformation and culturing the transformed host cell are well known in the art and are described, for example, Sambrook and others, 1989, see above.

The present invention relates to thermostable DNA polymerase, which together with ribonucleosides useful to perform numerous functions, including amplification of nucleic KIS is ucit using chain-terminating analogs, with the high cost, such as dNTP, and that is an important factor that facilitates the receipt of new amplification products suitable not only for the analysis of DNA sequences, but also for other types of analysis such as electrophoresis or hybridization, without the need for subsequent reactions performed DNA sequencing.

Previously, it was confirmed that the pyrophosphatase improves the sequencing results when used as a mesophilic polymerase, and thermostable DNA polymerases by reducing the number of accumulated products of the elongation of the resulting pyrophosphorolysis. However before cycle sequencing it is necessary to use methods that allow you to enter additional enzyme in the reaction sequence. However, the most significant advantage of the present izobreteniya is that for DNA sequencing is not required of pyrophosphatase. Thus, the use of new, presented in this description of the enzymes eliminates the need for additional costs for the introduction of the second enzyme in the mixture for the reaction sequence.

When using enzymes of the present invention reaction amplification and sequencing combine that sposobem, first of all, due to the fact that the inclusion of both conventional nucleotides, ribonucleotides and their analogues in the product of the elongation of the primer allows to obtain chimeric chain RNA/DNA-sensitive hydrolysis of RNA. The treatment has no effect on the skeleton's DNA and results in a population of nucleic acid fragments, each of which ends in a position where we have built ribonucleotide instead of dNTP. The hydrolysis is easily carried out in various ways, including, but not limited to, alkaline hydrolysis (for example, processing of NaOH at a final concentration of 0.2 M, as described below in example VI), heat or enzymatic treatment with RNase (edited by Vogel and others, Informational Macromolecular, New York, Academic Press, 1963, Chapter, written by Berg and others, entitled "The Synthesis of Mixed Polynucleotides Containing Ribo - and Deoxyribonucleotide by Purified Preparation of DNA Polymerase from E. coli", pages 467-483).

In a preferred embodiment, the present invention relates to new and improved compositions, the most suitable methods for DNA sequencing. New enzymes described in this description can be used for methods of sequencing nucleic acids, based on the application of either painted terminators, or colored primers, and the tion matrix for the extension of the primer in the presence of terminating chain of nucleotides, resulting in incomplete fragments, which are then separated by size. In standard dideoxy sequencing for termination circuit used dideoxynucleosides and DNA polymerase, such as a piece of maple from a strain of E. coli Pol I (Sanger and others, see above).

Thus, the main method of dideoxy-sequencing includes (I) annealing of oligonucleotide primers corresponding to the matrix; (II) extending the primer with DNA polymerase in four different reaction mixtures, each of which contains one labeled nucleotide or labeled primer, a mixture of unlabeled dNTP and one termination circuit ddntp; (III) the separation of the four sets of reaction products with, for example, gel electrophoresis on a denaturing polyacrylamide gel with high resolution sposobnostey/urea, capillary separation or other methods of separation; and (IV) obtaining autoradiographical image of the gel, which can be analyzed to obtain conclusions about the sequence. Alternatively, to obtain information about the DNA sequence can be applied mass spectrometric methods or methods based on hybridization using fluorescently macrocrania sequencing method (see U.S. patent 507216) and its variants, called cycle sequencing". When cyclic sequencing of repeated cycles of heating and cooling that allows you to get numerous products lengthening each target molecules (see Murray, 1989, Nucleic Acids Research, 17: 8889). This asymmetric amplification of sequences of the target-complementary sequences of the matrix, in the presence of dideoxy terminator circuit yields a family of products lengthening of all possible lengths.

After denaturation of the product of the extension reaction of the DNA-matrix multiple cycles of primer annealing and primer elongation is carried out in the presence of dideoxy-terminators. Thermostable DNA polymerases have several advantages when cyclic sequencing; they are resistant to harsh temperatures of annealing, which is required for specific hybridization of the primer with the nucleic acid target, and is also resistant to multiple cycles of high-temperature denaturation, which are carried out in each cycle, i.e., to a temperature of 90-95°C. For this reason, various forms of DNA polymerase AmpliTaqincluded in kits for cyclic sequencing using the Taq, which are being implemented across the state nucleotides, such as dNTP, connected with the problem of its use for cyclic sequencing, because ddntp or fluorescently labeled ddntp should be included as chain terminators. In General, before the creation of the present invention DNA sequencing with thermostable DNA polymerases require a mixture of terminating chain of nucleotides, usually dideoxynucleotides in a high concentration in order to guarantee that the population of products lengthening, which includes fragments, with all possible lengths up to a length of several hundred bases. Often to reduce the cost of this process was applied protocols using very low concentrations of conventional dNTP that did the reaction is inefficient. These reaction mixture comprising a low concentration dNTP and high concentration dNTP, created the conditions under which thermostable polymerase is considerably nucleotide substrates.

Despite the emergence of modified enzymes such as DNA polymerase AmpliTaqFS, allowing to increase the concentration of dNTP to more optimal levels for enzymes prototype were still required expensive ddntp for sequen is proven to increase the concentration of dNTP, but eliminate the use of expensive ddntp, because instead of them growing in the chain include RTF. The ability of new enzymes to effectively implement the partial replacement of ribonucleotides facilitates the obtaining steps for DNA sequencing in the absence of individual reactions to enable the terminating nucleotide.

Selection of unusual nucleotide analogues suitable for use in the methods DNA sequencing, previously defined as the ability of thermostable DNA polymerase to incorporate these analogues. Unfortunately, these nucleotide analogs are quite expensive. For example, the price ddntp approximately 25 times the price as RTF and dNTP. As previously thermostable DNA polymerase did not possess the ability to effectively enable MTF in the growing DNA chain using a matrix, such ribonucleotides, which are easily available and cheap, and could be used for DNA sequencing with the participation of thermostable DNA polymerase. The present invention eliminates the need for ddntp reactions in DNA sequencing. Thus, one of the objects of the invention is a method of DNA sequencing, which is significantly less derogatorily to influence the ability of the polymerase to accurately include the correct pairs of nucleotide bases. Manganese can be used to enhance the incorrect mating grounds or to reduce restrictions on the insertion of a nucleotide analogue. Previously, researchers used manganese with the intention to induce mutagenesis in replication or DNA amplification. Thus, the manganese can affect the correctness of the polymerization reaction, and the reaction product yield. The resulting sequence may be incorrect or same - in a method of DNA sequencing - derived information may be incorrect. The methods according to the present invention does not require divalent cation manganese in the mixture for the reaction sequence in order to enhance the ability of the polymerase to incorporate unnatural nucleotide. In contrast to the known from the prior art DNA polymerase in the present invention are described critical motif included in the polymerase domain that controls the ability of the enzyme to distinguish between the 2’-substituted and unsubstituted nucleotides, not using manganese.

The enzymes of the present invention does not require sequencing of high concentrations of unnatural analogues grounds. Before the creation of the present invention when performing sequencevalue and associated traditional base is typically present in a ratio (for example, datf:datf) approximately from 1.3:1 to 24:1. For comparison of thermostable polymerases described in the present invention, can reduce the ratio of unnatural analogues grounds to the usual bases in a hundred or several thousand times. Attitude RTF:dntf equal to 1:1 or below, in combination with new enzymes presented in this description is sufficient for the analysis of DNA sequences. In a preferred embodiment, the ratio RTF:dntf reduced to less than 1:8. The 2’-substituted nucleotides to the corresponding natural dNTP can be even 1:80 or 1:200, depending on the specific plan of the experiment and the required length of the fragments.

Thus, since the enzymes of the present invention can easily include nesvoistvennoe nucleotides such as 2’-substituted nucleotides, there is no need to strengthen their ability to enable RTF with high concentrations RTF and limit the concentration of the corresponding dNTP. Therefore, the methods of the present invention allow you to apply the optimal concentration dNTP in combination with small amounts of MTF.

When appropriate method, such as sequencing painted with primer, apply modificirovannaya each dNTP 50-500 μm. Preferably the concentration dNTP ranges from 100 to 300 μm. When using these intervals corresponding MTF may be present in approximately the same concentration as dNTP, or lower. Preferably the concentration MTF is approximately from 0.1 μm to 100 μm, most preferably from about 2.5 μm to 25 μm.

Concentration rntf suitable for application of the modified enzymes of the present invention, can be easily determined by conventional expert in this field of technology by titration and optimization experiments. The required number MTF or equivalent determined by the type of experiment, and it may influence the size and purity of the target, as well as the choice of buffer and specific types of enzyme.

Attitude RTF:dntf will determine the frequency with which rntf are incorporated into the growing oligonucleotide. Because each embedded RTF can happen hydrolysis, the ratio RTF:dNTP can be adjusted in such a way as to allow the user the flexibility to increase or decrease the size of the resulting fragments.

As is well known, DNA is a polymer synthesized from dNTP. Each deoxynucleosides vide also contain a hydroxyl group at the 3’-position of sugar. However rntf differ from dntf the fact that the 2’-position sugar hydrogen atom substituted on the second hydroxyl group. In this case rntf are an example, which reveals the ability of the enzymes according to the invention is precisely to include 2’-substituted nucleotides. However, the compounds according to the invention is not limited to the use of unusual nucleotides that are ribonucleotides. Modification of the sequence of thermostable polymerase in a critical domain described herein, allows the matrix is directed to include alternative 2’-substituted nucleotides such as 2’-hydroxyl,3’-deoxynucleotide and nucleotides substituted in the 2’-position by fluorine or amino group.

As described in the examples below, the inclusion of 3’-deoxy, 2’-hydroxyalp, which in this description is also marked as coordinational, facilitated by the presence of a second mutation in thermostable polymerase, the ability to limit the inclusion of nucleotide containing methoxypropyl 3’-position of ribose, is reduced. Such enzymes have been previously described, for example, in EP-A-655506 and in the application for U.S. patent 08/448223, filed may 23, 1995, the Deposit was ATSS 69820, deposited according to the Budapest Thermus aquaticus, the ability to limit the inclusion of analogues, such as dNTP reduced. Dideoxynucleotide have a substituent in the 3’-position as compared with the conventional dNTP. Thus, in combination with the present invention a double mutation, as exemplified in this description is 615G, F667Y mutant Taq polymerase, allows the use of nucleotide analogues that contain a substituent in the 3’- and 2’-positions of the ribose compared with dNTP (see examples III and V).

A specific application of the invention is a method of sequencing using MTF where sequenase primer is labeled using a detectable fluorescent or radioactive label. Unlike ddntp enable unmodified RTF does not lead to such action, as the termination circuit. The reaction mixture for DNA sequencing includes rntp and dNTP in combination with the enzyme according to the invention, resulting in a mixture of randomly substituted products of the elongation of the primer that is sensitive to cleavage at the 3’-5’-phosphodiester bond between ribonucleotides and neighboring deoxyribonucleotides. After extension of the primer, for example, during PCR amplification or cyclic sequencing and before separation of the products lengthening patiwat a ribonuclease, or other ways hydrolyzing products extension for each encountered ribonucleotide. For each labeled product elongation of the primer only the largest 5’-fragment, which is an intermediate product lengthening labeled primer, found on sequanorum gel. For this target, the analysis of the obtained securitysage gel gives step sequencing, i.e. the series of detected signals in G-, A-, T - and C-lines corresponding to the nucleotide sequence of the target. Received the degree of sequencing provide the same information as the conventional method involving the use of ddntp or based on the use of RTF and new thermostable polymerases presented in this description. Thus, when applying the present invention costly ddntp for DNA sequencing are no longer required (see example VI).

In an alternative method of sequencing used the termination circuit of the ribonucleotides. In this embodiment, as terminators using 2’-hydroxy,3’-deoxynucleotide, such as coordinational. These analogues MTF can be fluorescently labeled and used for DNA sequencing. In Lee and others, see above, describes the use is consistent enzymes for use with ddntp. Thermostable DNA polymerase that contains both the modification present in the DNA polymerase AmpliTaq FS(see above) and that presented in SEQ ID NO: 1, where X does not denote glutamic acid (E), as described previously, can be used for the effective inclusion of labeled analogues rntf in the implementation of the sequencing reaction, which is based on the termination circuit. This process can be automated and does not require the synthesis of dye-labeled primers. In addition, since the reaction with painted terminator enable in the same test tube, all four reactions, they are more convenient than the methods with the tinted primer. 2’-hydroxy,3’-deoxynucleotide can be synthesized from commercially available 3’-nucleotides (3’-Yes, 3’-DC, 3’-DG and 3’-dt, for example, supplied by the company Sigma Chemical Corporation, St. Louis, PCs Missouri) and adding 5’-triphosphate as described in Ludwig, Biophosphates and Their Synthesis, Structure, Metabolism and Activity, Ed. by Bruzik and Stec, Amsterdam, Elsevier Science Publishers, 1987, pp. 201-204.

In addition to the application of new sequencing methods, the modified enzymes presented in this description, can be used for various purposes in molecular biology. In one example assests normal, and unusual nucleotides, for example dNTP and at least one detectable in the tagging RTF, labels that include, for example, a fluorescent label or a radioisotope. Managed matrix synthesis of complementary chain results in a DNA product that contains ribonucleosides in various positions along its length. Heating and/or treatment with alkali hydrolyzing the product of the elongation of the nucleic acid at each ribonucleotide. Thus, get the collection of DNA segments, where each segment contains one labeled residue at its 3’end. The size of the resulting fragments of the nucleic acid can be modified by adjusting the relationship and the number rntf included in the response.

Amplification of the target using RTF and enzymes of the present invention offers many advantages, due to the specific use. In the above-described method using labeled rntp all received the collection of fragments is labeled with equal intensity: one label on one oligonucleotide fragment. To achieve optimal results by implementing methods such as detection of nucleic acids using the to amplificatory the target was randomly fragmented within a fixed and reproducible size in order to restrict the formation of secondary structures to control the kinetics of hybridization. In addition, to detect hybridization with the ordered arrangement of thousands of probes on a thin layer of silicone may be preferred that the nucleic acid fragments was in the state with equal intensity. According to the present invention has been developed ways of getting collections of fragments that satisfy this standard, thereby facilitating the use of alternative formats of identification, such as methods based on the use of thin layers, as described, for example, Cronin and others, 1996, Human Mutation, 7: 244-255.

In another example, assume the use of a single labeled primer and one unlabeled primer in the mixture for the amplification reaction, which includes termostabilno polymerase according to the invention, as well as RTF and dntf that allows you to simultaneously improve reaction amplification and sequencing. This method requires to make four different amplification reaction, one for each rntp. Thus, for example, because the enzyme according to the invention is suitable for amplification of the target, for example, by PCR or other methods of amplification, the resulting product, if present, can be detected by conventional means, such as gel electrophoresis or GI is enabled ribonucleotides, and the behavior of RNA/DNA chimeric chains will be the same as that expected for a normal product of nucleic acid amplification. If the desired product is found, the remaining portion of this reaction mixture can be treated with alkali and analyzed using gel electrophoresis to determine the sequence of nucleic acid. Thus, after detection of the product subsequent reaction sequencing is not required. This simplified method saves time and materials and is characterized by high accuracy by reducing stages: detected product is sequenced product.

In addition, it can also be used similar methodology with four labeled rntp and one biotinylated (labeled with Biotin) primer. After amplification, the product is decomposed with alkali hydrolysis, and primer associated with the products is removed by interaction with streptavidin coated granules. Captured granules products are then analyzed on sequanorum gel. This modification allows the sequencing reaction in a single tube, thereby eliminating the need for four different amplification.

In choosing the treatment RNA using a DNA template, or to obtain substituted DNA for Mediaroom alkali sterilization without the use of conventional sterilizing agents, such as uracil-N-glycosylase (UNG), as described in international application WO 92/01814.

In the examples of the invention, thermostable polymerase also contains a mutation in the 5’-3’-ectonucleoside domain, which leads to a significant deterioration ectonucleoside activity. The modified form of Taq polymerase is described in U.S. patent 5466591. In one of the embodiments of the invention, the codon encoding residue of glycine (G) at position 46 amino acid sequence was replaced by a codon coding for aspartic acid (D). Thus obtained enzyme has the advantage when used in reactions of cyclic sequencing due to reduced 5’-3’-ectonucleoside activity and is predpochteniem for use in the present invention. Compared with the wild-type enzyme, the presence of G46D mutation has no effect on polymerase domain amino acid sequences and polymerase activity.

According to the invention are also kits for sequencing nucleic acids containing thermostable polymerase of the present invention, which is an example of a commercial application of the invention.

These kits usually include dopolnilos unlabeled, can also be included labeled primer.

Below the invention is illustrated in the examples, not limiting its scope.

Example I

Gene expression is modified Taq polymerase with reduced ability to limit the inclusion of extraneous nucleotides

C-terminal part of the amino acid sequence of DNA polymerase Taq encodes a domain that includes the site of the polymerase activity (Lawyer and others, 1993, PCR Methods and Applications 2: 275-287). The DNA fragment containing this region was isolated from a full-sized gene Taq and mutagenesis was carried out using PCR amplification in the presence of manganese (Leung and others, Technique 1(1): 11-15). In this example, all restrictase were obtained from the company New England Biolabs, Beverly, pieces Massachusetts. Fragments containing the mutations were digested with restrictase > PST and BglII and cloned into the plasmid expressing Taq, in this case, the plasmid pLK102, which was pre-digested > PST and BglII. Plasmid pLK102 is a modified form of the plasmid pSYC1578, which expresses Taq (Lawyer and others, see above). The HincII fragment/EcoRV, localized at the 3’-region that encodes a polymerase, was removed to create the plasmid pLK101. > PST -BglII-fragment length 898 base pairs were then removed from pLK101 and were replaced by short oligonucleotide of Duplek the 3’-end of the pol gene Taq DNA with their replacement over a short section of DNA.

Received expressing plasmids transformed strain E. coli N1624 (described by Gottesman, 1973, J. Mol. Biol. 77: 531; it can also be obtained from E. coli Genetic Stock Center at Yale University, the number of strain CGSC 5066) and the obtained transformants were screened for the ability to more effectively compared with the wild-type enzyme to enable RTF. Using this technique, revealed the C1 mutant that can better enable RTF.

To determine which part of the gene Taq polymerase responsible for the altered phenotype containing the mutation plasmid expressing Taq named PC1 and isolated from mutant C1, uncoupled different restrictase and the resulting restriction fragments were subcloned into the plasmid pLK101 carrying the gene DNA Taq polymerase wild-type, replacing the restriction fragments that do not contain mutations. Analysis of the resulting subclones showed that the mutation responsible for the phenotype, were restriction fragment NheI-BamHI length 265 base pairs.

Carried out analysis of the DNA sequence of this region of the plasmid PC1 using the set ABI PRISMaDye Terminator Cycle Sequencing Core Kit, which includes DNA polymerase AmpliTaq FSsupplied by the company Applied Biosystems, foster city, California, and AI in the gene Taq polymerase between sites Nhel and BamHI. Mutation of amino acid at position 615 has led to the fact that the residue glutamic acid (E) was substituted by a residue of glycine (G), and the other mutation at position 653 led to the substitution of residue alanine (a) for threonine (T). The numbering starts with a codon encoding the first methionine residue of the Mature protein, as described in U.S. patent 5079352. Mutation E615G resulted in the replacement on GGG GAG at codon 615. Mutation AT resulted in the replacement of GCC to ACC in codon 653. The C1 plasmid in the strain-host E. coli T1624 was deposited according to the Budapest Treaty in ATSC 17 July 1996 and received a registration number 98107.

Two point mutations were analyzed separately by sublimirovanny each separately in the gene Taq polymerase wild-type, using recombinant PCR (under. ed Innis and others, PCR Protocols, San Diego, Academic Press, 1990, Chapter 22, entitled "Pecombinant PCR", the author Higuchi, pp. 177-183). The resulting expression profiles were analyzed to determine which mutations E615G or AT or both are responsible for the phenotype, characterized by the incorporation of ribonucleotides. The results of the experiment showed that the mutant phenotype is solely responsible mutation E615G.

For further analysis and quantitative assessment of the effect of the inclusion analogues nucleotidesequence pRDA3-2. The expression vector pRDA3-2 contained a full-sized gene Taq, functionally linked to the promoter of phage lambda PL. Ectonucleoside domain gene Taq this vector contained a point mutation in the codon encoding glycine, i.e., the amino acid residue at position 46, which reduces the 5’-3’-ectonucleoside activity. However, the sequence of a gene within the polymerase domain of the expression vector pRDA3-2 is identical to the gene sequence wild-type Taq. Plasmid pRDA3-2 is fully described in U.S. patent 5466591, where the plasmid is designated as "clone 3-2". Plasmid pRDA3-2 was digested with BamHI and NheI and PCR-fragment length 265 base pairs in standard ways ligated with the vector.

The obtained plasmid pLK108 transformed strain E. coli DG116 (Lawyer and others, 1993, see above, this strain can also be obtained from the American type culture collection under the number of ATSS 53606). Plasmid pLK108 encodes a thermostable DNA polymerase, indicated in the present description as G46D, E615G-Taq. Using recombinant PCR by combining mutations E615G and P667Y in the fragment BamHI-NheI received the mutant G46D, E615G, F667Y-Taq. This fragment was cloned in the plasmid pRDA3-2 to create the plasmid pLK109. Expressing plasmids pLK108 and pLK109 protein thermostable DNA polymerase was isolated influencers interest sequence was confirmed by analysis of DNA sequences. In box (insertions) pLK108 was discovered additional mutation in the sequence; however, this mutation did not change the amino acid sequence of the protein.

After partial purification activity of the modified enzyme was determined in accordance with the method of assessing the activity described by the Lawyer and others, 1989, J. Biol. Chem. 264: 6427-6437. The activity of the modified enzyme was estimated as follows: one unit of enzyme corresponds to 10 nolam product synthesized within 30 minutes. DNA polymerase activity of the enzyme wild type directly proportional to the concentration of the enzyme, providing up to 80-100 pmole DTMF (dilution of enzyme is 0.12 to 0.15 units per reaction). The activity of the mutants E615G, G46D and E615G, F667Y, G46D is directly proportional to the concentration that ensures the inclusion of up to 0.25-3 pmole DTMF (dilution of the enzyme ranges from 610-4to 510-3units per reaction). This enzyme preparation was used to assess inclusion and sequencing reactions described in examples III-V. For examples II and VI of the enzyme was purified in accordance with the methods described by the Lawyer and others (see above).

Example II

Analysis of the comparative effectiveness ://img.russianpatents.com/chr/945.gif">-32R]rntf that each of the enzymes may include DNA-matrix of activated salmon sperm with reduced concentration of this enzyme. To quantify enable RACF reaction mixture was prepared so that the final concentration in 50 μl of reaction mixture was 12.5 µg DNA activated salmon sperm obtained by the method described below, 200 μm each of dCTP, DSTF and dTTP (firm Perkin Elmer, Norwalk, PCs Connecticut), 100 μm [-32R]RATP, 1 mm-mercaptoethanol, 25 mm N-Tris[hydroxymethyl]methyl-3-aminopropiophenone acid (TAPS), a pH of 9.5 at 20°C, 50 mm KCl and 2.25 mm MgCl2.

A similar mixture for analysis were obtained to quantify enable rctf, FSRBs and Ruth. In each case rntf were radioactively labeled and was present at a concentration of 100 μm, and each of the other three dNTP (dATP, DSTF and dTTP for rctf, dATP, dCTP and dTTP for FSRBs and dATP, dCTP and DSTF for RUTF) was present at a concentration of 200 μm. As standard also measured the inclusion of each corresponding enzyme [-32R]dNTP. Mixture for analysis in these experiments was similar to that used in the experiment including Ameen 100 μm corresponding [-32R]dNTP. The crude DNA salmon sperm in the amount of 1 g/l obtained from the company Worthington Biochemical (Freehold, pc. new York), activated by incubation in 10 mm Tris-HCl, pH to 7.2, 5 mm MgCl2the temperature of 2-8°C for 96 hours. Then added add and NaCl to a concentration of 12.5 mm and 0.1 M, respectively. Then DNA was extracted with phenol/chloroform and then precipitated with ethanol and resuspendable in 10 mm Tris, 1 mm add, pH 7.5. Then the drug is activated DNA was subjected to dialysis against the same buffer.

45 μl of each reaction mixture was poured in the form of an aliquot quantities of five tubes with a volume of 0.5 ml (for example, in tubes Eppendorf), making each 5’-labeled precursors of nucleotides. Thus, each polymerase G46D-and Taq G46D, E615G-Taq were assessed twice, with one tube was left as a negative control. The mixture resulting from the polymerization reaction, from every experience in two test tubes were first mixed with 5 ál of either polymerase G46D-Taq (0.02 units) or G46D, E615G-Taq (0.002 units). As a control the background level to the reaction mixture, used as a negative control, instead of the enzyme was added to 5 μl of buffer for dilution of the enzyme.

Each reaction mixture for the tion was stopped, adding 10 μl of 60 mm etc, and kept on ice. For each sample, aliquots of 50 ál 60 ál of the reaction mixture was diluted with 1 ml of 2 mm etc, 50 μg/ml fragmented DNA salmon sperm. DNA was besieged by using trichloroacetic acid (THU) using standard methods and collecting on the filter discs of the type GF/C, Whatman, Kent, UK). The number included [-32P]-labeled nucleotide or ribonucleotide quantitatively assessed using a liquid scintillation spectrometry and then counted the number of active pmole. The number of pmoles each rntp included each enzyme was correlated with the number of pmoles the appropriate-32R]dNTP included each enzyme. In table.3 shows the data obtained.

These results indicate that polymerase G46D, E615G includes ribonucleotides more than 500 times more effective in comparison with how it can carry out polymerase G46D (for example, more effective for FSRBs in 181:0,36=502 times for rctf in 189:0,22=859 once and for RACF 210:0,18=1166 times).

Thus, a missense mutation in the polymerase gene at codon 615 results in a new phenotype: to receive termonuclear.

Example III

Analysis of the relative efficiency of incorporation of 3’-detoxif (cordycepin)

The relative ability of the polymerase G46D-; G46D, E615G-; G46D, E615G, F667Y and G46D, F667Y-Taq include 3’-deoxyadenosine-5’-triphosphate (coordinational) was determined by measuring the amount of [-32R]konicaminolta that each enzyme can include DNA-matrix of activated salmon sperm with reduced concentration of this enzyme. To quantify enable [-32R] konicaminolta the experiment was carried out so that the final concentration in 50 μl reaction mixture consisted of 12.5 µg DNA activated salmon sperm, 200 μm each of dCTP, DSTF and dTTP, 50 μm dATP (firm Perkin Elmer), 50 μm [-32P]-3 datf/3’-dATP (firm New England Nuclear, Sigma), 1 mm-mercaptoethanol, 25 mm N-Tris[hydroxymethyl]methyl-3-aminopropiophenone acid (TAPS), a pH of 9.5 at 20°C, 55 mm KCl and 2.25 mm MgCl2.

45 μl of each reaction mixture was poured in the form of an aliquot quantities in nine test tubes 0.5 ml, i.e., each reaction was carried out either with G46D-; G46D, E615G-; G46D, E615G, F667Y-; or G46D, F667Y-Taq in two replications, with one tube stop is for each experiment in two test tubes were first mixed with 5 μl (0,058 units) polymerase G46D-Taq. The same operation was performed with polymerase G46D, E615G-Taq (0,0025 units), polymerase G46D, E615G, F667Y-Taq (0,0034 units) or polymerase G46D, F667Y-Taq (0,083). As a control level of the background in the one remaining tube instead of the enzyme initially added to the buffer for dilution of the enzyme.

Each reaction mixture over a short time interval centrifuged, and incubated for 10 minutes at 75°C. the Reaction was stopped by adding 10 μl of 60 mm etc, and kept on ice. Each sample aliquots of 50 ál 60 ál of the reaction mixture was diluted with 1 ml of 2 mm etc, 50 μg/ml fragmented DNA salmon sperm. DNA was besieged by using THU, using standard methods and collecting on the filter discs of the type GF/C, Whatman, Kent, UK). The number included [-32P]-labeled nucleotide was assessed using a liquid scintillation spectrometry, and then counted the number of active pmole. The number of pmoles [-32R] konicaminolta included each enzyme was correlated with the number of units of each enzyme used in the experiment, to get the number of pmoles included unit of enzyme. In table.4 shows the obtained Danchenko include molecules of cordycepin in DNA.

Example IV

DNA sequencing is based on the alkaline decomposition using DNA polymerase G46D, E615G-Taq.

This example shows the use of the modified polymerase according to the invention for sequencing on the basis of alkaline decomposition using DNA, partially substituted rntf. Attitude RTF to dNTP in the reaction mixtures ranged from 1:80 to 1:8. The extension reaction of the primer was carried out in a buffer consisting of 50 mm Bicine (N,N-bis(2-hydroxyethyl) glycine; pH 8.3), 25 mm COAs and 2.5 mm MgCl2. Conducted four separate reactions, one for each of the four rntf. Each reaction mixture (50 μl) contained 200 μm each of dATP, dCTP, DSTF and dTTP (firm Perkin Elmer) and 0.09 pmole single-stranded DNA template MMR (firm Perkin Elmer), which was annealed with 5’-[32P]-labeled DG48 (Lawyer and others, 1993, PCR Methods and Applications 2: 275-287). The reaction mixture also contained 2,5, 2,5, 2,5, or 25 μm RATP, rctp, FSRBs or RUTF respectively.

Each of these four reactions were started by adding 7 units of DNA polymerase G46D, E615GB-Taq, and incubated for 10 minutes at a temperature of 75°C. the Reaction was stopped by adding 10 μl of 60 mm etc and placed on ice. 20 μl of each reaction mixture was added to 80 μl of 50 mm Bicine (pH 8.3), 25 mm COAs and 2.5 mm MgCl2. Has been neutralized by addition of 7 μl of 1 N. HCl. Each reaction mixture was besieged by adding 312 ál of 95% ethanol and 10 μl of 3 M sodium acetate (pH of 4.8). The reaction mixture was microcentrifuged for 15 minutes to collect the precipitate, the supernatant was removed, the debris was washed with 500 µl 70% ethanol and dried. Each debris resuspendable in 5 μl of 0.5 × stop buffer (supplied by the company Perkin Elmer, Norwalk, PCs Connecticut, which contains 95% formamide, 20 mm etc and 0.05% Bromphenol blue), kept at a temperature of 98°C for 3 minutes and was directly loaded on sequenase gel for DNA, consisting of previously subjected to electrophoresis on 6% polyacrylamide/8 M urea, and was carried out by electrophoresis. The gel was dried and removed to the x-ray film. The obtained film showed a pronounced sequeiros staircase, which leads to an excess of 100 bases of correct sequence.

Example V

DNA sequencing using DNA polymerase G46D, E615G, F667Y-Taq and 3’-deoxynucleotides

This example describes the use of a modified polymerase G46D, E615G, F667Y-for Taq DNA sequencing using 3’-deoxynucleotides. This experiment was performed using 3’-detoxif; however, it can also be assistam of 50 mm Bicine (pH 8.3), 25 mm COAs and 2.5 mm MgCl2. Each reaction mixture (50 μl) contained 200 μm each of dATP, dCTP, DSTF and dTTP (firm Perkin Elmer) and 0.09 pmole single-stranded DNA template MMR (firm Perkin Elmer), which was annealed with 5’-[32P]-labeled DG48 (Lawyer and others, 1993, PCR Methods and Applications 2: 275-287). The reaction mixture also contained 0, 0,1, 0,25, 0,5, 1 or 5 μm 3’-detoxif.

Each of the reactions were started by adding 7 units of DNA polymerase G46D, E615G, F667Y-Taq, and incubated for 10 minutes at 75°C. the Reaction was stopped by adding 10 μl of 60 mm etc and placed on ice. 30 μl of each reaction mixture was precipitated with ethanol and resuspendable in the stop buffer, kept at a temperature of 98°C for 3 minutes and was directly loaded on sequenase gel for DNA, consisting of previously subjected to electrophoresis on 6% polyacrylamide/8 M urea, and was carried out by electrophoresis. The gel was dried and removed to the x-ray film. The band, which contained the products of a reaction conducted in the presence of cordycepin, testified to the presence of explicit stairs termination. The band containing the largest number of cordycepin, i.e., 5 µm, showed a ladder termination, in which the average band were shorter in length compared to the Polo is authorized in the absence of cordycepin, testified to the presence of the product, having full length, and showed no stairs termination. These results indicate that the mutant enzyme is able to include korditsepin, and the inclusion of this molecule in the product of the elongation of the primer causes the termination. This method can also be used to create ladder DNA sequencing using 3’-detoxing, 3’-desoxy and 3’-detoxif.

Example VI

PCR-sequencing painted with primer using a DNA polymerase G46D, E615G-Taq

This method illustrates the use of a modified polymerase according to the invention for sequencing painted with primer using ribonucleosides (MTF) in PCR with a ratio of MTF:dntf not more than 1:30. Conducted four separate reactions, each using RTF. The reaction sequence-based PCR was performed in a buffer consisting of 25 mm Tris-Hcl (pH 9), 5.0 mm MgCl2and 10% glycerol (volume/volume). Each reaction mixture contained 500 μm each of dATP, dCTP, DSTF and dTTP (firm Perkin Elmer), the matrix representing 5106copies/ál plasmid pBSM13+ (firm Stratagene), linearized in the result of processing by the restriction enzyme XmnI, and 0.05 um is Raymer JOE M13 Reverse Dye Primer (firm Perkin Elmer) and 0.1 μm of primer ASC46 (5’-CGCCATTCGCCATTCAG). The reaction mixture of RIBO-TTF (10 μl) contained 2.5 μm TTF (firm Pharmacia Biotech), 0.1 µl of primer FAM M13 Reverse Dye Primer (firm Perkin Elmer) and 0.1 μm of primer ASC46. The reaction mixture of RIBO-GTP (20 μl) contained 2.5 μm GTP (firm Pharmacia Biotech), 0.1 µl of primer TAMRA M13 Reverse Dye Primer (firm Perkin Elmer) and 0.1 μm of primer ASC46. The reaction mixture of RIBO-UTP (20 µl) contained 16 μm UTP (firm Pharmacia Biotech), 0.1 µl of primer ROX M13 Reverse Dye Primer (firm Perkin Elmer) and 0.1 μm of primer ASC46.

Each of the four reaction mixtures were placed in a preheated (75°C) thermoacetica for PCR type Perkin Elmer GeneAMPPCR System 9600 and subjected to 30 cycles: aging at a temperature of 95°C for 10 seconds at a temperature of 55°C for 10 seconds, gradually lowering the temperature to 65°C for 1 minute and holding at 65°C for 5 minutes. The result is that each of the reactions involving RTF and rctf received 61011copies of the dye-labeled amplified product length 300 base pairs, and as a result of reactions with participation of FSRBs and UTP received 1,2x1012copies of the dye-labeled amplified product length 300 base pairs.

To determine the DNA sequence of the amplified PCR products without the use of lye and heated, neutralized and precipitated as follows. United 4 μl each of the reaction mixtures with the use of ATP and TTF and 8 μl each of the reaction mixtures using GTP and UTP. To the combined reaction mixtures were added 2 μl of 0.25 M EDD (pH 8.0) (final concentration 10 mm), 10 μl 1 M NaOH (final concentration 200 mm) and 14 μl of N2Oh, and then incubated at 95°C for 5 minutes in thermoacetica for PCR type Perkin Elmer GeneAMPPCR System 9600 and neutralized 10 ám 1 M HCl. Then the combined reaction mixture was besieged by adding 150 ál of 95% ethanol followed by incubation at 4°C for 15 minutes. Then it was microcentrifuged for 15 minutes at 4°C to collect the sediment and the supernatant was removed by aspiration. Debris washed with 300 ál of 70% ethanol was microcentrifuged within 5 minutes, the supernatant was removed by aspiration and debris was dried. Debris resuspendable in 6 μl of formamide, 50 mg/ml blue dextran (25 mm etc, 5:1 (volume/volume) and kept at a temperature of 90°C for 3 minutes. 1,5 µl resuspending debris directly downloaded sequenase gel, consisting of previously subjected to electrophoresis 5I on DNA-sequencing machine type Perkin Elmer ABI Prism377 DNA Sequencer according to the manufacturer's instructions. Automatic determination of the grounds on the basis of software for sequence analysis Perkin Elmer ABI Prism377 Sequencing Analysis showed more than 99% accuracy determine the DNA sequence product length 300 base pairs amplified using PCR.

Claims

1. Thermostable DNA polymerase, representing a derivative of natural thermostable DNA polymerase comprising the amino acid sequence motif SerGlnIleGluLeuArgXaa (SEQ ID NO: 2), where XAA at position 7 of this sequence indicates the residue of valine (Val) or a residue of isoleucine (IIE), which result from the replacement of a glutamic acid residue (Glu) at position 4 of the sequence and selection of mutant (modified) form, characterised by decreased compared with the corresponding natural enzyme ability to limit the inclusion of extraneous nucleotides, preferably rntf.

2. Thermostable DNA polymerase under item 1, characterized in that it is a recombinant derivative of natural thermostable DNA polymerase.

3. Of the components is of Leonidov compared with the ability of the corresponding natural polymerases include unusual nucleotide increases at least 20 times.

4. Thermostable DNA polymerase according to any one of paragraphs.1-3, characterized in that it has sufficient activity for use in the reaction, DNA sequencing, involving the use of unusual nucleotides, preferably rntp, and the corresponding normal nucleotide at a ratio of 1:1 or less.

5. Thermostable DNA polymerase according to any one of paragraphs.1-3, characterized in that it has sufficient activity for use in the reaction, DNA sequencing, involving the use of unusual nucleotides, preferably RTF present in a concentration of less than approximately 100 μm, and the corresponding normal nucleotide is present in a concentration of more than about 100 microns.

6. Thermostable DNA polymerase according to any one of paragraphs.2-5, which is a recombinant derivative of natural thermostable DNA polymerase from an organism selected from the group comprising Thermus aquaticus, Thermus caldophilus, Thermus chliarophilus, Thermus filiformis, Thermus flavus, Thermus oshimai, Thermus ruber, Thermus scotoductus, Thermus silvanus, Thermus species Z05, Thermus species spsl7, Thermus thermophilus, Thermotoga maritima, Thermotoga neapolitana, Thermosipho africanus, Anaerocellum thermophilum, Bacillus caldotenax and Bacillus stearothermophilus.

7. Thermostable DNA polymerase according to any one of paragraphs.2-5, which is the but thermostable DNA polymerase, that includes the amino acid sequence LeuAspTyrSerGlnIleGluLeuArgValLeualahisleuser (SEQ ID NO: 5).

8. Thermostable DNA polymerase according to any one of paragraphs.1-5, a sequence which is homologous amino acid sequences of DNA Taq polymerase (SEQ ID NO: 7), at least approximately 39%, preferably at least about 60%, more preferably at least about 80%.

9. The nucleotide sequence encoding a thermostable DNA polymerase according to any one of paragraphs.1-8.

10. Vector providing the expression of a thermostable DNA polymerase and containing the nucleotide sequence under item 9.

11. Bacterial strain E. Li ATSS No. 98107 producing thermostable DNA polymerase.

12. The method of obtaining thermostable DNA polymerase, including

(a) culturing the strain on p. 11 under conditions that increase the expression of a thermostable DNA polymerase, and (b) isolation of thermostable DNA polymerase.

13. Thermostable DNA polymerase obtained by the method according to p. 12.

14. Composition for use in the reaction, DNA sequencing, which contains a nucleic acid matrix; oligonucleotide primer complementary to the matrix; thermostable DNA polymerase according to any one of paragraphs.1-8; with the public nucleotide corresponding normal nucleotide is 1:1 or less.

15. The composition according to p. 14, where unusual nucleotide is ribonucleotide, and this ribonucleotide preferably present in concentrations lower than approximately 100 μm, and the corresponding normal nucleotide is present at a concentration higher than about 100 microns.

16. The composition according to p. 15, characterized in that the unusual nucleotide is unlabeled.

17. The method of sequencing a nucleic acid target, comprising the following stages:

(a) preparation of unnatural nucleotides, preferably rntp, and the corresponding normal nucleotide reaction DNA sequencing, and unusual nucleotide and corresponding normal nucleotide present in a ratio of less than about 1:1; (b) treating the reaction mixture from stage (a) in the presence of thermostable DNA polymerase according to any one of paragraphs.1-8 under conditions suitable for extension of the primer, to produce elongation of the primer, containing unusual nucleotide; (C) processing the products of the elongation of the primer from step (b) under conditions suitable for hydrolysis of these products extend the primer; (d) separating the reaction products from step (C) and (d) sequencing the nucleic acid target.t ribonucleotide preferably present in a concentration of about 0.1-100 microns.

19. The method of sequencing by p. 17, where the corresponding normal nucleotide is preferably present in a concentration of about 50-500 μm.

20. A kit for sequencing a nucleic acid containing a thermostable DNA polymerase according to any one of paragraphs.1-8; buffers used in the method sequence; one or more oligonucleotide primers; the mixture dNTP and at least one unnatural nucleotide, preferably rntp, and the unusual relationship between the nucleotide and the corresponding normal nucleotide, preferably less than 1.

Convention priority is selected from 06.08.1996 on PP.1-20 in accordance with the application 60-023.376, filed in the U.S. patent office.

 

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