WO2011142492A1 - Dna polymerase capable of long pcr, and genes thereof - Google Patents

Abstract

The present invention relates to a mutant DNA polymerase capable of long PCR, and to the genes thereof, and more specifically, to a DNA polymerase that is useful in long PCR polymerization obtained by mutating a wild-type TNA1_pol DNA polymerase derived from the Thermococcus sp. strain, to a gene encoding the mutant DNA polymerase, and to a method for preparing a polymerase using same. The mutant DNA polymerase of the present invention exhibits excellent performance in the PCR of long-chain DNA, compared to conventional wild-types or mutant-types, and thus may be widely applied in various fields that require the amplification of long chains, such as human gene vector analysis, etc.

Description

DNA polymerase and its genes capable of long PCR

The present invention relates to a mutant DNA polymerase capable of performing long PCR, a gene sequence encoding the same, a preparation method, and a PCR polymerization using the same. More specifically, the present invention relates to a specific DNA derived from Thermococcus sp. The present invention relates to a DNA polymerase causing a mutation at a site, an amino acid sequence thereof, a gene encoding the mutant DNA polymerase, and a method for producing a polymerase using the same.

Recent advances in genomic research have yielded enormous amounts of gene sequence information. With the general combination of conventional genetic engineering and genomic research techniques, the genomic sequences of some highly thermophilic microorganisms are of interest because they are characterized by heat-resistant enzymes in the field of biotechnology, and various thermally stable enzymes are used for biotechnological purposes. Is being developed.

Such thermostable thermophilic DNA polymerase and polymerase chain reaction (PCR) using the same have important contributions in protein and gene research, and are widely used in biological applications. In fact, more than 50 DNA polymerase genes have been cloned from various organisms, including thermophilic and archaea, and recently, highly thermophilic bacteria with higher fidelity in PCR based on proofreading activity than common Taq polymerase. B family DNA polymerase was isolated and widely used from Pyrococcus sp. And Thermococcus sp. Strains. Nevertheless, high fidelity polymerases are low in DNA elongation ability and require many improvements despite the above advantages.

We have already isolated new highly thermophilic strains from the deep-sea hydrothermal spout area of PACMANUS field and analyzed the 16S rDNA sequence for thermococcus. Genus (Thermococcussp.) strain, and then analyzed the entire genome sequence to find a heat stable enzyme from the strain. Analysis of the genomic information confirmed that the strain had a DNA polymerase of type B, cloned the gene corresponding to the DNA polymerase from the strain and expressed this gene in E. coli, and recombinant DNA polymerase There was a patent registration for a DNA polymerase having high-extension and high-fidelity by pure separation (see Korean Patent Registration No. 10-0777227).

 Therefore, the present inventors continued efforts to develop a DNA polymerase suitable for long PCR polymerization reaction using wild-type TNA1_pol DNA polymerase of Korea Patent Registration No. 10-0777227, Thermococus Genus (Thermococcussp.) DNA polymerases isolated from the strains were mutated to select DNA polymerases suitable for long PCR polymerization, and they were found to be useful for long chain DNA amplification reactions in human genome research. This invention was completed.

An object of the present invention is to provide a mutant DNA polymerase capable of long PCR, genes thereof.

For this purpose, the present invention is a DNA polymerase derived from Thermococcus sp. Strain and mutated to wild type TNA1_pol DNA polymerase of Korea Patent Registration No. 10-0777227 to be useful for long PCR polymerization. To provide.

A first aspect of the invention provides a DNA polymerase comprising the amino acid sequence of SEQ ID NO.

As used herein, "DNA polymerase" refers to DNA in the 5 '-> 3' direction from deoxynucleotide triphosphate by successively adding nucleotides to the growing 3'hydroxy group using complementary template DNA strands and primers. It is an enzyme that synthesizes. The template strand determines the order of nucleotides added by Watson-Crick base pairs.

Mutant DNA polymerases of the present invention may also include their "functional equivalents." Functional equivalents herein include amino acid sequence variants in which some or all of the amino acid sequences of the mutant DNA polymerase are substituted or a portion of the amino acid is deleted or added. At this time, the substitution of the amino acid is preferably a conservative substitution. Examples of conservative substitutions of amino acids present in nature are as follows; Aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn ) And sulfur containing amino acids (Cys, Met). In this case, the deletion of the amino acid is preferably located at a portion not directly involved in the activity of the DNA polymerase.

A second aspect of the invention provides a DNA polymerase gene encoding the amino acid sequence of SEQ ID NO. The DNA polymerase gene encoding the amino acid sequence of SEQ ID NO: 2 may have various types of sequences by degeneration of heredity. The gene sequence is preferably a DNA polymerase gene represented by SEQ ID NO: 1. One skilled in the art can make functional equivalents to the DNA polymerase sequence of SEQ ID NO: 2, and the scope of the present invention includes gene sequences encoding such functional equivalents.

A third aspect of the invention provides a recombinant vector comprising the DNA polymerase gene.

The term vector, as used in the present invention, refers to a nucleic acid molecule capable of binding and transferring another nucleic acid thereto. The term expression vector includes plasmids, cosmids or phages capable of synthesizing proteins encoded by each recombinant gene carried by the vector. Preferred vectors are vectors capable of self-replicating and expressing the nucleic acid to which they are bound. The vector may be a vector of plasmid, cosmid, phagemid, phage, viral form. Methods of making recombinant vectors comprising the DNA polymerase gene of the present invention using such vectors are known in the art.

A fourth aspect of the present invention provides a host cell transformed with the recombinant vector. The term 'transformation' used in the present invention means that foreign DNA or RNA is absorbed into a cell and the genotype of the cell is changed.

A fifth aspect of the present invention is characterized by culturing a host cell transformed with a recombinant vector comprising a DNA polymerase gene encoding the amino acid sequence of SEQ ID NO: 2 and inducing the expression of the recombinant protein to isolate the polymerase protein. It provides a method for producing a polymerase.

The mutant DNA polymerase of the present invention exhibits superior performance in PCR of long chain DNA compared to the wild type or mutant type, and is widely applied to various fields requiring long chain amplification such as human genome analysis. Can be.

Figure 1 shows the comparison of PCR amplification of the human genomic DNA of the wild type polymerase, mutant polymerase and mutant DNA polymerase of the present invention. M, standard marker; Lane 1, hprt 2.7 kb; Lane 2, hprt 6.25 kb; Lane 3, hprt 10.4 kb; Lane 4, β-globin 13.5 kb; Lane 5, mitochondria 16.2 kb; Ex Taq, common Taq polymerase; TNA1, wild type polymerase; TLA, mutant polymerase; U7, which shows the mutant DNA polymerase of the present invention.

2 shows a cleavage map of the recombinant plasmid vector.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Reference Example 1. Cloning and Primary Sequence Analysis of the Wild-Type Polymerase TNA1_pol Gene

Thermococcus genus (Thermococcus sp.) NA1 strains were isolated from deep-sea hydrothermal spouts in the Western Pacific region of Papua New Guinea. YPS Badges It was used to culture DNA of genus NA1 strain, Culture and strain maintenance of genus NA1 were performed by standard methods. Thermococcus In order to prepare genus NA1 seed culture, YPS medium in 25 ml serum container was inoculated with a single colony formed on a pattagel plate and incubated at 90 ° C. for 20 hours. Seed culture was used to inoculate 700 ml of YPS medium in an anaerobic jar and incubated at 90 ° C. for 20 hours.

Example 1 Preparation of Wild-type Polymerase TNA1_pol Gene and Mutation thereof

Thermococcus genus (Thermococcus sp.) Escherichia coli for plasmid proliferation and nucleic acid sequencing including polymerase TNA1_pol gene of NA1 strainE. coli DH5α strain was used. E. coli BL21-Codonplus (DE3) -RIL cells (stratazine, Lazola, Calif.) And plasmid pET-24a (+) (Novagen, Madison, Wisconsin) were used for gene expression. E. coli strains were cultured using Luria-Bertani medium at 37 ° C. and added to the medium so that kanamycin was at a final concentration of 50 μg / ml.

In addition, DNA manipulations were done in standard ways as described by Sambrok and Russell. Genomic DNA in the thermococcus was isolated by standard methods. Restriction enzymes and other modified enzymes were purchased from Promega (Madison, Wisconsin). Small amounts of plasmid DNA from E. coli cells were made using the plasmid mini kit (Qiagen, Hilden, Germany). DNA sequencing was performed using an automated sequencer (AB3100) using the Big Die Terminator Kit (PE Applied Biosystems, Foster City, CA).

Analysis of the genomic sequence revealed an open reading frame (3,927 bp) encoding a protein consisting of 1,308 amino acids and showed very high similarity with the B family type DNA polymerase. The molecular weight of the protein obtained from the inferred amino acid sequence was 151.9 kDa, which was much larger than expected for the average thermostable DNA polymerase. In addition, sequence analysis revealed that the DNA polymerase gene was conserved between the putative 3'-5 'exonuclease domain, the α-like DNA polymerase domain, and the α-like DNA polymerase of eukaryotes and archaea. It was confirmed to include one intervening sequence of 1605 bp (535 amino acids) located in Pol III). Also, the amino acid sequence of inferred intein was very similar to that of other archaea polymerases, and pol_1 intein 1 (537 amino acids originating from DNA polymerase from Thermococcus sp. Strain GE8, AJ25033). At 81.0% homology, 69.0% homology to IVS-B (537 amino acids of KOD DNA polymerase origin, D29671) and 67.0% of phases to intein (537 amino acids of deep vent DNA polymerase origin, U00707) I showed same sex.

In addition, the splicing site of intein could be predicted by sequencing because His-Asn-Cys / Thr was well conserved at Cys or Ser and C-terminal splice junctions at the N-terminus of intein. Thus, a gene of mature form of polymerase TNA1_pol without intein could be expected, which was determined to be 2,322 bp encoding a protein consisting of 773 amino acid residues. The estimated sequence of polymerase TNA1_pol was compared with those of other DNA polymerases. In the pairwise alignment, the deduced mature polymerase TNA1_pol amino acid sequence was 91.0% homologous with KOD DNA polymerase (gi: 52696275), 82.0% homology with deep vent DNA polymerase (gi: 436495) and pfu DNA polymerization. It showed 79.0% homology with enzyme (gi: 18892147). To confirm the performance of polymerase TNA1_pol in PCR amplification, TNA1_pol DNA was prepared by removing intein from the polymerase full length as mentioned above.

Mature forms of DNA polymerase containing no intein were prepared as follows. Using primers designed to include overlapping sequences, the N-terminal and C-terminal portions of TNA1-pol were amplified, respectively. The full length of the mature TNA1_pol gene flanked by restriction enzymes Nde I and Xho I sites was then amplified using a mixture of the N-terminal and C-terminal partial amplified PCR segments as two primers and templates. It became. The amplified sequences were digested with restriction enzymes Nde I and Xho I, was linked to the restriction enzyme Nde I, and the plasmid vector pET-24a digested with Xho I (+). The conjugate was transformed into E. coli DH5α. Candidates with the correct structure were identified by restriction enzymes, and the DNA sequences of the clones were analyzed to confirm that they contained the genes of the mature DNA polymerase. Thus, wild-type DNA polymerase TNA1_pol was prepared. In order to mutate the specific site of the TNA_pol, site-directed mutagenesis using polymerase chain reaction (PCR) using various synthetic primers corresponding to each specific site according to the existing protocol. Induced. By examining the activity of the mutated DNA polymerase and searching for DNA polymerase capable of long chain amplification, the DNA polymerase of SEQ ID NO: 2 was invented.

Example 2. Long PCR Activity Verification

 In order to verify the activity of the DNA polymerase prepared according to the present invention, DNA polymerizing activity was verified using the primers of Table 1 below in human genomic DNA PCR.

Table 1

Top	Bottom	Size
HpbF (SEQ ID NO: 5)	HpeR (SEQ ID NO: 6)	2.7
HpaF (SEQ ID NO: 3)	HpeR (SEQ ID NO: 6)	6.25
HpbF (SEQ ID NO: 5)	HpgR (SEQ ID NO: 7)	10.4
RH1098F (SEQ ID NO: 4)	HpeR (SEQ ID NO: 6)	15.4
RH1022F (SEQ ID NO: 8)	RH1053R (SEQ ID NO: 9)	13.5

As a result of the verification, as shown in FIG. 1, it was confirmed that the DNA polymerase of the present invention was effective for amplifying a long genome of the human genome.

Attached a total of 9 sequences. DNA polymerase gene sequence SEQ ID NO: 1, DNA polymerase amino acid sequence SEQ ID NO: 2, SEQ ID NO: 3 to 9 is a primer sequence.

HpaF (SEQ ID NO: 3)

RH1098F (SEQ ID NO: 4)

HpbF (SEQ ID NO: 5)

HpeR (SEQ ID NO: 6

HpgR (SEQ ID NO: 7)

RH1022F (SEQ ID NO: 8)

RH1053R (SEQ ID NO: 9)

Claims (6)

1. A DNA polymerase comprising the amino acid sequence of SEQ ID NO.
2. A DNA polymerase gene encoding the amino acid sequence of SEQ ID NO.
3. The DNA polymerase gene according to claim 2, wherein the DNA polymerase gene is SEQ ID NO: 1.
4. Recombinant vector comprising a DNA polymerase gene of claim 2.
5. A host cell transformed with the recombinant vector of claim 4.
6. A method for producing a polymerase, comprising culturing the host cell of claim 5 and inducing the expression of the recombinant protein to separate the polymerase protein.

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