KR20100053011A - Preparation of pcr premix using nitrogen gas - Google Patents

Abstract

PURPOSE: A method for manufacturing PCR mixture using nitrogen gas is provided to save preparation time and to prevent error and contamination. CONSTITUTION: A method for manufacturing PCR reaction mixture comprises: a step of putting PCT reaction mixture into a tube; and a step of injecting nitrogen gas to the tube to dry mixture solution. The reaction mixture contains high temperature stable DNA polymerase, dNTP, reverse transcriptase, reaction buffer, trehalose, RNase inhibitor, diethylpyrocarbonate(DEPC) water, primer or mixture thereof. The drying is performing for 10-25 minutes.

Description

Method for preparing a polymerase chain reaction mixture using nitrogen gas and a polymerase chain reaction mixture prepared by such method {preparation of PCR premix using nitrogen gas}

RT-PCT (Reverse Transcription) in which a polymerase chain reaction (PCR), a cDNA synthesis reaction using a reverse transcriptase, a PCR reaction and a cDNA synthesis reaction are carried out in one reaction vessel. PCR reaction is a representative method for obtaining or analyzing genes. However, in order to use these techniques, reaction enzymes, reaction buffers, dNTP mixtures, template DNA and primers, RNA, oligo dT, and the like are required. Many reactive elements are required, including precipitation agents and water-soluble dyes.

Due to the many reaction factors, it takes a lot of time to make the reaction mixture before the experiment, and there is a possibility of contamination and error of the mixing capacity during the preparation of the mixture, these problems are a big error in the subsequent analysis using PCR May appear.

In order to solve the problems as described above, by adding a stabilizing material to the DNA polymerase reaction mixture containing a reaction buffer solution, magnesium chloride, four dNTP mixtures and DNA polymerase and lyophilized, A technique for preparing a reaction mixture of a polymerase '(Korean Patent No. 162270) has been attempted, but such a lyophilization method requires a separate centrifugal separation procedure, thereby increasing production time and production cost. There was a problem of increased activity and loss of activity due to exposure of the enzyme to ice crystals during freeze-drying.

In addition, Korean Patent No. 0730364 describes a method for preparing a PCR reaction mixture by adding a DNA polymerase, a reaction buffer, a dNTP mixture, and a gelling buffer to dry at room temperature or warming. Although this has been attempted, it is required to proceed for about 4 hours at room temperature and about 1 hour in warm conditions, which requires a long production time, and the reaction mixture may be contaminated or the enzyme may lose activity when exposed to air for a long time. There was a problem.

In addition, a PCR reaction mixture comprising a reaction buffer containing magnesium chloride, four dNTPs (dATP, dGTP, dCTP, dTTP) and a heat-stable DNA polymerase is dried. The technique for dehydrated PCR premix (prepared Korean Patent Publication No. 10-2006- 0081575) was also attempted, but this is a method of dehydration after drying for 20 days or more at room temperature. However, there is a problem that the production time is excessively long, and the same problem exists that the reaction mixture may be contaminated or the enzyme activity may be lost due to prolonged exposure to air.

The present invention is a method for producing a reaction mixture containing a DNA polymerase, reverse transcriptase, etc. required for a polymerase chain reaction (PCR) to amplify a specific DNA or RNA site in a dried state using nitrogen gas And to provide a reaction mixture and the like produced by such a production method. In the case of using the preparation method and the reaction mixture prepared according to the present invention, the user can save time spent in the preparation procedure for performing PCR, and contamination and mixing capacity errors occur during the preparation of the mixture. This can solve the problem.

As mentioned above, the conventional polymerase chain reaction mixture undergoes warm drying or lyophilization in the manufacturing process, and thus, the related enzyme may lose activity. Even if it is dried at room temperature, air may be kept for a long time without any additional process. There was a problem in that the reactant is contaminated as it is exposed to, the production time takes a long time.

The inventors of the present invention to prepare a reaction mixture used in the polymerase chain reaction (PCR) in a dry state to shorten the manufacturing time, to prevent contamination of the reaction mixture, and to prevent the loss of activity of the reaction enzyme, After researching a method applying various techniques other than the prior art, by drying the reaction mixture using nitrogen gas, a manufacturing method was developed in which the production time was shortened and the reaction mixture was not contaminated while maintaining the enzyme activity. Was done.

Nitrogen is the most abundant element containing 78% in air. Nitrogen gas is colorless, tasteless, and odorless. It is inert, nonflammable, and corresponds to a minimum temperature of -196 ° C at atmospheric pressure. Since such nitrogen gas is inert under normal conditions, it is widely used as an atomizing atmosphere gas, and is importantly used in the semiconductor and electronic industries that require a delicate working environment. Nitrogen gas can be used in large quantities by installing a storage tank at the user's place according to the amount of use, and has a related industrial base so that it can be used in a small amount using a nitrogen generator, and can be easily obtained and used in the manufacturing process. .

The present invention is characterized in that the use of such nitrogen gas in the drying step in the polymerase chain reaction mixture manufacturing process. Nitrogen gas is non-reactive unlike other gases, so it does not affect the composition of the polymerase chain reaction mixture, and it is injected into the nitrogen gas, unlike simply drying and drying the polymerase chain reaction mixture at room temperature, warming and freezing conditions. In this case, the drying time can be shortened.

Specifically, the present invention comprises the steps of dispensing the reaction mixture used in the polymerase chain reaction (Polymerase Chain Reaction; PCR) in the tube; And it provides a method of producing a reaction mixture used in the polymerase chain reaction (Polymerase Chain Reaction (PCR)) comprising the step of drying the mixed solution by injecting nitrogen gas into the tube in which the mixture is dispensed.

In this process, the reaction mixture dispensed into the tube is a high temperature safety DNA polymerase, dNTP, reverse transcriptase, reaction buffer, trehalose, RNAase inhibitor, Diethylpyrocarbonate (hereinafter referred to as " Sterile distilled water, primers, or combinations thereof).

That is, in preparing a reaction mixture used for a general PCR reaction, a high safety DNA polymerase, a reaction buffer, trehalose, a water-soluble dye, sterile distilled water, etc. are used, and a reaction mixture used for reverse transcription for cDNA synthesis is prepared. In the preparation, reverse transcriptase, reaction buffer, trehalose, RNAase inhibitor, RNase inhibitor, dNTP, and sterile distilled water treated with DEPC were used, and PCR reaction and cDNA synthesis reaction were carried out in one reaction container. In preparing reaction mixtures for RT-PCR (Reverse Transcription-PCR) reactions, sterile distilled water treated with high temperature safety DNA polymerase, dNTP, reverse transcriptase, reaction buffer, trehalose, RNase inhibitor, DEPC can be used. It is. In addition, a specific primer (primer) is added to the reaction mixture as needed to dry it together can be used as a kit for amplifying the gene of a specific site.

According to the present invention, since nitrogen gas is injected, drying can be easily performed in a short time without undergoing a drying step in a freezing condition or a heating condition. Therefore, in the present invention, the temperature of the drying step is not particularly limited, but considering that the enzyme can lose activity by breaking protein structure at a high temperature, it is preferable to dry at room temperature conditions.

In the present invention, the time of the drying step is preferably 10-25 minutes. If it dries for less than 10 minutes, the drying is not sufficient, and if it dries for more than 25 minutes, the reactivity of the enzyme is lowered.

By using the method of preparing a reaction mixture used in the polymerase chain reaction (PCR) as described above, a reaction mixture used in the polymerase chain reaction (PCR) can be obtained, and such a reaction mixture May be used in the manufacture of a sequencing kit or disease diagnostic kit comprising the same as an active ingredient.

The preparation method of the reaction mixture used for the polymerase chain reaction using nitrogen gas according to the present invention and the reaction mixture prepared by the present invention significantly reduce the production time and manufacturing cost, and at the same time, Activity can be usefully used in industry as a reaction mixture that can be maintained equal or superior to conventional premixes. Therefore, the user performing the PCR can use the reaction mixture according to the present invention, thereby reducing the time spent in the preparation procedure for performing the PCR, and the problem of contamination of the mixture during the preparation of the mixture and an error in the mixing capacity. This can solve the problem.

Hereinafter, the present invention will be described in detail with reference to examples, but these examples are for explaining the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples.

Example  One: DNA  Preparation of Polymerase Chain Reaction Mixture

To prepare a DNA polymerase chain reaction mixture, a high temperature stable DNA polymerase, a reaction solution, a dNTP mixture, a protein stabilizing agent trehalose, a gel loading buffer were mixed in the amounts shown in Table 1, and then dispensed into a PCR tube. DNA polymerase chain reaction mixture was prepared.

TABLE 1

Furtherance Amount High Temperature Safety DNA Polymerase 1 ~ 2 unit 10x reaction buffer 2 μl 10 mM dNTP mixture 2 μl 2M Trehalose 0.5 ~ 2μl Water soluble dyes 0.005-0.02% (v / v) Sterile distilled water variable total 10 μl

Example  2: cDNA  Preparation and Drying of Synthetic Reaction Mixtures

reverse transcriptase, the reaction solution in order to prepare the cDNA synthesis reaction mixture, dNTP mix, oligo dT _{16 ~ 18,} RNA degrading enzyme inhibitor (RNase inhibitor), a mixture of the protein stabilizing material trehalose (Trehalose) in an amount shown in Table 2 PCR Dispense into a tube to prepare a cDNA synthesis reaction mixture.

TABLE 2

Furtherance Amount Reverse transcriptase 10 to 200 units 10x reaction buffer 2 μl 10 mM dNTP mixture 2 μl 2M Trehalose 0.5 ~ 2μl RNAase inhibitors 5 ~ 20units 100 mM oligo dT 1 to 2 μl Sterile Distilled Water Treated with DEPC variable total 10 μl

Example  3: cDNA  Synthetic and DNA  Preparation of reaction mixture containing all polymerase chain reaction mixture

In order to allow the cDNA synthesis and the DNA polymerase chain reaction to proceed in one reaction vessel, various substances including both the cDNA synthesis and DNA polymerase chain reaction mixtures are mixed in the amounts shown in Table 3 and dispensed into a PCR tube for reaction mixture. Was prepared.

TABLE 3

Furtherance Amount High Temperature Safety DNA Polymerase 1 ~ 2 units Reverse transcriptase 10 to 200 units 10x reaction buffer 2 μl 10 mM dNTP mixture 2 μl 2M Trehalose 0.5 ~ 2μl RNAase inhibitors 5 ~ 20units Sterile Distilled Water Treated with DEPC variable total 10 μl

Example  4: DNA Examination of Reactivity of Reaction Mixtures by Drying Time of Polymerase Chain Reaction Mixtures

After preparing the DNA polymerase chain reaction mixture as in Example 1, 10uL of the reaction mixture was dispensed into a 0.2mL tube for PCR. Nitrogen gas was injected into the dispensed DNA polymerase chain reaction mixture tube at a pressure of 0.1 to 0.5 bar at room temperature for 10 minutes, 15 minutes, 20 minutes, 25 minutes, and 30 minutes, respectively, to prepare the dried DNA polymerase chain reaction mixture. Prepared. After changing only drying time, the same template DNA and primers were used to examine whether the reaction mixture was degraded with drying time using the dried reaction mixture and the liquid reaction mixture before drying.

       CDNA was synthesized using 20 ng of human total RNA, and DNA amplification was performed using 1 ul of the synthesized cDNA reactant as template DNA. Primers used for DNA amplification were used to amplify the gene region of Actin, GAPDH, and the experiment was performed by varying the size of each amplification.

Figure 1 was used as a liquid reaction mixture before drying as a control, after which the liquid reaction mixture was dried using nitrogen gas for 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes and then amplified using each primer described above. Is the result of experiment.

Lane M is 100bp DNA Ladder, Lanes 1 and 2 are the results by Actin amplification size, Lanes 3 and 4 are the results by GAPDH amplification size.

As shown in FIG. 1, the reaction mixture prepared through the drying step of 10 minutes to 25 minutes showed the same activity as the liquid reaction mixture before drying, but the reaction mixture of 30 minutes exceeding 25 minutes. In case of the band 2 and 4 is not clear, the activity was found to decrease a lot. Therefore, in the preparation of the reaction mixture of the present invention, it can be seen that the drying step is performed for 10 to 25 minutes does not affect the activity of the enzyme.

Example  5: DNA Examination of Reactivity of Reaction Mixture with Drying Temperature of Polymerase Chain Reaction Mixture

The reaction mixture was prepared by the preparation method of the DNA polymerase chain reaction mixture shown in Example 1, and then 10 uL was dispensed into a 0.2 mL tube for PCR. Nitrogen gas was injected into the dispensed DNA polymerase chain reaction mixture tube at a pressure of 0.1 to 0.5 bar at 1 ° C., 10 ° C., 20 ° C., and 30 ° C. for 15 minutes to prepare a dried DNA polymerase chain reaction mixture. After changing only the drying temperature, the same template DNA and primers were used, respectively, using the dried reaction mixture and the dried reaction mixture before drying to examine whether the reaction mixture was degraded with drying time.

       Template DNA was used for 20ng lambda DNA, DNA amplification was performed so that the amplification size was 1kb, 2kb, 3kb with different primers.

Figure 2 used the liquid premix before drying as a control, and after drying the liquid premix using nitrogen gas at 1 ℃, 10 ℃, 20 ℃, 30 ℃, the results of experiments to test the degree of amplification using each primer described above to be.

Lane M is a 100bp DNA Ladder, Lane 1, 2, 3 is the result of amplification size of 1kb, 2kb, 3kb respectively.

As can be seen in Figure 2, when drying the enzyme reactants using the enzyme reactant drying method of the present invention it can be seen that in the drying temperature of 1 ~ 30 ℃ no significant decrease in activity of the reaction mixture according to the drying temperature. Therefore, in the method of the present invention it can be seen that the temperature of the drying step can be taken over a wide temperature range from the refrigeration temperature to room temperature.

Example  6: dried DNA Reactivity difference test between polymerase chain reaction mixture and undried enzyme reactant

A DNA polymerase chain reaction mixture was prepared by the method shown in Example 1, and nitrogen gas was injected at room temperature for 15 minutes to prepare a dried reaction mixture. After that, the reaction mixture of the conventional manual method was prepared and PCR was performed using the same template DNA and primer. After PCR, the performance of the dried reaction mixture was examined by electrophoresis.

Template DNA was used as lambda bacteriophage DNA 20pg, 2pg, 200fg, 20fg, Bacterial genomic DNA was used 2ng, 200pg, 20pg, 2pg, Human genomic DNA was used 20ng, 2ng, 200pg, 20pg.

In FIG. 3, Panel A is a result of DNA amplification by preparing a reaction mixture in a manual manner, and Panel B is a result of DNA amplification using a dried reaction mixture.

Lane M is 100bp DNA Ladder, Lanes 1 to 4 are the results of DNA amplification by concentration of lambda bacteriophage DNA, Lanes 5 to 8 are the results of DNA amplification by concentration of Bacterial genomic DNA, and lanes 9 to 12 are the concentrations of human genomic DNA. Amplification results are shown.

As can be seen in Figure 3, it can be seen that the enzyme reaction drying method of the present invention does not show a decrease in performance as compared to the enzyme reaction preparation method according to the manual method generally used.

Example  7: prepared by the drying method of the present invention DNA  Polymerase reactants and conventional lyophilization methods Warming drying method  Comparative experiment with the enzyme reaction mixture used

A DNA polymerase chain reaction mixture was prepared by the method shown in Example 1, and nitrogen gas was injected at room temperature for 15 minutes to prepare a dried reaction mixture. The dried reaction mixtures and the reaction mixtures prepared by conventional lyophilization and warm-drying methods were compared using the same template DNA and primers. The reaction mixture prepared by the lyophilization method is used by purchasing a product of a manufacturer manufactured by a conventional patent method (Korean Registered Patent No. 162270) (Bionia, Inc., K-2012, product name: AccuPower PCR Premix). It was. The reaction mixture prepared by the warm-drying method is manufactured by the manufacturer's product (Intron Biotecology, Inc., product number: 25025, product name: Maxime PCR Premix) manufactured by the existing patent method (Korean Patent No. 0730364). It was purchased and used.

Template DNA used lambda bacteriophage DNA 2pg, 200fg, Bacterial genomic DNA 2ng, 200pg, Human genomic DNA 20ng, 2ng was used.

In Figure 4, panel A is a DNA polymerase chain reaction mixture prepared by the enzyme reactant drying method of the present invention, panels B and C are DNA polymerase chain reaction mixtures prepared by lyophilization and warm drying, respectively. Experimental results used.

Lane M is 100bp DNA Ladder, Lanes 1 and 2 are the results of DNA amplification by lambda bacteriophage DNA concentration, Lanes 3 and 4 are the results of DNA amplification by concentration of Bacterial genomic DNA, Lanes 5 and 6 are the concentration of human genomic DNA Amplification results are shown.

As can be seen in Figure 4 in the case of the DNA polymerase chain reaction mixture prepared by the conventional freeze-drying method and warm-drying method, this band and band 6 appears unlike the DNA polymerase chain reaction mixture according to the patent invention It was found that performance was worse.

Example  8: prepared by the drying method of the present invention cDNA  Comparative Experiment with Synthetic Reaction Mixture and Enzyme Reaction Mixture Using Conventional Lyophilization

A cDNA synthesis reaction mixture was prepared by the method shown in Example 2, and a dried reaction mixture was prepared by injecting nitrogen gas at room temperature for 15 minutes. The dried reaction mixture and the cDNA synthesis reaction mixture prepared using the conventional lyophilization method were compared using the same RNA and primers. The reaction mixture prepared by the lyophilization method is used by purchasing the manufacturer's product (Bionia, Inc .: K-2041, product name: AccuPower RT Premix) manufactured by the existing patent method (Korean Patent No. 162270). It was.

CDNA was synthesized using 20 ng of human total RNA, and DNA amplification was performed using 1 ul of the synthesized cDNA reactant as template DNA. Primers used for DNA amplification were those capable of amplifying the gene regions of Actin, GAPDH, Protein Phosphatase and Hypoxanthine Phosphoribosyltransferase 1, and experiments were performed at different amplification sizes. Was carried out.

In FIG. 5, Panel A is the one prepared by the drying method of the cDNA synthesis reaction mixture of the present invention, and Panel B is the experimental result using the cDNA synthesis reaction mixture prepared by the lyophilization method.

Lane M is 100bp DNA Ladder, Lanes 1 ~ 3 are results by Actin amplification size, Lanes 4 ~ 6 are results by GAPDH amplification size, Lanes 7 ~ 9 are results by Protein Phosphatase amplification size, Lane 10 ~ 12 Shows the results of amplification size of hypoxanthine phosphoribosyltransferase 1 (Hypoxanthine Phosphoribosyltransferase 1).

As can be seen in Figure 5 cDNA synthesis reaction mixture of the present invention compared to the cDNA synthesis reaction mixture prepared by lyophilization method, 3, 7, 8 and 9 band cDNA synthesis reaction by the conventional method Unlike the case of using the mixture, it can be seen that more vivid.

Example  9: prepared by the drying method of the present invention cDNA  Synthetic and DNA  Comparison experiments with reaction mixtures containing all polymerase chain reaction mixtures and enzyme reaction mixtures using conventional lyophilization

A reaction mixture including both cDNA synthesis and DNA polymerase chain reaction mixture was prepared by the method shown in Example 3, and a dried reaction mixture was prepared by injecting nitrogen gas at room temperature for 15 minutes, and prepared by conventional lyophilization. The reaction mixtures were compared using the same RNA and primers. The reaction mixture prepared by the lyophilization method was purchased from the manufacturer (Bionia Co., Ltd., product number: K-2055, product name: AccuPower RT / PCR Premix) manufactured by the existing patent method (Korean Patent No. 162270). Was used.

The reaction was performed at 42 degrees for 30 minutes using primers that could amplify 20 ng of human total RNA and the gene sites of Actin, GAPDH, Protein Phosphatase and Hypoxanthine Phosphoribosyltransferase 1 DNA polymerization was performed using a tube. In addition, experiments were carried out with different amplification sizes.

In Figure 6, Panel A is a reaction mixture containing both the cDNA synthesis and DNA polymerase chain reaction mixture prepared by the drying method presented in the present invention, Panel B is an experimental result using the reaction mixture prepared by the lyophilization method.

Lane M is 100bp DNA Ladder, Lane 1 ~ 3 are results by Actin amplification size, Lane 4 ~ 6 are results by GAPDH amplification size, Lane 7 ~ 9 are results by Protein Phosphatase, Lane 10 ~ 12 is Results of amplification size of hypoxanthine phosphoribosyltransferase 1 (Hypoxanthine Phosphoribosyltransferase 1) are shown.

As can be seen in Figure 6, unlike the case of the conventional freeze-drying method when using the product according to the drying method of the present invention it can be seen that the 6th to 12th band more clearly appear.

Example  10: Stability test of the enzyme reaction mixture prepared by the drying method of the present invention

A DNA polymerase chain reaction mixture was prepared by the method described in Example 1, nitrogen gas was injected at room temperature for 15 minutes, and the dried reaction mixture was stored at 4 ° C. for 6 months to carry out stability experiments of the reaction mixture.

Template DNA was lambda bacteriophage DNA 200fg was used, the experiment was performed by varying the size of the amplified DNA.

      In Figure 7, Panel A is a DNA polymerase chain reaction mixture was prepared immediately used, Panel B was used after storing the dried reaction mixture at 4 degrees for 6 months.

Lane M is a 1kb DNA Ladder, and Lanes 1-4 are reaction products of different sizes of amplified DNA.

As can be seen in Figure 7 it can be seen that even if the enzyme reactant prepared by the enzyme reactant drying method of the present invention stored for 6 months at 4 ℃ does not show a decrease in activity.

1 is a result of the reaction mixture prepared by the method of Example 1 was prepared for each drying time, and then the DNA amplification and electrophoresis using the same template DNA and primer using the reaction mixture in the liquid state as a control .

       2 is a result of electrophoresis after DNA amplification using the same template DNA and primers by preparing the reaction mixture prepared by the method of Example 1 for each drying temperature and then controlling the reaction mixture in a liquid state as a control. .

       FIG. 3 shows a dry reaction mixture prepared by drying the reaction mixture prepared by the method of Example 1 at room temperature for 15 minutes and a reaction mixture prepared by a manual method generally used (manufactured directly in an experiment). As a result of electrophoresis by DNA amplification using the same template DNA and primers, Panel A was the result of the reaction mixture prepared by the manual method, and Panel B was added to the reaction mixture prepared by the method of Example 1. Result.

Figure 4, Panel A is a dry reaction mixture for DNA polymerase prepared by the method of the present invention, Panel B is a freeze-drying method, Panel C is a PCR using a reaction mixture prepared by a warm drying method electrophoresis One result.

5 is a result of electrophoresis of panel A using the cDNA synthesis reaction mixture prepared by the method of the present invention and panel B using the cDNA synthesis reaction mixture prepared by the lyophilization method.

Figure 6, Panel A is a reaction mixture containing both cDNA synthesis and DNA polymerase chain reaction mixture prepared by the method of the present invention, Panel B was RT-PCR using a reaction mixture prepared by the lyophilization method It is the result of electrophoresis.

FIG. 7 shows that Panel A was used immediately after preparation of the reaction mixture prepared by the method of Example 1, and Panel B was stored at 4 ° C. for 6 months after the preparation of the reaction mixture prepared by the method of Example 1. PCR result using electrophoresis.

Claims (6)

Dispensing the reaction mixture used in the polymerase chain reaction (PCR) into tubes; And Drying the mixed solution by injecting nitrogen gas into the tube where the mixture is dispensed Method for producing a reaction mixture used in the polymerase chain reaction (Polymerase Chain Reaction; PCR) comprising a According to claim 1, wherein the reaction mixture used in the polymerase chain reaction is a high temperature safety DNA polymerase, dNTP, reverse transcriptase, reaction buffer, trehalose (trehalose), RNAase inhibitor (RNase inhibitor), diethylpyrocarbonate ( Method for producing a reaction mixture used in a polymerase chain reaction (PCR), characterized in that it comprises any one selected from the group consisting of sterile distilled water, primers or a combination thereof treated with Diethylpyrocarbonate (DEPC) The method of claim 1, wherein the drying step is a method for producing a reaction mixture used for a polymerase chain reaction (PCR), characterized in that 10 minutes to 25 minutes. A reaction mixture used in a polymerase chain reaction (PCR) prepared by a method for preparing a reaction mixture used in a polymerase chain reaction (PCR) according to claim 1. Kit for sequencing analysis comprising the reaction mixture used in claim 4 polymerase chain reaction (PCR) as an active ingredient Disease diagnosis kit comprising the reaction mixture used in claim 4 polymerase chain reaction (PCR) as an active ingredient

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