JP2017108735A - Reagent for nucleic acid amplification and nucleic acid amplification method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition capable of improving nucleic acid amplification efficiency and detection sensitivity and performing effective nucleic acid amplification.SOLUTION: A composition for nucleic acid amplification has no inhibitory effects on enzymes, has extremely low reduction in operability, can be stored for the long term, and has high stability in a mixed solution state, the composition having a DNA polymerase resistant to tetramethylammonium salt coexist with tetramethylammonium salt, in particular at least one compound selected from the group consisting of tetramethylammonium acetate and tetramethylammonium chloride.SELECTED DRAWING: None

Description

The present invention relates to a composition and a nucleic acid amplification method capable of improving nucleic acid amplification efficiency and detection sensitivity and performing effective nucleic acid amplification.

Nucleic acid amplification is a technology that amplifies several copies of target nucleic acid to a level that can be visualized, that is, several hundred million copies or more, and not only in the life science research field, but also in the medical field such as genetic diagnosis and clinical testing, or in food and the environment. It is also widely used in the microbiological examination of the A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR includes (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of primer to template single-stranded DNA, (3) the primer using DNA polymerase This is a method of amplifying a target nucleic acid in a sample by repeating three cycles of three steps of extension as one cycle.

When the nucleic acid to be detected is RNA, for example, when the target is an RNA virus in detecting pathogenic microorganisms, or when the expression level of a gene is measured by quantifying mRNA, RNA is converted into cDNA by reverse transcriptase. The reaction (reverse transcription reaction) is performed before PCR.

In many cases, the reverse transcription reaction using reverse transcriptase and the PCR using DNA polymerase are performed by performing the reverse transcription reaction first and then transferring to another reaction solution because of the difference in the optimum conditions of each enzyme. In order to eliminate this complexity, a one-step RT-PCR method, in which two reactions are successively performed in the same reaction solution, has been developed in recent years (Patent Document 1).

In recent years, the real-time PCR method has been widely implemented among PCR. The real-time PCR method can analyze the amplified nucleic acid over time, ensure high quantitativeness, and perform highly sensitive detection.

However, in such nucleic acid amplification, when the amount of nucleic acid to be detected is very small, the amplification varies and there are cases where amplification is not successful. In particular, when the nucleic acid to be detected is RNA, a small amount of RNA is quantified because the reverse transcription reaction is likely to cause sample loss by PCR before PCR and may be degraded by RNase in the environment. It is difficult to do. In addition, when detecting viral RNA from a biological sample such as a serum sample of a patient, the amount of viral RNA in the sample at the initial stage of virus infection is small and often cannot be detected.

From such a background, in order to improve the efficiency and sensitivity of the nucleic acid amplification method, a method of adding various reagents to the nucleic acid amplification reaction solution has been attempted.

For example, dimethyl sulfoxide (DMSO), formamide, glycerol, betaine and the like have been reported as methods for improving nucleic acid amplification efficiency (Patent Documents 2, 3, and 4). These agents are known to improve nucleic acid amplification efficiency by lowering the melting temperature of the nucleic acid. However, all of these substances are known to have disadvantages. For example, dimethyl sulfoxide and formamide have the property of inhibiting the action of anti-DNA polymerase antibodies for hot start that are frequently used to increase the specificity of PCR (Non-patent Document 1). Further, formamide has low stability in its own aqueous solution, and long-term storage in a mixed state with a PCR reaction solution is difficult. On the other hand, glycerol and betaine have few problems in terms of reaction inhibition and stability, but their action to lower the melting temperature is relatively low, so it is necessary to add a very high concentration when mixing into a PCR reaction solution. . Glycerol in particular has a high viscosity, and the addition of a high concentration significantly reduces the operability when preparing a reaction solution, or there is a problem that an addition error tends to occur when preparing a reaction solution with a small amount. It was.

Non-Patent Document 2 specifies that tetramethylammonium salt is an enhancer that improves amplification efficiency and specificity, and discusses the effect and optimum concentration of tetramethylammonium salt in PCR reaction using Taq DNA polymerase. ing. According to Non-Patent Document 2, the concentration at which the amplification efficiency is maximum is 5 mM for tetramethylammonium chloride, 10 mM or less for tetramethylammonium acetate, and the concentration at which the specificity is maximum is 20 mM for both tetramethylammonium chloride and tetramethylammonium acetate. is there. Furthermore, tetramethylammonium salts are known to inhibit enzymes at high concentrations. According to Non-Patent Document 2, when Taq DNA polymerase is used, tetramethylammonium chloride is 35 mM and tetramethylammonium acetate is 40 mM. If exceeded, nucleic acid amplification is inhibited by 90%. Thus, tetramethylammonium salts have been conventionally used for nucleic acid amplification at concentrations generally lower than 100 mM.

Japanese Patent No. 2968585 Japanese Patent No. 4761265 Japanese Patent No. 4300321 JP 2014-27934 A Clontech, TaqStart Antibody manual (issued in March 2005) Nucleic Acids Research, Vol. 28, no. 13 (issued in 2000)

In nucleic acid amplification methods, studies on nucleic acid amplification methods that improve nucleic acid amplification efficiency and detection sensitivity have been underway. However, the conventional studies have been disadvantageous in use, such as those that lower stability, those that inhibit DNA polymerase, and those that are complicated and time-consuming.

Therefore, the present invention provides a nucleic acid amplification reagent that (1) operation is not complicated, (2) nucleic acid amplification efficiency is improved and highly sensitive detection can be performed, and (3) operation is simple and risk of contamination. It aims at providing the composition which satisfy | fills that it is low.

In view of the above-mentioned problems, the present inventors have made extensive studies, and are resistant to tetramethylammonium salts, particularly at least one compound selected from the group consisting of tetramethylammonium acetate and tetramethylammonium chloride. When nucleic acid amplification is carried out in the presence of DNA polymerase which is difficult to be inhibited, it has been found that amplification efficiency is improved and highly sensitive detection is possible. Based on this finding, it has been found that a composition for nucleic acid amplification can be provided that has no inhibitory action on enzymes, has very little operability reduction, can be stored for a long period of time, and has high stability in a mixed solution state. It was. Further, the inventors have found that this knowledge can be applied to PCR, real-time PCR, RT-PCR, and real-time RT-PCR, particularly one-step RT-PCR, and have completed the present invention.

That is, the outline of the present invention is as follows.
Item 1. A nucleic acid amplification composition comprising a tetramethylammonium salt having a final concentration of 50 mM or more and a tetramethylammonium resistant DNA polymerase.
Item 2. Item 2. The nucleic acid amplification composition according to Item 1, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase that is not inhibited in the presence of a tetramethylammonium salt having a final concentration of 50 mM or more.
Item 3. Item 3. The nucleic acid amplification composition according to Item 1 or 2, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase having 90% or more identity with SEQ ID NO: 10.
Item 4. Item 3. The nucleic acid amplification composition according to Item 1 or 2, wherein the tetramethylammonium resistant DNA polymerase is Tth DNA polymerase.
Item 5. Item 3. The nucleic acid amplification composition according to Item 1 or 2, wherein the tetramethylammonium resistant DNA polymerase is Z05 DNA polymerase.
Item 6. Item 3. The nucleic acid amplification composition according to Item 1 or 2, wherein the tetramethylammonium resistant DNA polymerase is a variant of SEQ ID NO: 11.
Item 7. Item 7. The nucleic acid amplification composition according to Item 6, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase in which at least E507 position or E742 position of SEQ ID NO: 11 is modified.
Item 8. Item 8. The nucleic acid amplification composition according to any one of Items 1 to 7, wherein the tetramethylammonium salt is at least one compound selected from the group consisting of tetramethylammonium acetate, tetramethylammonium chloride, and tetramethylammonium hydroxide. object.
Item 9. Item 9. The nucleic acid amplification composition according to any one of Items 1 to 8, wherein the final concentration of the tetramethylammonium salt is 100 to 210 mM.
Item 10. Item 10. The nucleic acid amplification composition according to any one of Items 1 to 9, further comprising at least an antibody, a reaction buffer, and a metal ion.
Item 11. Item 11. The nucleic acid amplification composition according to any one of Items 1 to 10, comprising at least a reverse transcriptase, an antibody, a reaction buffer, and a metal ion.
Item 12. Item 12. The composition for nucleic acid amplification according to any one of Items 1 to 11, wherein the nucleic acid amplification reaction is PCR.
Item 13. Item 13. The nucleic acid amplification composition according to any one of Items 1 to 12, wherein the nucleic acid amplification reaction is one-step RT-PCR.
Item 14. A nucleic acid amplification method comprising a tetramethylammonium salt and a DNA polymerase having a tetramethylammonium resistance having a final concentration of 50 mM or more.
Item 15. Item 15. The nucleic acid amplification method according to Item 14, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase that is not inhibited in the presence of 50 mM tetramethylammonium salt.
Item 16. Item 16. The nucleic acid amplification method according to Item 14 or 15, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase having 90% or more identity with SEQ ID NO: 10.
Item 17.
The nucleic acid amplification method according to any one of claims 14 to 16, wherein the tetramethylammonium resistant DNA polymerase is Tth DNA polymerase.
Item 18. Item 18. The nucleic acid amplification method according to any one of Items 14 to 17, wherein the tetramethylammonium resistant DNA polymerase is Z05 DNA polymerase.
Item 19. Item 19. The nucleic acid amplification method according to any one of Items 14 to 18, wherein the tetramethylammonium resistant DNA polymerase is a variant of SEQ ID NO: 11.
Item 20. Item 20. The nucleic acid amplification method according to any one of Items 14 to 19, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase in which at least E507 position or E742 position of SEQ ID NO: 11 is modified.
Item 21. Item 21. The nucleic acid amplification method according to any one of Items 14 to 20, wherein the tetramethylammonium salt is at least one compound selected from the group consisting of tetramethylammonium acetate, tetramethylammonium chloride, and tetramethylammonium hydroxide.
Item 22. Item 22. The nucleic acid amplification method according to any one of Items 14 to 21, wherein the final concentration of the tetramethylammonium salt is 100 to 210 mM.
Item 23. Item 23. The nucleic acid amplification method according to any one of Items 14 to 22, comprising at least an antibody, a reaction buffer, and a metal ion.
Item 24. Item 24. The nucleic acid amplification method according to any one of Items 14 to 23, comprising at least a reverse transcriptase, an antibody, a reaction buffer, and a metal ion.
Item 25. Item 25. The nucleic acid amplification method according to any one of Items 14 to 24, wherein the nucleic acid amplification reaction is PCR.
Item 26. Item 26. The nucleic acid amplification method according to any one of Items 14 to 25, wherein the nucleic acid amplification reaction is one-step RT-PCR.

According to the present invention, nucleic acid amplification efficiency and sensitivity can be improved. The method of the present invention can amplify a nucleic acid easily without using a reagent that requires special handling and without depending on the type of sample or target.

The sensitivity when cDNA was synthesized with a cDNA synthesis reagent and the concentration of tetramethylammonium acetate was added from 0 mM to 160 mM was examined in real-time PCR. A is a summary of Ct values, and B is an amplification curve. The sensitivity when the concentration of tetramethylammonium acetate is added from 0 mM to 170 mM is examined in real-time RT-PCR, and the Ct values are summarized. In the table, black painting indicates that the Ct value cannot be measured (ND). The sensitivity when the concentration of tetramethylammonium acetate is added from 180 mM to 200 mM is examined in real-time RT-PCR, and the Ct values are summarized. In the table, black painting indicates that the Ct value cannot be measured (ND). It is the figure which examined the sensitivity when the density | concentration of tetramethylammonium acetate was added from 0 mM to 200 mM in real-time RT-PCR, took the Ct value on the vertical axis | shaft, and took the logarithm value of total amount RNA on the horizontal axis, and was made into the graph. Buffers with and without tetramethylammonium acetate were prepared. It is the figure which implemented real-time RT-PCR with each buffer, and plotted the Ct value on the vertical axis | shaft, and the logarithm value of RNA on the horizontal axis | shaft. Buffers with and without tetramethylammonium acetate were prepared. It is an amplification curve when real-time RT-PCR is implemented with each buffer. A is a buffer not containing tetramethylammonium acetate, and B is a buffer containing tetramethylammonium acetate. Buffers with and without tetramethylammonium acetate were prepared. It is the figure which implemented real-time RT-PCR with Tth DNA polymerase and Taq DNA polymerase with the prepared buffer, and plotted the logarithm value of RNA with the Ct value on the vertical axis and the horizontal axis. Buffers with and without tetramethylammonium acetate were prepared. It is an amplification curve when real-time RT-PCR is performed with the prepared buffer using Tth DNA polymerase and Taq DNA polymerase. A is a diagram when Tth DNA polymerase is used, and B is a diagram when Taq DNA polymerase is used. A buffer added with tetramethylammonium acetate and tetramethylammonium chloride was prepared. It is the figure which implemented real-time RT-PCR with each prepared buffer, took the Ct value on the vertical axis | shaft, and plotted the logarithm value of RNA on the horizontal axis | shaft. A buffer added with tetramethylammonium acetate and tetramethylammonium chloride was prepared. It is an amplification curve when the prepared buffer and real-time RT-PCR are implemented. A is a diagram when tetramethylammonium acetate is used, and B is a diagram when tetramethylammonium chloride is used. It is the figure which confirmed by adding tetramethylammonium acetate to the reaction system of PCR whether it was resistant to tetramethylammonium salt. Buffers with and without tetramethylammonium acetate were prepared. These are the amplification curve and Ct value when real-time PCR was performed from blood using Taq DNA polymerase and Taq DNA polymerase mutants using the prepared buffer. It is the figure which confirmed the amino acid sequence identity of Tth DNA polymerase and Z05 DNA polymerase.

The first aspect of the present invention is a nucleic acid amplification method capable of improving nucleic acid amplification efficiency and detection sensitivity in detection of nucleic acids. This nucleic acid amplification method is a reagent for nucleic acid amplification characterized in that it contains tetramethylammonium salt and DNA polymerase resistant to tetramethylammonium salt.

Generally, prior to nucleic acid amplification, it is necessary to separate cells, bacteria, viruses and the like from a test substance and extract nucleic acids. A method of degrading cells and bacteria with an enzyme, a surfactant, a chaotropic agent, etc., and extracting nucleic acid from the degraded product with phenol or phenol / chloroform has been conventionally used. In the nucleic acid extraction method of the present invention, when a nucleic acid to be amplified, such as an organ or a cell, is present in a tissue of a sample, the action of destroying the tissue to extract the nucleic acid (disruption by physical treatment, surfactant) Etc.) and is not limited to these. The nucleic acid purification method in the present invention is a method for separating DNA in a biological sample from contaminants such as tissues and cell walls of the biological sample, and a method for separating DNA using phenol or phenol / chloroform, Including, but not limited to, methods of separating DNA using an ion exchange resin, a glass filter or a reagent having a protein aggregation action.

The nucleic acid amplification reaction to which the nucleic acid amplification reagent of the present invention is applied refers to a reaction for synthesizing a nucleic acid having a complementary sequence to a template nucleic acid in a sequence-dependent manner, and the mode is not particularly limited. Specifically, a method of amplifying a specific target sequence exponentially like the polymerase chain reaction (PCR) method is exemplified. The PCR may be real time PCR. Other forms include quantitative PCR (qPCR), LA-PCR (Long and Accurate PCR), competitive PCR, In situ PCR, RNA-primed PCR, multiplex PCR, shuttle PCR, PCR / GC-calmp method, stretch PCR, Methods applying the PCR method such as Alu PCR, megaprimer PCR, and Immuno PCR are also included in the nucleic acid amplification method.

The nucleic acid amplification method of the present invention can also be applied to RT-PCR. The reverse transcription step and the PCR step in RT-PCR may be performed separately in sequence, or a one-step method that is performed continuously may be employed. Real-time RT-PCR may also be used.

As the tetramethylammonium salt in the present invention, any quaternary ammonium salt may be used, but tetramethylammonium hydroxide, tetramethylammonium chloride, and tetramethylammonium acetate are preferably used. More preferably, tetramethylammonium acetate is used. Although these tetramethylammonium salts have different production methods depending on the structure, industrial production methods have been established for them, and they can be easily obtained at low cost. Only one kind of these tetramethylammonium salts may be used, and even if two or more kinds are used in any combination, the effects of the present invention are exhibited.

The tetramethylammonium salt in the present invention is preferably mixed in advance with a reagent for nucleic acid amplification reaction. In the present invention, the lower limit of the tetramethylammonium salt in the nucleic acid amplification reaction solution is preferably 100 mM as the final concentration. On the other hand, the upper limit of the tetramethylammonium salt is preferably 210 mM as the final concentration.

The DNA polymerase used in the nucleic acid amplification reagent of the present invention is preferably a DNA polymerase resistant to tetramethylammonium salt. The DNA polymerase resistant to tetramethylammonium salt refers to a DNA polymerase that is not inhibited even if tetramethylammonium salt is contained in the PCR reaction system, and preferably resistant to 50 mM tetramethylammonium salt. It is desirable. More preferably, it should be resistant to 100 mM tetramethylammonium salt, and more preferably it should be resistant to 200 mM tetramethylammonium salt.

Whether or not the tetramethylammonium salt is resistant can be confirmed as follows.
Evaluation of Tetramethylammonium Salt Resistance of DNA Polymerase Using 10 × PCR Buffer attached to BlendTaq (Toyobo), 1 × PCR Buffer, 0.2 mM dNTPs, 50 ng human genome, 15 pmol of β-actin 1.3 kb, SEQ ID NO: 12 and Into 50 μl of the reaction solution containing the primer described in 13, add the enzyme to be determined for resistance to 2.5 U. Tetramethylammonium acetate was added to a final concentration of 0, 50, 100, 150, and 200 mM, and after 94 ° C for 30 seconds pre-reaction, 94 ° C, 30 seconds → 60 ° C, 30 seconds → 72 ° C, 90 ° C. Perform PCR on a schedule that repeats seconds for 30 cycles. Confirm the amplification product by electrophoresis, compare the one with added tetramethylammonium acetate and the one without added tetramethylammonium acetate, and if the amount of amplification does not change, it is resistant to tetramethylammonium salt up to its concentration It can be said.

Specifically, the DNA polymerase resistant to tetramethylammonium salt is preferably Tth DNA polymerase or Z05 DNA polymerase. The DNA polymerase may be a wild type or may be modified as long as the DNA polymerase activity is maintained. Furthermore, they may be produced by recombinant production using an appropriate expression system. Moreover, in order to make tetramethylammonium salt resistant, Taq DNA polymerase may be mutated. More preferably, mutations may be made at positions E507 and E742 in SEQ ID NO: 11, but other modifications may be made within the range that maintains DNA polymerase activity, and there is no particular limitation. In the case of Tth DNA polymerase, it may be a DNA polymerase having the identity of 90% or more, preferably 95% or more with SEQ ID NO: 10.

In the nucleic acid amplification reagent of the present invention, in the nucleic acid amplification reaction solution, in addition to a tetramethylammonium salt and a DNA polymerase resistant to a tetramethylammonium salt, a template nucleic acid, RNA polymerase, reverse transcriptase, Oligonucleotides, dideoxynucleoside triphosphates (dNTPs), reaction buffers, and magnesium ions such as magnesium ions as primers are present as required by the nucleic acid amplification method to be performed.

As an example, in nucleic acid amplification using the PCR method, template nucleic acid, DNA polymerase, oligonucleotide, dNTPs, and reaction buffer are generally required.

In the case of real-time PCR, in order to confirm nucleic acid amplification over time, a fluorescent dye such as SYBR Green I (registered trademark) or Eva Green (registered trademark) or a probe with a fluorescent label is present as necessary.

In the case of RT-PCR, a reverse transcriptase is further present as necessary for reverse transcription.

In the case of real-time RT-PCR, a reverse transcriptase is further present as necessary for reverse transcription. Further, in order to confirm nucleic acid amplification over time, a fluorescent dye such as SYBR Green I (registered trademark) or Eva Green (registered trademark) or a probe labeled with a fluorescent label is also present if necessary.

In the nucleic acid amplification reagent of the present invention, the nucleic acid amplification reaction solution may contain an anti-DNA polymerase antibody in order to perform the hot start PCR method. In the past, when DMSO or formaldehyde was added for the purpose of adjusting the melting temperature of nucleic acids, these substances inhibited the action of anti-DNA polymerase antibodies. In the present invention, 100 mM to 210 mM tetramethylammonium salt is added as a final concentration, but this does not inhibit the action of the anti-DNA polymerase antibody, so the hot start PCR method is extremely useful.

The form of the anti-DNA polymerase antibody herein is not particularly limited, but preferably binds to a heat-resistant DNA polymerase and inhibits activity by 80% or more at a low temperature of 50 ° C. or less, more preferably 90% or more. Things can be used. The anti-thermostable DNA polymerase antibody used here may be a single type of monoclonal antibody, a plurality of monoclonal antibodies, or a polyclonal antibody. Such an antibody can be produced by a known monoclonal antibody production method such as hybridoma production using mouse, rat, hamster or the like.

In the nucleic acid amplification reagent of the present invention, conditions such as temperature, time and reaction cycle for nucleic acid amplification vary depending on the type of nucleic acid to be amplified, base sequence, chain length, etc., but those skilled in the art can appropriately set them. . However, in order to enjoy the effects of the present invention, the set temperature and set time in the denaturation reaction are preferably set lower than usual or shorter, but are not limited thereto.

Another aspect of the present invention is a nucleic acid amplification reagent having high amplification efficiency and sensitivity, comprising a tetramethylammonium salt and a DNA polymerase resistant to tetramethylammonium salt. The reagent referred to here is not limited to a commercially available product such as a kit, but includes any one prepared in-house, and may be in any form. In another aspect, aspects such as a PCR reagent, RT-PCR reagent, one-step RT-PCR reagent and the like can be mentioned. The composition of the composition contained in the reagent for nucleic acid amplification of the present invention and details of each component are in accordance with the description of the nucleic acid amplification method described above.

In the reagent for nucleic acid amplification of the present invention, the tetramethylammonium salt and the DNA polymerase resistant to the tetramethylammonium salt may be contained in the same container, or those held in separate containers are used. You may mix before. In particular, in the case of one-step RT-PCR, reverse transcriptase needs to be present, but this reverse transcriptase may also be contained in the same container as the above-mentioned reagents or be held in a separate container. You may mix before using what was used.

Hereinafter, the present invention will be described in more detail based on examples. In addition, this invention is not specifically limited to an Example.

Example 1
(1) RNA sample Enterovirus RNA (Vircell) was used as an RNA sample. 1570 copies of enterovirus RNA was used as a template.
(2) cDNA synthesis cDNA was synthesized using enterovirus RNA as a template.
River Tra Ace® qPCR RT Master Mix (Toyobo) 5 × RT Master Mix 2 μL,
5 μL of RNA,
10 μL of the reaction solution containing was reacted under the following temperature conditions.
37 ° C. 10 minutes 50 ° C. 5 minutes 98 ° C. 5 minutes (3) PCR reaction A buffer to which tetramethylammonium acetate was added from 0 to 160 mM was prepared, and the cDNA obtained in (2) was added to perform real-time PCR.
rTth DNA Polymerase (Toyobo) 1 unit,
0.4 μg of anti-Tth antibody,
10 × Buffer (Toyobo rTth DNA Polymerase Buffer) 2 μL,
10 mM dNTPs (Toyobo) 0.4 μL,
10 μM enterovirus forwarded primer (SEQ ID NO: 1) 0.6 μL,
10 μM enterovirus probe (SEQ ID NO: 2) 0.4 μL,
0.6 μL of 10 μM enterovirus reverse primer (SEQ ID NO: 3),
5 μL of RNA,
Tetramethylammonium acetate concentration for each condition,
20 μL of the reaction solution containing was reacted in the following temperature cycle.
95 ° C 1 minute 95 ° C 15 seconds -60 ° C 45 seconds 45 cycles

(4) Results The results are shown in FIG. FIG. 1A summarizes the Ct values for each copy number determined by real-time PCR. FIG. 1B is a diagram of an amplification curve obtained by real-time PCR. Surprisingly, as tetramethylammonium acetate is added from 0 to 160 mM, the rise of the amplification curve becomes faster (Ct value becomes smaller). This shows that the amplification efficiency of the PCR reaction is improved by increasing the concentration of tetramethylammonium acetate.

A similar experiment was performed with Z05 DNA polymerase (Roche) as follows. Like Tth DNA polymerase, the addition of tetramethylammonium acetate from 0 to 160 mM accelerated the rise of the amplification curve (the Ct value was small). became).
Z05 DNA Polymerase (Roche) 1 unit,
0.4 μg of anti-Tth antibody,
10 × Buffer (Toyobo rTth DNA Polymerase Buffer) 2 μL,
10 mM dNTPs (Toyobo) 0.4 μL,
10 μM enterovirus forwarded primer (SEQ ID NO: 1) 0.6 μL,
10 μM enterovirus probe (SEQ ID NO: 2) 0.4 μL,
0.6 μL of 10 μM enterovirus reverse primer (SEQ ID NO: 3),
5 μL of RNA,
Tetramethylammonium acetate concentration for each condition,
20 μL of the reaction solution containing was reacted in the following temperature cycle.
95 ° C 1 minute 95 ° C 15 seconds -60 ° C 45 seconds 45 cycles

Taq DNA polymerase, which is used for general purposes, is not resistant to tetramethylammonium salt and cannot fully exert the effect of tetramethylammonium that promotes the reaction. Tth DNA polymerase and Z05 DNA polymerase are resistant to tetramethylammonium, and it is considered that the reaction can be carried out at a concentration at which the effect of the tetramethylammonium salt can be sufficiently exerted.

Example 2
(1) RNA sample As an RNA sample, RNA derived from bacteriophage MS2 (Roche Diagnostics (product number: 10165948001)) was used. MS2 RNA was serially diluted 1/10 from 1,000,000 copies to 1 copy and used as a template.
The sensitivity when tetramethylammonium acetate was added from 0 to 300 mM was compared in real-time RT-PCR.
(2) RT-PCR reaction RT-PCR targeting MS2 RNA was performed as an index of the reaction efficiency and sensitivity of PCR.
RiverTra Ace (Toyobo) 1 unit,
RNase inhibitor (Toyobo) 1 unit,
rTth DNA Polymerase (Toyobo) 1 unit,
0.4 μg of anti-Tth antibody,
10 × Buffer (Toyobo rTth DNA Polymerase Buffer) 2 μL,
10 mM dNTPs (Toyobo) 0.4 μL,
10 μM MS2 forward primer (SEQ ID NO: 4) 0.6 μL,
10 μM MS2 probe (SEQ ID NO: 5) 0.4 μL,
10 μM MS2 reverse primer (SEQ ID NO: 6) 0.6 μL,
5 μL of RNA,
Tetramethylammonium acetate concentration for each condition,
20 μL of the reaction solution containing was reacted in the following temperature cycle.
42 ° C. 10 minutes 95 ° C. 5 minutes 95 ° C. 15 seconds-60 ° C. 45 seconds 45 cycles The conditions of tetramethylammonium acetate concentration from 0 to 300 mM (final concentration) were compared.

(3) Results The results are shown in FIG. 2, FIG. 3, and FIG. 2 and 3 summarize the Ct values for each copy number determined by real-time RT-PCR. FIG. 4 is a graph showing the logarithmic value of the copy number of MS2RNA on the x-axis and the Ct value on the y-axis. Surprisingly, as tetramethylammonium acetate is added from 0 to 100 mM, the rise of the amplification curve becomes faster (Ct value becomes smaller), and the detection sensitivity is improved. In addition, the rise of the amplification curve is the fastest at about 100 to 210 mM of tetramethylammonium acetate (Ct value is minimum), the detection sensitivity is maximized, and if it exceeds 220 mM, the reaction is inhibited and cannot be detected.

Example 3
A buffer containing 120 mM (final concentration) tetramethylammonium acetate and a buffer not containing 120 mM (final concentration) tetramethylammonium acetate were prepared. Real-time RT-PCR was performed with each prepared buffer.
(1) RNA sample Mumps virus RNA (Vircell) was used as an RNA sample. Mumps virus RNA was serially diluted by 1/4 from 6250 copies to 24 copies and used as a template.
(2) RT-PCR reaction RT-PCR targeting mumps virus RNA was performed as an index for studying the reaction efficiency and sensitivity of PCR. Compositions other than tetramethylammonium acetate are shown below.
RiverTra Ace (Toyobo) 1 unit,
RNase inhibitor (Toyobo) 1 unit,
rTth DNA Polymerase (Toyobo) 1 unit,
0.4 μg of anti-Tth antibody,
10 × Buffer (Toyobo rTth DNA Polymerase Buffer) 2 μL,
10 mM dNTPs (Toyobo) 0.4 μL,
10 μM mumps virus forward primer (SEQ ID NO: 7) 0.6 μL,
10 μM mumps virus probe (SEQ ID NO: 8) 0.6 μL,
0.4 μL of 10 μM mumps virus reverse primer (SEQ ID NO: 9),
5 μL of RNA,
20 μL of the reaction solution containing was reacted in the following temperature cycle.
50 ° C 10 minutes 95 ° C 1 minute 95 ° C 15 seconds -60 ° C 45 seconds 45 cycles

(3) Results The results are shown in FIGS. FIG. 5 is a graph in which real-time RT-PCR was performed with a buffer not containing 120 mM tetramethylammonium acetate and a buffer containing 120 mM tetramethylammonium acetate, with the Ct value on the vertical axis and the logarithm of RNA on the horizontal axis. It is. FIG. 6 is an amplification curve when real-time RT-PCR was performed with each prepared buffer. FIG. A shows a buffer without 120 mM tetramethylammonium acetate, and FIG. B shows a buffer with 120 mM tetramethylammonium acetate. A buffer containing no tetramethylammonium acetate was able to amplify up to about 100 copies. However, when tetramethylammonium acetate was added, it was surprisingly possible to detect 30 copies or less.

Example 4
Real-time RT-PCR was performed with Tth DNA polymerase and Taq DNA polymerase in a buffer containing 120 mM (final concentration) tetramethylammonium acetate.
(1) RNA sample Mumps virus RNA (Vircell) was used as an RNA sample. Mumps virus RNA was serially diluted by 1/4 from 6250 copies to 24 copies and used as a template.
(2) RT-PCR reaction RT-PCR targeting mumps virus RNA was performed as an index for studying the reaction efficiency and sensitivity of PCR. Compositions other than tetramethylammonium acetate are shown below.
RiverTra Ace (Toyobo) 1 unit,
RNase inhibitor (Toyobo) 1 unit,
rTth DNA Polymerase (Toyobo) or rTaq DNA Polymerase (Toyobo) 1 unit,
0.4 μg of anti-Taq antibody or anti-Tth antibody,
10 × Buffer (Toyobo rTth DNA Polymerase Buffer) 2 μL,
10 mM dNTPs (Toyobo) 0.4 μL,
10 μM mumps virus forward primer (SEQ ID NO: 7) 0.6 μL,
10 μM mumps virus probe (SEQ ID NO: 8) 0.6 μL,
0.4 μL of 10 μM mumps virus reverse primer (SEQ ID NO: 9),
0.3 M carnitine 4% trimethylene glycol RNA 5 μL,
20 μL of the reaction solution containing was reacted in the following temperature cycle.
50 ° C 10 minutes 95 ° C 1 minute 95 ° C 15 seconds -60 ° C 45 seconds 45 cycles

(3) Results The results are shown in FIGS. FIG. 7 is a graph in which the Ct value is plotted on the vertical axis and the logarithmic value of RNA is plotted on the horizontal axis. 8A is an amplification curve when real-time RT-PCR is performed with Tth DNA polymerase, and FIG. 8B is Taq DNA polymerase. Surprisingly, the detection sensitivity of the buffer containing tetramethylammonium acetate was improved by combination with Tth DNA polymerase. On the other hand, Taq DNA polymerase was inhibited and could not be detected.

Example 5
Buffers were prepared by adding tetramethylammonium chloride in place of 120 mM (final concentration) tetramethylammonium acetate and 120 mM (final concentration) tetramethylammonium acetate, respectively. Real-time RT-PCR was performed with each prepared buffer.
(1) RNA sample Mumps virus RNA (Vircell) was used as an RNA sample. Mumps virus RNA was serially diluted by 1/4 from 6250 copies to 24 copies and used as a template.
(2) RT-PCR reaction RT-PCR targeting mumps virus RNA was performed as an index for studying the reaction efficiency and sensitivity of PCR. Compositions other than tetramethylammonium acetate or tetramethylammonium chloride are shown below.
RiverTra Ace (Toyobo) 1 unit,
RNase inhibitor (Toyobo) 1 unit,
rTth DNA Polymerase (Toyobo) 1 unit,
0.4 μg of anti-Tth antibody,
10 × Buffer (Toyobo rTth DNA Polymerase Buffer) 2 μL,
10 mM dNTPs (Toyobo) 0.4 μL,
10 μM mumps virus forward primer (SEQ ID NO: 7) 0.6 μL,
10 μM mumps virus probe (SEQ ID NO: 8) 0.6 μL,
0.4 μL of 10 μM mumps virus reverse primer (SEQ ID NO: 9),
0.3 M carnitine 4% trimethylene glycol RNA 5 μL,
20 μL of the reaction solution containing was reacted in the following temperature cycle.
50 ° C 10 minutes 95 ° C 1 minute 95 ° C 15 seconds -60 ° C 45 seconds 45 cycles

(3) Results The results are shown in FIGS. FIG. 9 is a graph showing the Ct value on the vertical axis and the logarithmic value of RNA on the horizontal axis. FIG. 10A is an amplification curve when real-time RT-PCR is performed with tetramethylammonium acetate and B is tetramethylammonium chloride. Surprisingly, when tetramethylammonium chloride was used, the detection sensitivity was improved as in the case of tetramethylammonium acetate.

Example 6
Preparation of Taq variants Plasmids containing the modified thermostable DNA polymerase gene (SEQ ID NO: 14) derived from Thermus aquaticus, pTaq E507K, pTaq E507R, pTaq E742K, and pTaq E742R were prepared.

  The DNA template used for mutagenesis was a thermostable DNA polymerase gene (SEQ ID NO: 14) (pTaq) derived from Thermusaquaticus cloned in pBluescript. Mutation was introduced using KOD-Plus-Mutageness Kit (Toyobo) according to the instruction manual. As primers for mutagenesis, the primers described in SEQ ID NOs: 15 and 16 for pTaq E507K, the primers described in SEQ ID NOs: 17 and 18 for pTaq E507R, and the SEQ ID NOs: 19 and 20 for pTaq E742K. For the pTaq E742R, the primers described in SEQ ID NOs: 21 and 22 were used. The mutant was confirmed by decoding the base sequence. The resulting plasmid was transformed into Escherichia coli JM109 and used for enzyme preparation.

Example 7
Preparation of Tth mutants Plasmids containing modified thermostable DNA polymerase gene (SEQ ID NO: 26) derived from Thermus thermophilus, pTth Q509K, pTth Q509R, pTth E744K, and pTth E744R were prepared.

  As a DNA template used for mutagenesis, a thermostable DNA polymerase gene (SEQ ID NO: 26) (pTth) derived from Thermusthermophilus cloned in pBluescript was used. Mutation was introduced using KOD-Plus-Mutageness Kit (Toyobo) according to the instruction manual. As the primers for generating mutations, the primers described in SEQ ID NOs: 27 and 28 for pTth Q509K, the primers described in SEQ ID NOs: 29 and 30 for pTth Q509R, and the SEQ ID NOs: 31 and 32 for pTth E744K are described. Primers described in SEQ ID NOs: 33 and 34 were used for pTth E744R. The mutant was confirmed by decoding the base sequence. The resulting plasmid was transformed into Escherichia coli JM109 and used for enzyme preparation.

Example 8
Production of thermostable DNA polymerase The cells obtained in Examples 6 and 7 were cultured as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing sterilized 100 μg / ml ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia cultivated at 37 ° C. for 16 hours in 3 ml of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / ml ampicillin in advance. Coli JM109 (plasmid transformant) (using a test tube) was inoculated and cultured at 37 ° C. for 16 hours with aeration. The cells are collected from the culture solution by centrifugation, suspended in 50 ml of disruption buffer (30 mM Tris-HCl buffer (pH 8.0), 30 mM NaCl, 0.1 mM EDTA), and then subjected to sonication. By crushing, a cell lysate was obtained. Next, the cell lysate was treated at 80 ° C. for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid treatment using polyethyleneimine, ammonium sulfate precipitation, and heparin sepharose chromatography were performed. Finally, storage buffer (50 mM Tris-HCl buffer (pH 8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0 1% Tween 20, 0.1% nonidet P40, 50% glycerin) to obtain a thermostable DNA polymerase.

  The DNA polymerase activity measurement in the purification step was performed by the following operation. When the enzyme activity was high, the sample was diluted for measurement.

(reagent)
Solution A: 40 mM Tris-HCl buffer (pH 7.5), 16 mM magnesium chloride, 15 mM dithiothreitol, 100 μg / ml BSA
Solution B: 1.5 μg / μl activated calf thymus DNA
Solution C: 1.5 mM dNTP (250 cpm / pmol [3H] dTTP)
Liquid D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
Solution E: 1 mg / ml Calf thymus DNA

(Method)
Add 25 μl of solution A, 5 μl of solution B, 5 μl of solution C, and 10 μl of sterilized water to the microtube, stir and mix, add 5 μl of the purified enzyme dilution solution, and react at 75 ° C. for 10 minutes. After cooling, 50 μl of E solution and 100 μl of D solution are added, and after stirring, the mixture is ice-cooled for 10 minutes. The solution is filtered through a glass filter (Whatman GF / C filter), washed thoroughly with 0.1N hydrochloric acid and ethanol, and the radioactivity of the filter is measured using a liquid scintillation counter (Packard Tri-Carb2810 TR). The nucleotide incorporation into the template DNA was measured. One unit of enzyme activity was defined as the amount of enzyme that incorporated 10 nmol of nucleotide per 30 minutes into the acid-insoluble fraction under these conditions.

Example 9
Evaluation of resistance to tetramethylammonium salt of DNA polymerase By the above-described method for evaluating resistance to tetramethylammonium salt, modified Taq DNA polymerase, wild-type Taq DNA polymerase (Toyobo), Tth DNA polymerase obtained in Example 7 (Toyobo), Z05 DNA polymerase (Roche) was evaluated.

The results are shown in FIG. In the wild type Taq DNA polymerase, inhibition was caused by addition of 50 mM tetramethylammonium acetate, and amplification was not observed. On the other hand, the modified Taq DNA polymerase, Tth DNA polymerase, and Z05 DNA polymerase did not inhibit amplification even when 200 mM tetramethylammonium acetate was added. It can be seen that the modification can make the tetramethylammonium salt resistant. It can also be seen that Tth DNA polymerase and Z05 DNA polymerase are resistant to tetramethylammonium salts. FIG. 13 shows the identity of the amino acid sequences of Tth DNA polymerase and Z05 DNA polymerase. Tth DNA polymerase and Z05 DNA polymerase have an identity of 95% or more, and it is considered that the same performance was obtained from this high identity.

Similar tetraammonium salt resistance evaluation was performed with modified Tth DNA polymerase. As a result, even in the case of Q509K, Q509R, E744K, and E744R mutants of Tth DNA polymerase, addition of 200 mM tetramethylammonium acetate did not inhibit amplification as in the wild type.

Example 10
Real-time PCR was carried out using Taq DNA polymerase and modified Taq DNA polymerase (Taq E507K, Taq E507R) with blood as a template in a buffer containing 120 mM (final concentration) tetramethylammonium acetate.
(1) 1 μl of blood was used as a template.
(2) PCR reaction The composition other than tetramethylammonium acetate is shown below.
Taq DNA Polymerase (Toyobo) or modified Taq DNA Polymerase 1 unit,
Anti-Taq antibody 0.4 μg,
10 × Buffer (Toyobo rTth DNA Polymerase Buffer) 2 μL,
10 mM dNTPs (Toyobo) 0.4 μL,
0.6 μL of 10 μM primer (SEQ ID NO: 23),
10 μM probe (SEQ ID NO: 24) 0.6 μL,
0.4 μL of 10 μM primer (SEQ ID NO: 25),
0.3 M carnitine 4% trimethylene glycol blood 1 μL,
20 μL of the reaction solution containing was reacted in the following temperature cycle.
95 ° C 1 minute 95 ° C 15 seconds-60 ° C 60 seconds 40 cycles

(3) Results The results are shown in FIG. FIG. 12 shows amplification curves performed with each enzyme. Although no amplification was observed with Taq DNA polymerase, rising was observed with modified Taq DNA polymerases (E507K, E742K), indicating that amplification was possible directly from blood without purification. Similar results were obtained with E507R and E742R. The efficiency of PCR is improved by the effect of the tetramethylammonium salt, and it is considered that PCR was successful without receiving blood inhibition.

Similar evaluation was performed using wild type Tth DNA polymerase and modified Tth DNA polymerase (Q509K, Q509R, E744K, E744R) instead of Taq DNA polymerase. As a result, Tth DNA polymerase showed rise in both wild type and modified type, indicating that it can be amplified directly from blood without purification. If the tetramethylammonium salt is resistant, not only wild-type but also modified DNA polymerase can be amplified from blood.

Taq DNA polymerase, which has been used for general purposes until now, has a greater inhibition effect of tetramethylammonium salt, and the effect of promoting PCR could not be sufficiently observed. It was found that by using a DNA polymerase resistant to tetramethylammonium salt, the PCR promoting effect of the tetramethylammonium salt can be sufficiently exerted, and direct PCR from blood, which was normally considered impossible, can also be realized.

The present invention provides a composition useful in the field of molecular biology, particularly a composition useful in the production of DNA from a template nucleic acid and further DNA amplification. According to the present invention, there is no reaction inhibition in nucleic acid amplification, the operability is not significantly reduced, and the reaction can be carried out at low cost. In particular, it is more convenient for use in mixing with a premix reagent for nucleic acid amplification reaction. High reagent form can be supplied. The present invention is particularly useful for gene expression analysis, and can be used not only for research purposes but also for clinical diagnosis and environmental testing.

Claims (26)

A nucleic acid amplification composition comprising a tetramethylammonium salt having a final concentration of 50 mM or more and a tetramethylammonium resistant DNA polymerase. The nucleic acid amplification composition according to claim 1, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase that is not inhibited in the presence of a tetramethylammonium salt having a final concentration of 50 mM or more. The nucleic acid amplification composition according to claim 1 or 2, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase having 90% or more identity with SEQ ID NO: 10. The nucleic acid amplification composition according to claim 1 or 2, wherein the tetramethylammonium resistant DNA polymerase is Tth DNA polymerase. The nucleic acid amplification composition according to claim 1 or 2, wherein the tetramethylammonium resistant DNA polymerase is Z05 DNA polymerase. The nucleic acid amplification composition according to claim 1 or 2, wherein the tetramethylammonium resistant DNA polymerase is a variant of SEQ ID NO: 11. The composition for nucleic acid amplification according to claim 6, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase in which at least E507 position or E742 position of SEQ ID NO: 11 is modified. The nucleic acid amplification product according to any one of claims 1 to 7, wherein the tetramethylammonium salt is at least one compound selected from the group consisting of tetramethylammonium acetate, tetramethylammonium chloride, and tetramethylammonium hydroxide. Composition. The composition for nucleic acid amplification according to any one of claims 1 to 8, wherein the final concentration of the tetramethylammonium salt is 100 to 210 mM. The composition for nucleic acid amplification according to any one of claims 1 to 9, further comprising at least an antibody, a reaction buffer and a metal ion. The composition for nucleic acid amplification according to any one of claims 1 to 10, comprising at least a reverse transcriptase, an antibody, a reaction buffer, and a metal ion. The nucleic acid amplification composition according to any one of claims 1 to 11, wherein the nucleic acid amplification reaction is PCR. The nucleic acid amplification composition according to any one of claims 1 to 12, wherein the nucleic acid amplification reaction is one-step RT-PCR. A nucleic acid amplification method comprising a tetramethylammonium salt and a DNA polymerase having a tetramethylammonium resistance having a final concentration of 50 mM or more. The nucleic acid amplification method according to claim 14, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase that is not inhibited in the presence of 50 mM tetramethylammonium salt. The nucleic acid amplification method according to claim 14 or 15, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase having at least 90% identity to SEQ ID NO: 10. The nucleic acid amplification method according to any one of claims 14 to 16, wherein the tetramethylammonium resistant DNA polymerase is Tth DNA polymerase. The nucleic acid amplification method according to any one of claims 14 to 17, wherein the tetramethylammonium resistant DNA polymerase is Z05 DNA polymerase. The nucleic acid amplification method according to any one of claims 14 to 18, wherein the tetramethylammonium resistant DNA polymerase is a variant of SEQ ID NO: 11. The nucleic acid amplification method according to any one of claims 14 to 19, wherein the tetramethylammonium resistant DNA polymerase is a DNA polymerase in which at least the E507 position or the E742 position of SEQ ID NO: 11 is modified. The nucleic acid amplification method according to any one of claims 14 to 20, wherein the tetramethylammonium salt is at least one compound selected from the group consisting of tetramethylammonium acetate, tetramethylammonium chloride, and tetramethylammonium hydroxide. . The nucleic acid amplification method according to any one of claims 14 to 21, wherein the final concentration of the tetramethylammonium salt is 100 to 210 mM. The nucleic acid amplification method according to any one of claims 14 to 22, comprising at least an antibody, a reaction buffer and a metal ion. The nucleic acid amplification method according to any one of claims 14 to 23, comprising at least a reverse transcriptase, an antibody, a reaction buffer, and a metal ion. The nucleic acid amplification method according to any one of claims 14 to 24, wherein the nucleic acid amplification reaction is PCR. The nucleic acid amplification method according to any one of claims 14 to 25, wherein the nucleic acid amplification reaction is one-step RT-PCR.