KR101155368B1 - Nanoarchaeum equitans plus DNA polymerase, method for preparing the same and its PCR method using dUTP and uracil-DNA glycosylase - Google Patents

Abstract

The present invention is a nano-aqueum Iquitans DNA polymerase ( Nanoarchaeum on the application of equitans DNA polymerase, hereinafter "Neq DNA polymerase, ") manufactured and Neq DNA polymerase for PCR (polymerase chain reaction, hereinafter referred to as" PCR ") using the novel PCR reaction buffer solution of will be. Neq DNA polymerase can amplify [lambda] -DNA up to 10 kb in the novel buffer solution of the present invention and perform PCR using deoxyutif (dUTP) and deoxyitif (dITP). The present invention also relates to the preparation of Neq plus (DNA) polymerase. Neq plus DNA polymerase is a Taq to Neq DNA polymerase It was prepared by mixing DNA polymerase, which can amplify λ-DNA fragments of 20 kb or more by PCR, and perform PCR 2-3 times more effectively than using Taq DNA polymerase even in the presence of deoxyutif (dUTP). Can be done. Also Taq It has a higher fidelity than DNA polymerase. In addition, the present invention is more useful than Taq DNA polymerase in the technology of preventing carry-over contamination of nucleic acid samples using deoxyutypi (dUTP) together with uracil-DNA glycosylase (UDG). Will be able to be used.

DNA polymerase, Neq plus DNA polymerase, polymerase chain reaction, cross contamination, dUTP, dITP, UDG.

Description

Nanoarchaeum equitans plus DNA polymerase, preparation method thereof and polymerase chain reaction (PCR) method using deoxyutif (DXTP) and uracil-DNA glycosylase (XD) the same and its PCR method using dUTP and uracil-DNA glycosylase}

The invention nanoah keum Iquique Tansu Plus DNA polymerase (Nanoarchaeum equitans plus DNA polymerase, Neq plus DNA polymerase, hereinafter referred to as "Neq plus DNA polymerase"), its production method and a PCR (polymerase chain reaction, or less The present invention relates to an application in 'PCR', and the present invention relates to deoxyuridine 5'-triphosphate (2'-deoxyuridine) with uracil DNA glycosylase (hereinafter referred to as 'UDG'). 5'-triphosphate (hereinafter referred to as 'dUTP') can be usefully used in the technology of preventing carry-over contamination of nucleic acid samples.

DNA polymerase (deoxyribonucleic acid polymerase, DNA polymerase, EC number 2.7.7.7) is an enzyme that synthesizes DNA in the 5 '→ 3' direction depending on the template DNA. It is the enzyme that plays the most important role in repairs (Lehninger, AL et al. , 1993, Principles of Biochemistry , 2nd ed., Worth Publishers). DNA polymerases can be divided into at least six families (A, B, C, D, X, Y) based on amino acid sequences (Todo, GC et al., 2001, The Y-family of DNA). polymerases , Mol Cell 8, 7-8).

The thermostable DNA polymerase was introduced by Chien et al. In 1976 by Thermophile, Thermos Aquaticus YT-1 ( Thermus). first isolated from aquaticus YT-1) and its properties are revealed (hereinafter, Taq) DNA polymerase, see Chien, A. et al. , 1976, J. Bacteriol 127, 1550-1557). Then, in 1988, Saiki et al. Developed a PCR technology using heat-resistant DNA polymerase (see Saiki, RK et al. , 1988, Science 239, 487-491), and heat-resistant DNA polymerases began to attract attention. In addition, heat-resistant DNA polymerases have been developed from several high temperature and hyperthermophile archaea. These archaea-derived DNA polymerases are involved in the replication of eukaryotic DNA polymerases and Escherichia coli. coli ), such as DNA polymerase II, are classified as family B. In particular, they are combined with DNA polymerization activity, called 3 ′ → 5 ′ nucleic acid terminal hydrolase activity (3 ′ → It also has a 5´ exonuclease activity and has been used more efficiently than Taq DNA polymerase for PCRs that require high accuracy (Mattila, P. et al., 1991, Nucleic Acids Res, 19, 4967-4973; Lundberg). , KS et al., 1991, Gene 108, 1-6). Another characteristic of archaea-derived DNA polymerases is the presence of uracil-pockets in the N-terminal domain of DNA polymerases (Fogg, MJ et al ., 2002, Structural basis for uracil recognition by archaeal family B DNA polymerases , Nat Struct Biol 9: 922-7.). The uracil-pocket recognizes uracil bases in single-stranded DNA templates and stops DNA polymerase progression. In other words, archaea-derived DNA polymerases have limited DNA polymerizing activity in the presence of uracil and cannot perform PCR (Gill, S. et al ., 2007, Interaction of the family-B DNA polymerase from the archaeon Pyrococcus furiosus with deaminated bases, J Mol Biol 372: 855-63.).

Hyperthermophilic nanoarchaeon nanoaqueum equitanes are extremely small, nano-size anaerobic microorganisms isolated from submarine hot vents in Kolbeinsey, Iceland. (anaerobe) (Huber, H. et al. , 2002, Nature 417, 63-67). The strain is grown on the surface of Ignicoccus sp. Strain KIN4 / I ( Ignicoccus sp.), Which is a specific host under strict anaerobic conditions at 70-98 ° C (optimal growth temperature of 90 ° C). The complete sequencing of the nanoaqueum Iquitans genome (490,885 base pairs, bp), one of the smallest microbial genomes known to date, has recently been discovered (Waters, E. et. al. , 2003, Proc. Natl . Acad . Sci . USA 100, 12984-12988).

Neq DNA polymerase is encoded by two genes 83,295 bp apart on the NanoAqueum Iquitans genome, each of which contains sequences that are believed to encode a split mini-intein. , E. et al. , 2003, Proc Natl Acad Sci USA 100, 12984-12988. The two proteins that were split were transspliced to remove the inteins and only the exteins were linked to peptide bonds. The protein formed was called Neq C (protein trans- spliced form of Neq DNA polymerase). a DNA polymerase by recombinant a dichroic stain encrypted part and the octane dichroic stain encrypted portion of the Neq DNA polymerase small fragment gene, remove the encrypted portion of the large fragment gene Neq DNA polymerase obtained by expression of a single polypeptide form remove Neq P (genetically protein splicing-processed form of Neq DNA polymerase). And although Neq C and Neq P were prepared by different methods, they were found to be enzymes that not only have the same activity but also show the same biochemical properties (see Choi, JJ et al ., 2006, Protein trans-splicing and characterization). of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeumequitans, J Mol Biol 356: 1093-106; Kwon, Seok-Tae et al., 2008, A method for preparing an active nanoarqueum Iquitans DNA polymerase and an active DNA polymerase prepared by the method, Korean Patent Registration No. 10-0793007).

Polymerase chain reaction (PCR) is a technique that isolates or identifies useful genes by amplifying large quantities of specific nucleic acid sites in vitro using DNA polymerases derived from heat-resistant bacteria. Erlich, HA, 1989, J Clin Immunol 9, 437-447; Shin, HJ et al ., 2005, J Microbiol Biotechnol 15, 1359-136). Such PCR has contributed to greatly lowering the detection limit required for nucleic acid detection, and is currently very useful for identifying and determining diseases of viruses and pathogenic bacteria. However, when the concentration of nucleic acid is very low, there is a problem that is difficult to detect, there is a disadvantage that the reaction efficiency is different from reactor to reactor. In particular, carry-over contamination may occur during the selection of samples, nucleic acid separation, transfer of samples, PCR of samples, storage of samples and recovery of samples after electrophoresis. Sources of contamination in the PCR process include cross contamination between samples, DNA contamination in the laboratory, and carry-over by primers of amplification products and previous PCR products (Sobek, H. et. al ., FEBS Lett 388, 1996), even if the cross-contamination is amplified with the target sample, even if the trace amount is a problem that can not distinguish whether the sample is contaminated by conventional PCR method.

Therefore, as a method for preventing cross-contamination occurring in recent years, dUTP instead of 2'-deoxythymidine 5'-triphosphate (hereinafter referred to as 'dTTP') A method for performing PCR has been suggested by Longo et al . (Longo, MC et al ., 1990, Gene 93, 125-128). In addition, the UDG was treated to remove extremely contaminated uracil-containing DNA (uracil-DNA) with template DNA in the sample before initiation of PCR, inactivation of the UDG by heating, and then again by adding dUTP instead of dTTP. Methods have been reported (Udaykumar., Et al ., 1993, Nucleic Acids Res . 21, 3917-3918; Taggart et al ., 2002, J. Virol . Methods 105, 57-65). For this reason, recently, PCR products including UDGs or UDGs are being sold in the PCR process. In particular, Taq DNA polymerase has been used as an enzyme used to perform PCR by adding dUTP instead of dTTP.

When performing PCR, Taq DNA polymerase is generally used. However, PCR applications using Taq DNA polymerase show some disadvantages, such as high error-rate and short amplification distance. Efforts have been made to overcome this shortcoming, which has been accomplished by mixing Taq DNA polymerase with DNA polymerase with proofreading activity (Barnes, WM 1994. PCR amplification of up to 35-). kb DNA with high fidelity and high yield from lambda bacteriophage templates .Proc Natl Acad Sci USA 91,2216-20.). The enzymatic mixture of the calibrated DNA polymerase and the Taq DNA polymerase has a lower error rate than the original Taq DNA polymerase and enables long range PCR.

Recently, the inventors have reported the cloning, expression and purification of Neq DNA polymerase genes, enzyme characterization and the possibility of using PCR (see Patent Registration No. 10-0793007). ^{The 2+} and KCl concentrations, the use of dITP in PCR reactions, etc., were not reported in detail.

Under these circumstances , the present inventors have provided a method for preparing a buffer having optimal conditions for PCR of Neq DNA polymerase and a PCR application method using a deaminated substrate. After mixing Neq DNA polymerase with Taq DNA polymerase in various ratios and finding the optimal PCR amplification rate, it was named ' Neq plus DNA polymerase' and used to determine PCR efficiency in the presence of dITP or dUTP. Compared with the polymerases, and based on this, it was confirmed that it can be superior to the existing Taq DNA polymerase in the experiment of preventing carry-over contamination using dUTP and UDG, and completed the present invention.

An object of the present invention is to provide a PCR application method of various purposes using Neq DNA polymerase.

Another object of the present invention is to prepare Neq plus DNA polymerase having improved PCR amplification efficiency than Neq DNA polymerase.

Another object of the present invention is prepared Neq It provides a long-range DNA amplification using plus DNA polymerase and PCR application method using dUTP and UDG.

Another object of the present invention is the Neq The present invention provides a method for removing cross-contamination of a PCR product using plus DNA polymerase and dUTP and UDG.

In order to achieve the above object, the present invention is a nano-aqueum Iquitans family B DNA polymerase ( Nanoarchaeum It provides a PCR method using the Applied equitans family B DNA polymerase, Neq DNA polymerase, hereinafter referred to as "Neq DNA polymerase").

Neq PCR method using DNA polymerase has already been patented (refer to Kwon Seok-tae et al., 2, 2008, preparation method of active nano-aqueum Iquitans DNA polymerase and active DNA polymerase prepared by the method, Korea patent registration 10-0793007), the present specification provides an improved PCR optimal buffer solution and a PCR application method using the same by reference to the above patent. According to the prior art identified technical Neq DNA polymerase were able to amplify the lambda DNA to 4 kb under optimal reaction buffer (50 mM Tris-HCl (pH 7.5) / 1 mM MgCl 2/90 mM KCl / 0.01% BSA) , Using the buffer solution provided in the present invention can be amplified up to 10 kb (see Figure 2).

The present invention provides a Neq DNA polymerase PCR reaction buffer comprising 20 mM Tris-HCl pH 7.5-8.5, MgCl ₂ 0.5-4.5 mM, KCl 10-130 mM and 0.01% BSA. More preferably, the present invention provides an Neq DNA polymerase PCR optimal reaction buffer comprising 20 mM Tris-HCl pH 8.0, MgCl ₂ 1.5 mM, KCl 50 mM and 0.01% BSA.

In addition, the present invention is a PCR method using Neq DNA polymerase, the pH of the reaction pH 7.5-8.5, the reaction Mg ^{2 +} concentration 0.5-4.5 mM, the reaction KCl concentration 10-130 mM PCR method To provide. The Neq DNA polymerase was found to be able to perform PCR using dUTP (2'-deoxyuridine 5'-triphosphate), but in addition, the present invention is deoxyinosine 5'-triphosphate (deoxyinosine 5'-triphosphate). , Hereinafter referred to as 'dITP'.

In addition,Neq DNA polymerase (Nanoarchaeum equitans DNA polymerase)Taq DNA polymerase (Thermus aquaticus DNA polymerase) is mixed at a concentration ratio of 1: 1 to 1: 100 to provide an enzyme mixture prepared. As used herein, the term "DNA polymerase" refers to a deoxyribonucleoside 5'- having a new DNA single-catalyzed catalytic ability using a strand of DNA as a template, that is, complementary to the DNA template. By triphosphate (deoxyribonucleoside 5'-triphosphate, hereinafter 'dNTP') means an enzyme capable of polymerizing (polymerization) to 3'-OH of the primer (primer). According to the known artNeq DNA polymerase is a heat-resistant DNA polymerase derived from archaea belonging to the family B having a polymerization activity and a correction activity,Taq DNA polymerases do not have corrective activity. According to the conventional method, a long range of PCR is possible by mixing a DNA polymerase having a corrective activity and a polymerase which is not (see Barnes, WM 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield). from lambda bacteriophage templates.Proc Natl Acad Sci ,U.S.A. 91: 2216-20), a mixture of DNA polymerases (eg,Taq Lower polymer error than DNA polymerase) (Cline, J.et al, 1996, PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases, Nucleic Acids Res 24: 3546-51). Based on thisNeq DNA polymeraseTaq As a result of comparative experiments in which DNA polymerase was mixed at various concentration ratios (1:10 to 100), PCR efficiency was found to be the best at the concentration ratio of 1:10.Neq It was named plus DNA polymerase (see FIG. 4).

Accordingly, the present invention provides a novel Neq plus DNA polymerase and its preparation method.

In another aspect, the present invention provides a method for performing a nucleic acid polymerization reaction using Neq plus DNA polymerase. Neq plus DNA polymerase of the present invention can be used for all reactions based on polymerizing dNTP complementary to the template with a strand of DNA to the 3'-OH of the primer. As a representative example, the present invention provides a polymerase chain reaction (PCR) method using Neq plus DNA polymerase. PCR is one of the representative nucleic acid amplification techniques (NATs) for amplifying large quantities of specific DNA regions in vitro using DNA polymerases. Mullis, K. et al., 1986, Cold Spring Harbor Symp. Biol. 51, 263-273) and US Pat. Nos. 4,683,195, 4,683,202 and 4,965,188 and the like. PCR denatures the template DNA into single strands; Binding a primer to a target gene sequence; DNA synthesis is performed using a DNA polymerase. As the above steps are repeated, the DNA is amplified. Usually repeats 25 to 30 times. The temperature at which denaturation, binding and synthesis reactions occur can be controlled. If the binding temperature is too low, non-specific amplification may occur, so take this into consideration. Various modifications and applications in the PCR method are possible and are included within the scope of the present invention. For example, RT-PCR, Touch down PCR, Differential Display PCR, Gradient PCR, Real time PCR, and the like.

Neq plus DNA polymerase named by the inventors Neq DNA polymerase and Taq DNA polymerase are mixed at a ratio of 1:10 based on the enzyme active unit. That is, by mixing a DNA polymerase having a corrective activity and a polymerase which is not, a long-range PCR (long-range PCR) is possible. Experiments were performed to target amplified 20 kb bacteriophage lambda DNA fragments (see FIG. 4). According to the known art (see Patent No. 10-0793007), both Neq DNA polymerase and Taq DNA polymerase can perform PCR in the presence of dUTP. Neq plus DNA polymerase may also perform PCR in the presence of dUTP, so PCR was performed in the presence of dUTP using the two polymerases and Neq plus DNA polymerase, and Neq plus DNA polymerase was more effective than the two DNA polymerases. 2 to 3 times more excellent efficiency (see FIG. 5).

In another aspect, the present invention relates to a method of using the Neq plus DNA polymerase in cross-contamination prevention PCR using dUTP and UDG together. In particular, as described above, the Neq plus DNA polymerase provided in the present invention has a DNA polymerizing activity in the presence of dUTP, so the combination of the electric enzyme and the low-temperature UDG will be well suited for PCR performed for diagnostic purposes. Since UDG has an activity of specifically degrading only uracil bases in DNA substrates, UDG is used to remove cross contamination of uracil DNA generated during PCR. The method for removing cross-contamination of a PCR product according to the present invention is to decompose contaminated uracil DNA by reacting the substrate containing uracil base and the composition for PCR containing the UDG enzyme for 10 minutes at 25 ° C., followed by PCR. By doing so, cross-contamination in the PCR product can be eliminated. Neq PCR was performed using the plus DNA polymerase and Taq DNA polymerase, respectively, by 20, 25, and 30 cycles by the above method, and as a result, Neq was compared to Taq DNA polymerase. plus DNA polymerase also showed higher efficiency in PCR to prevent cross-contamination (see Figure 6).

In addition, Neq plus DNA polymerase prepared by the method of the present invention may be provided as a PCR kit (kit) together with dUTP, UDG and the like. Neq plus DNA polymerase of the present invention or a PCR kit comprising the same can be more useful than Taq DNA polymerase for genetic engineering and molecular biology experiments, clinical diagnosis, forensic research and the like.

The present invention provides a PCR buffer of Neq DNA polymerase improved over the prior art. Under the buffer, Neq DNA polymerase can efficiently use dUTP and dITP, which are deaminated substrates. In another aspect, the present invention provides Neq plus DNA polymerase and its preparation method. Neq plus DNA polymerase can also use dUTP more efficiently and can be used for long-range PCR. Neq plus DNA polymerase has a higher fidelity than Taq DNA polymerase and, among other things, showed greater efficiency than Taq DNA polymerase in PCR for preventing carry-over contamination with UDG. Therefore, the Neq plus DNA polymerase of the present invention may be usefully used for clinical diagnosis, forensic research, and the like.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited to the following examples.

Example  One: Neq  DNA polymerase PCR  Reaction Buffer

Active PCR was performed using the following buffer solution to determine the optimal pH of PCR using Neq DNA polymerase (50 μl of reaction solution: 20 mM Tris-HCl buffer, 2 mM MgCl ₂ , 50 mM KCl). , 0.1% BSA (bovine serum albumin), and Tris-HCl buffer solution was prepared using lambda DNA fragments of 1 kb using λ-1 primers (see Table 1) in the forward and reverse directions at pH 7.0-9.0. As a result of performing PCR, PCR amplification was confirmed in Tris-HCl buffer pH 7.5-8.5, and the optimum pH was determined as Tris-HCl buffer, pH 8.0 Figure 1 (a) shows Neq DNA polymerase. The results according to pH are shown in PCR using Lane M is loaded with a 1 kb DNA size marker.

PCR was performed using the following buffer solution to determine the optimal MgCl ₂ concentration of PCR using an active Neq DNA polymerase. 50 μl of the reaction solution was composed of 20 mM Tris-HCl buffer (pH 8.0), 50 mM KCl, 0.1% BSA (bovine serum albumin), and PCR was carried out as described above in the presence of various concentrations of MgCl _2. , PCR amplification was confirmed in the presence of 0.5-4.5 mM MgCl ₂ , the optimum MgCl ₂ concentration was determined to be 1.5 mM. Figure 1 (b) shows the results according to the concentration of MgCl _{2 in} PCR using the active Neq DNA polymerase. Lane M was loaded with 1 kb DNA size marker.

To determine the optimal KCl concentration of PCR using the active Neq DNA polymerase, 50 μl of the reaction solution was added with 20 mM Tris-HCl buffer (pH 8.0), 1.5 mM MgCl ₂ , and 0.1% BSA (bovine serum albumin). As a result of performing PCR in the presence of various concentrations of KCl, PCR amplification was confirmed in the presence of 10-130 mM KCl, and the optimum KCl concentration was determined to be 50 mM. Figure 1 (c) shows the results according to the KCl concentration in PCR using the active Neq DNA polymerase. Lane M was loaded with 1 kb DNA size marker.

Example  2: Neq  DNA polymerase PCR

Optimal reaction buffer (20 mM Tris-HCl (pH 8.0) / 1.5 mM MgCl 2/50 mM KCl / 0.01% BSA) under a lambda phage genomic DNA (hereinafter referred to as lambda DNA ") template determined as described above PCR was performed by adding various primers, respectively, to amplify products of various sizes (1 kb, 2 kb, 4 kb, 6 kb, 8 kb, 10 kb) using purified active Neq DNA polymerases. 0.4 U / μl of Neq DNA polymerase, 30 ng lambda DNA, 250 to PCR tubes with 5 pmole forward and reverse λ-1, 2, 4, 6, 8, 10 primers (see Table 1), respectively Prepare a PCR reaction mixture (20 μl) to which μM dNTP and 1X Neq DNA polymerase optimal reaction buffer were added, and perform DNA denaturation at 95 ° C. for 3 minutes, followed by DNA denaturation (94 ° C., 30 seconds), primer After repeating three steps of annealing (56 ° C., 30 seconds) and DNA amplification (72 ° C., 12 minutes) 25 times, a further DNA amplification process was performed at 72 ° C. for 15 minutes. As a result of electrophoresis of the PCR reaction solution on the agarose gel with the DNA size marker, it was found that PCR using Neq DNA polymerase was possible to amplify up to 10 kb under the optimal reaction buffer solution (see FIG. 2). .

Example  3: dUTP Wow dITP Using PCR

The experiment using the deamination base was performed according to the PCR method performed in Example 2. Agarose gel electrophoresis by PCR using dUTP having uracil base deamined with thymine and dITP having hypoxanthine base dehydrated with guanine This is Fig. 3 (a). In dUTP PCR, all dTTPs were replaced with dUTP, and in dITP PCR, 10% of dGTP was replaced with dITP. Taq , including Neq DNA polymerase (RexJin Biotech, E1001B), Pfu (Stratagene, 600135), Vent (New England Biolabs, M0254S), and KOD (Novagen, 71085-3) DNA polymerases were used to compare the deaminized substrates by PCR. Perform DNA denaturation at 94 ° C for 1 minute, then repeat 25 steps of DNA denaturation (94 ° C, 20 seconds), primer annealing (56 ° C, 20 seconds) and DNA amplification (72 ° C, 1 minute). Afterwards, DNA amplification was performed for 5 minutes at 72 ° C. In order to amplify the 1 kb lambda DNA fragment, λ-1 primers in the forward and reverse directions (see Table 1) were used. As shown in FIG. 3 (a), PCR of the family B DNA polymerase Pfu , Vent, and KOD DNA polymerase derived from archaea was not performed using dUTP and dITP, which are deaminated substrates. On the other hand, Neq and Taq DNA polymerases were able to perform PCR using both dUTP and dITP.

Further experiments were performed to investigate the dITP availability of Neq and Taq DNA polymerase in detail. PCR was performed using a substrate mixture having a concentration of 250 μM mixed with dGTP while increasing the ratio of dITP to 0, 25, 50, 75, and 100%. Perform DNA denaturation at 94 ° C for 1 minute, then repeat 25 steps of DNA denaturation (94 ° C, 20 seconds), primer annealing (56 ° C, 20 seconds) and DNA amplification (72 ° C, 1 minute). Then, agarose gel electrophoresis by performing a DNA amplification process at 72 ℃ for 5 minutes is Figure 3 (b). In order to amplify the 1 kb lambda DNA fragment, λ-1 primers in the forward and reverse directions were used. Neq as shown in the result of Fig. 3 (b) DNA polymerase was able to use dITP up to 50% concentration, and Taq DNA polymerase was available up to 75%.

Example  4: Neq  Preparation of Plus DNA Polymerase and PCR Application of

Neq DNA polymerase having corrective activity and Taq DNA polymerase (rexgene Biotech Co., Ltd., Super Taq polymerase, E1001B) without corrective activity are 1:10 to 1: based on the active unit of the enzyme. Mix at the rate of 100. That is, Neq DNA polymerase was used at a concentration of 0.2 U / μl to 0.02 U / μl, and Taq DNA polymerase was used at a concentration of 2 U / μl to prepare a DNA polymerase mixture as follows. Add 2 U / μl of Taq DNA Polymerase to 0.02 U / μl-0.2 U / μl of Neq DNA Polymerase, respectively, and add 30 ng of lambda DNA as a template to 10 different enzyme mixtures. Added. In addition, a primer for λ-10 PCR in the forward and reverse directions (see Table 1) and 250 μM of dNTP and Super Taq Buffer II (RexJin Biotech, Super Taq Buffer,) aimed at amplifying 10 kb DNA fragments into the mixture. Each was added to finally prepare a PCR reaction mixture (50 μl total). The PCR mixtures were subjected to DNA denaturation at 95 ° C. for 3 minutes, followed by three steps of DNA denaturation (94 ° C., 1 minute), primer annealing (56 ° C., 1 minute), and DNA amplification (72 ° C., 8 minutes). After repeated 25 times, the final amplification time of 10 minutes at 72 ℃ was given. In the PCR aimed at 20 kb, λ-20 primers (see Table 1) in the forward and reverse directions were used, and PCR was performed by giving 15 minutes instead of 8 minutes at 72 ° C. under the above conditions.

Figure 4 shows the results of the electrophoresis of the PCR product of the lambda DNA fragment according to the mixing ratio of Neq DNA polymerase and Taq DNA polymerase on 0.8% agarose gel (agarose gel). 4 (a) shows an amplification result of 10 kb, Figure 4 (b) shows an amplification result of 20 kb. M1 and M2 represent 1 kb DNA markers, and M2 is a DNA marker obtained by cleaving lambda DNA with Hin dIII restriction enzymes. Lane 1 shows the results of using only Taq DNA polymerase, lanes 2 to 11 show the results of mixing Taq and Neq polymerase according to the ratio shown in FIG. Based on these results, the most amplified efficiency of 10: 1 ( Taq polymerase: Neq polymerase) ratio of enzyme mixture is Neq. It was named plus DNA polymerase. Neq plus DNA polymerase showed a superior amplification efficiency than Taq DNA polymerase at 10 kb, as well as successfully performed PCR at 20 kb.

Example  5: dUTP  In existence Neq  plus DNA polymerase PCR Done of

To determine the amplification efficiency of Neq plus DNA polymerase in the presence of dUTP, 1 kb to a mixture containing 250 μM of dNTP (replacement of dTTP with dUTP), 0.22 U / μl of Neq plus DNA Polymerase and Super Taq Buffer II, respectively. PCR was performed by adding primers for amplifying various DNA fragment lengths of 2 kb, 4 kb, 6 kb, and 8 kb, respectively (for forward and reverse λ-1, 2, 4, 6, 8 primers, Table 1 Reference). In addition, in order to compare the amount of amplification, the PCR reaction mixture was added with only Neq DNA polymerase (0.4 U / μl) or Taq DNA polymerase (0.2 U / μl) instead of Neq plus DNA polymerase. . PCR conditions were carried out for 3 minutes DNA denaturation process at 95 ℃, followed by three steps of DNA denaturation (94 ℃, 20 seconds), primer annealing (56 ℃, 20 seconds) and DNA amplification (72 ℃, 8 minutes) After 25 repetitions, an additional amplification time of 10 minutes was given at 72 ° C. Yields of PCR were compared using the LabWorks 4.6 program. Figure 5 is the result of electrophoresis of the above PCR product on 0.8% agarose gel (agarose gel). As shown in Figure 5, Taq DNA polymerase was able to amplify up to 6 kb. In contrast, Neq plus DNA polymerase showed improved PCR efficiency at all amplification lengths (1-8 kb). Compared to 4 kb DNA fragments, Neq plus DNA polymerase showed 2.3 times higher amplification efficiency than Neq DNA polymerase and 3.4 times higher than Taq DNA polymerase.

PCR primers to amplify various DNA fragment lengths order
number Prime name
(target size)
Sequence Lambda DNA
sequence landmarks
(bp) One λ-1
(1 kb) λ-1F CAAAGGCGGTTAAGGTGGTA 20791 ~ 21787
2 λ-1R GGCTGTACCGGACAATGAGT 3 λ-2
(2 kb) λ-2F AGAAGTTCAGGAAGCGGTGA 4212 ~ 6221
4 λ-2R ACGGAAAAAGAGACGCAGAA 5 λ-4
(4 kb) λ-4F CCGGTAATGGTGAGTTTGCT 9643 ~ 13652
6 λ-4R GTACTGGTGCCTTTGCCATT 7 λ-6
(6 kb) λ-6F TTTACAGCGTGATGGAGCAG 3587-9577
8 λ-6R GACACGGTGGCTTTTGTTTT 9 λ-8
(8 kb) λ-8F AGCTGAGTTTTGCCCTGAAA 10128-18130
10 λ-8R AATGTTTGCCGTCTTTGGTC 11 λ-10
(10 kb) λ-10F AGGATGACTGGTGGCGTAAC 14556-24548
12 λ-10R GTGCCTAGCAAACTCGGAAG 13 λ-20
(20 kb) λ-20F AAGGCAGAGGCTGCGTATAA 11520 ~ 31526
14 λ-20R ATGCCTGGTACTTTGCCAAC

All the primers have a direction of 5 '→ 3'.

Example  6: Neq  plus of DNA polymerase PCR  Fidelity comparison

PCR fidelity comparisons using heat resistant DNA polymerases were performed with a slight modification of the Lundberg method (Lundberg et al ., 1991, Gene 108, 1-6). First, the lacZα fragment (835bp) of the expression vector pJR-lacZ containing lacZ (β-galactosidase gene) was used as a template, and 94 ° C 30 seconds, 60 ° C using Neq , Neq plus DNA, Taq , and Pfu DNA polymerase, respectively. After repeating 25 times at 30 ° C. for 1 minute at 72 ° C., the PCR product was digested with restriction enzymes Bam HI and Cla I, respectively, and then transformed after conjugation to an electric vector digested with the same restriction enzyme. IPTG, X-gal and antibiotic ampicillin were evenly spread on a solid medium and incubated at 37 ° C. for 16 hours. After that, white and blue colonies were counted. Based on this, mutation frequency and error rate were examined (see Table 2). Neq plus DNA polymerase showed an error rate of 7.96 × 10 ⁻⁶ . The error rate of Taq DNA polymerase is 11.96 x 10 ^-6 , which is Neq 1.5 times higher than the plus DNA polymerase. Neq on the other hand The DNA polymerase showed an error rate of 6.67 × 10 ⁻⁶ and the Pfu DNA polymerase showed an error rate of 2.67 × 10 ⁻⁶ .

Neq , Comparison of PCR fidelity of Neq plus, Pfu and Taq DNA polymerase Blue White Mutation frequency ^a Template doublings ^b Error rate ^c
(Ⅹ10 ^-6 ) Fold improvement over Taq Neq 8767 410 0.0539 9.68 6.67 1.8 Neq plus 7039 536 0.0707 10.64 7.96 1.5 Pfu 8821 208 0.0230 10.32 2.66 4.5 Taq 6349 744 0.1048 10.49 11.96 One

^a Mutation frequency is the total number of colonies divided by the number of white colonies.

^b Template doubling (d) used the equation '2 ^d = (amount of PCR product) / (amount of starting DNA template)'.

^{c The} error rate was calculated using the equation 'error rate = mutation rate / ( bp ⅹ d )'. bp is the length of the LacZα gene 835, d is a template doubling value.

Example  7: Uracil DNA With glycosylase Neq  plus DNA polymerase PCR Application of

The inventor Neq plus DNA polymerase was applied with UDG (uracil-DNA glycosylase) to perform PCR to prevent cross contamination. First, 2 kb uracil-DNA (dirty DNA) substrates and 4 kb DNA (normal DNA) substrates were prepared using lambda DNA as a template. Λ-2 and λ-4 primers were used to amplify the 2 kb uracil-DNA substrate and the 4 kb DNA substrate, respectively.

First, PCR conditions for 2 kb uracil-DNA substrate synthesis are as follows. 50 μl of the PCR mixture was added dATP, dCTP, dGTP and dUTP to the final concentration of 250 μM, respectively, 23 ng lambda DNA, 5 pmol of forward and reverse λ-2 primers, 0.5 U / μl Super Taq DNA Polymerase and IX Super Taq Buffer II were added. PCR was repeated 30 times at 94 degreeC for 30 second, 58 degreeC 30 second, and 72 degreeC 1 minute 30 second. To reduce possible DNA incorporation, a small amount of this amplified uracil-DNA was taken and amplified once again in the same manner.

PCR conditions for 4 kb DNA substrate synthesis were the same as the 2 kb uracil-DNA substrate synthesis method described above except that the forward and reverse λ-4 primers were added and dTTP was added instead of dUTP. Subsequently, two kinds of substrates were prepared by PCR, and then PCR products were purely separated using a PCR purification kit and used as each substrate.

After digestion of artificially contaminated 2 kb uracil-DNA (contaminated DNA) using UDG, 4 kb of normal DNA using 0.22 U / μl of Neq plus DNA polymerase and 0.2 U / μl of Taq DNA polymerase. This experiment compares how efficiently each amplifies. PCR reaction mixture was prepared as follows. 2 kb uracil-DNA (75 pg / μl) substrate and 4 kb DNA (150 pg / μl) substrate prepared above, 1 U / μl of BMTU 3346 UDG (Roche) and 1 × Super Taq buffer II, 250 μM dATP, dCTP, dGTP and dUTP, 5 pmol primers [λ-2F and λ-2R (2 kb DNA fragment amplification), λ-4F and λ-4R (4 kb DNA fragment amplification)] and Neq plus DNA polymerase Alternatively, PCR was performed by adding Taq DNA polymerase, respectively. The PCR was repeated 20 times, 25 times, 30 times for 20 seconds at 94 ° C, 20 seconds at 56 ° C, 3 minutes 30 seconds at 72 ° C, and PCR products were electrophoresed on 0.8% agarose gel. 6 is shown.

Lane 1 in Figure 6 shows Neq without the UDG as a control. As a result of PCR by plus DNA polymerase, it can be seen that both contaminated 2 kb uracil-DNA and 4 kb of normal DNA were amplified. Lane 2 to 4 using the Taq DNA polymerase, the results of PCR in 20, 25, 30 cycles. Lanes 5-7 are Neq Using a plus DNA polymerase, PCR is the result of 20, 25, 30 cycles. Neq in the comparison of samples that were PCR with the same cycle as shown in the results. Plus DNA polymerase was found to be significantly more efficient in PCR using UDG and dUTP than Taq DNA polymerase.

While the invention has been described above with reference to specific embodiments, various modifications, changes or modifications are possible in the art within the spirit and scope of the appended claims, and thus the foregoing description and drawings illustrate the invention. It should not be construed as limiting the technical spirit of the present invention but as illustrating the present invention. All documents cited in the present invention are incorporated herein by reference.

Figure 1 shows the various pH (Fig. 1a), MgCl ₂ to determine the PCR optimum conditions of Neq DNA polymerase 1 kb DNA fragments were amplified under concentrations (FIG. 1b) and KCl concentrations (FIG. 1c).

Figure 2 shows the PCR efficiency of Neq DNA polymerase according to the length of DNA amplification.

Figure 3 (a) is a comparison of the PCR performance of the four DNA polymerases ( Taq , Pfu , Vent , KOD ) other than Neq DNA polymerase in the presence of dNTP and deaminated substrates (dUTP, dITP), respectively . 3 (b) shows Neq and Taq according to the dITP ratio This is the result of comparing PCR amplification efficiency of DNA polymerase.

4 is a result of amplifying Neq DNA polymerase and Taq DNA polymerase according to the ratios indicated, and then amplifying 10 kb DNA fragments (FIG. 4a) and 20 kb DNA fragments (FIG. 4b) by PCR.

Figure 5 shows the comparison of PCR efficiency according to the length of the synthesis product of Neq DNA polymerase, Neq plus DNA polymerase, Taq DNA polymerase in the presence of dUTP, respectively.

Figure 6 shows the comparison of PCR efficiency of Neq plus DNA polymerase and Taq DNA polymerase by PCR cycle (uracil-DNA glycosylase) using dUTP and uracil-DNA glycosylase.

<110> SUNGKYUNKWAN UNIVERSITY Foundation for Corporate Collaboration <120> Nanoarchaeum equitans plus DNA polymerase, method for preparing          the same and its PCR method using dUTP and uracil-DNA glycosylase <130> IPDC35222 <160> 14 <170> Kopatentin 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-1 forward primer <400> 1 caaaggcggt taaggtggta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-1 reverse primer <400> 2 ggctgtaccg gacaatgagt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-2 forward primer <400> 3 agaagttcag gaagcggtga 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-2 reverse primer <400> 4 acggaaaaag agacgcagaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-4 forward primer <400> 5 ccggtaatgg tgagtttgct 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-4 reverse primer <400> 6 gtactggtgc ctttgccatt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-6 forward primer <400> 7 tttacagcgt gatggagcag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-6 reverse primer <400> 8 gacacggtgg cttttgtttt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-8 forward primer <400> 9 agctgagttt tgccctgaaa 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-8 reverse primer <400> 10 aatgtttgcc gtctttggtc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-10 forward primer <400> 11 aggatgactg gtggcgtaac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-10 reverse primer <400> 12 gtgcctagca aactcggaag 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-20 forward primer <400> 13 aaggcagagg ctgcgtataa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Lambda-20 reverse primer <400> 14 atgcctggta ctttgccaac 20  

Claims (15)

The Neq DNA polymerase (Nanoarchaeum equitans DNA polymerase) and Taq DNA polymerase (Thermus aquaticus DNA polymerase) for the prepared mixture at a concentration ratio of 1:10 dUTP (2'-deoxyuridine 5'- triphosphate) PCR polymerase reaction in the presence Neq plus DNA polymerase to perform. Enzyme mixture for performing PCR polymerization in the presence of dUTP (2'-deoxyuridine 5'-triphosphate) prepared by mixing Neq DNA polymerase and Taq DNA polymerase in a concentration ratio of 1:10 to 1: 100. Neq DNA polymerase and Taq DNA polymerase are mixed at a concentration ratio of 1:10 to prepare Neq plus DNA polymerase for performing PCR polymerization in the presence of dUTP (2'-deoxyuridine 5'-triphosphate). Way. A method of performing a nucleic acid polymerization reaction using the Neq plus (DNA) polymerase of claim 1. delete delete The method of claim 4, further comprising uracil-DNA glycosylase (UDG). delete Neq plus DNA polymerase of claim 1, a DNA substrate comprising a uracil base, dUTP and uracil-DNA glycosylase (UDG) composition for a PCR reaction. A method of removing cross-contamination of a PCR reaction product by performing PCR after decomposing contaminated uracil DNA by reacting the composition for a PCR reaction of claim 9 for 10 minutes at 25 ° C. delete delete delete delete delete

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