JP7709262B2 - Modified DNA polymerase - Google Patents

Description

The present invention relates to a mutant of DNA polymerase having reverse transcription activity used in polymerase chain reaction (PCR) and the like. The present invention can be used not only in the research field but also in clinical diagnosis, environmental testing, and the like.

Nucleic acid amplification is a technique that amplifies a few copies of a target nucleic acid to a level where it can be visualized, i.e., hundreds of millions of copies or more. It is widely used not only in the field of life science research, but also in the medical field, such as genetic diagnosis and clinical testing, and in microbial testing in food and the environment.

A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR is a method that amplifies a target nucleic acid in a sample by repeating three cycles: (1) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) extension of the primer using DNA polymerase.

When the nucleic acid to be detected is RNA, for example when the target is an RNA virus in the detection of pathogenic microorganisms, or when the expression level of a gene is measured by quantifying mRNA, RT-PCR is also widely used, in which a reaction to convert RNA to cDNA using reverse transcriptase (reverse transcription reaction) is carried out before PCR.

In RT-PCR, two types of enzymes, reverse transcriptase and DNA polymerase, are generally used. However, some DNA polymerases also have reverse transcription activity, and in recent years, such DNA polymerases with reverse transcription activity are sometimes used. Examples of DNA polymerases with reverse transcription activity include DNA polymerase (Tth) derived from Thermus thermophilus HB8, DNA polymerase (Z05) derived from Themus sp Z05, and DNA polymerase (Tma) derived from Thermotoga maritima. However, the reverse transcription activity of these DNA polymerases is not high, and further improvement has been required. In addition, due to the low reverse transcription activity, the reaction time for reverse transcription in RT-PCR using these DNA polymerases generally requires about 20 minutes. In situations where rapid testing or diagnosis is required, it is desirable to shorten the reaction time, and there has been a demand for improved reverse transcription activity.

Various mutants of DNA polymerases with reverse transcription activity have been investigated to date (Patent Documents 1, 2, and 3). However, even these mutants may not be sufficient to carry out the reaction efficiently, and there is a need to develop polymerases with even higher performance.

Patent No. 5189101 International Publication No. 2010/050418 International Publication No. 2018/096961

The present invention was made in consideration of the above-mentioned conventional techniques, and aims to provide a DNA polymerase that has reverse transcription activity and high amplification efficiency.

In view of the above problems, the inventors conducted extensive research and discovered that efficient amplification was possible by modifying amino acids at specific sites in DNA polymerase derived from Thermus thermophilus, thus arriving at the present invention.

That is, the present invention mainly comprises the following components.
[Item 1] A DNA polymerase having reverse transcription activity and consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, characterized in that the amino acid at the site corresponding to position 751 is modified.
[Item 2] A DNA polymerase having reverse transcription activity and characterized in that the amino acid at the site corresponding to position 751 in the amino acid sequence of SEQ ID NO: 1 is modified by deleting, substituting, and/or adding one or several amino acids.
[Item 3] A DNA polymerase having reverse transcription activity and comprising the amino acid sequence of SEQ ID NO:1, wherein the amino acid at the site corresponding to position 751 has been modified.
[Item 4] The DNA polymerase according to any one of Items 1 to 3, wherein the amino acid modification at the site corresponding to position 751 is a substitution with a neutral amino acid having a polar side chain selected from the group consisting of tyrosine, cysteine, glutamine, serine, threonine, and asparagine.
[Item 5] The DNA polymerase according to any one of Items 1 to 4, wherein the amino acid modification at the site corresponding to position 751 is a substitution with tyrosine.
[Item 6] The DNA polymerase according to any one of Items 1 to 5, wherein the reverse transcription reaction is completed in 5 minutes or less.
[Item 7] The DNA polymerase according to any one of Items 1 to 6, wherein the reverse transcription reaction is completed in 1 minute or less.
[Item 8] The DNA polymerase according to any one of Items 1 to 7, further comprising an amino acid modification at the site corresponding to position 509.
[Item 9] The DNA polymerase according to Item 8, wherein the amino acid modification at the site corresponding to position 509 is a substitution with a basic amino acid selected from the group consisting of histidine, lysine and arginine.
[Item 10] The DNA polymerase according to Item 8 or 9, wherein the amino acid modification at the site corresponding to position 509 is a substitution with arginine.
[Item 11] A nucleic acid amplification reagent comprising the DNA polymerase according to any one of Items 1 to 10.
[Item 12] The nucleic acid amplification reagent according to Item 11, which is used for amplifying nucleic acid from RNA.
[Item 13] The nucleic acid amplification reagent according to Item 11 or 12, which is used in an RT-PCR method.
[Item 14] A kit for nucleic acid amplification comprising the nucleic acid amplification reagent according to any one of Items 11 to 13.
[Item 15] A method for amplifying nucleic acid, comprising using the DNA polymerase according to any one of Items 1 to 10, the reagent for nucleic acid amplification according to any one of Items 11 to 13, or the kit for nucleic acid amplification according to Item 14.
[Item 16] The nucleic acid amplification method according to Item 15, wherein RNA is used as the nucleic acid to be detected.
[Item 17] The nucleic acid amplification method according to Item 15 or 16, wherein the reverse transcription reaction time is 5 minutes or less.
[Item 18] The nucleic acid amplification method according to any one of Items 15 to 17, wherein the reverse transcription reaction time is 1 minute or less.
[Item 19] The method for amplifying nucleic acid according to any one of Items 15 to 18, which comprises a step of carrying out an RT-PCR reaction.

The present invention provides a novel DNA polymerase that has reverse transcription activity and has improved amplification efficiency. By using the DNA polymerase of the present invention, it becomes possible to efficiently amplify nucleic acids from RNA.

The present invention is described in detail below, but is not limited to these.

The present invention relates to a modified DNA polymerase having reverse transcription activity. A DNA polymerase having reverse transcription activity is a DNA polymerase that has both the ability to convert RNA to cDNA (also called "reverse transcription activity" (RT activity)) and the ability to amplify DNA (also called "DNA polymerase activity"). A DNA polymerase refers to an enzyme that uses a single-stranded nucleic acid as a template to synthesize a DNA strand having a complementary base sequence. The presence or absence of reverse transcription activity can be determined, for example, by whether or not a nucleic acid amplification reaction is established in RT-PCR using RNA as a template, and specifically, can be measured by the procedure described in the method for evaluating reverse transcription activity (RT activity) described below. The presence or absence of DNA polymerase activity can be measured according to the method described in the method for measuring DNA polymerase activity described below.

In a specific embodiment, the present invention provides a mutant DNA polymerase derived from Thermus thermophilus, which has an amino acid modification at a site corresponding to position 751. The full-length amino acid sequence of this DNA polymerase derived from Thermus thermophilus is shown in SEQ ID NO: 1. The gene sequence derived from Thermus thermophilus that encodes the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

In one embodiment, the DNA polymerase of the present invention is characterized in that it is a modified DNA polymerase that contains a mutation (preferably a substitution with another amino acid) in the amino acid at the site corresponding to position 751 in the protein consisting of the amino acid sequence shown in SEQ ID NO: 1. The amino acid sequence before modification is not limited to being completely identical to SEQ ID NO: 1, and is not particularly limited as long as the reverse transcription activity and DNA polymerase activity are maintained. For example, it is preferable to have an amino acid sequence that has an identity of 90% or more, preferably 95% or more, more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and especially 99% or more with the amino acid sequence described in SEQ ID NO: 1. Furthermore, the amino acid sequence before modification may be an amino acid sequence in which one or several amino acids are deleted, substituted, and/or added in the amino acid sequence described in SEQ ID NO: 1. Here, "one or several" is not particularly limited as long as the reverse transcription activity and DNA polymerase activity are maintained, and is, for example, 1 to 20, preferably 1 to 10, and more preferably 1 to 5. The amino acid sequence before modification as described above may be, for example, one artificially produced by genetic engineering techniques, or may be the amino acid sequence of a naturally derived protein. Such naturally derived amino acid sequences are not particularly limited, but include, for example, DNA polymerase (Tth) derived from Thermus thermophilus HB8, DNA polymerase (Z05) derived from Themus sp Z05, and DNA polymerase (Tma) derived from Thermotoga maritima. Preferably, it is an amino acid sequence derived from Tth or Z05, and among them, an amino acid sequence derived from Tth is particularly preferred.

In a specific embodiment, the modified DNA polymerase of the present invention has an amino acid modification at a site corresponding to phenylalanine at position 751 (F751) in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having a specific relationship with SEQ ID NO: 1 as described above. The modified DNA polymerase of the present invention may contain any amino acid modification at other amino acid sites in addition to the amino acid modification at the site corresponding to F751, as long as the effect of the present invention is achieved. From the viewpoint of being able to perform nucleic acid amplification from RNA more reliably and efficiently, it is preferable that the modified DNA polymerase further has an amino acid modification at a site corresponding to glutamine at position 509 (position Q509) in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having a specific relationship with SEQ ID NO: 1 as described above.

In this specification, simplified symbols using alphabetical notation may be used for base sequences, amino acid sequences, and their individual components, but all follow the conventions in the fields of molecular biology and genetic engineering. In this specification, in order to simply indicate mutations in amino acid sequences, notations such as "F751Y" are used. "F751Y" indicates that the 751st phenylalanine has been replaced with tyrosine, that is, it indicates the type of amino acid residue before the replacement, its location, and the type of amino acid residue after the replacement. In addition, unless otherwise specified, the sequence number corresponds to the sequence number listed in the sequence table. In addition, in the case of multiple mutants, the above notations are connected with "/". For example, "Q509R/F751Y" indicates that the 509th glutamine has been replaced with arginine, and the 751st phenylalanine has been replaced with tyrosine. In this specification, a site in an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO:1 that corresponds to a certain position (order) in SEQ ID NO:1 refers to a position that corresponds to the position in SEQ ID NO:1 when the primary structures of the sequences are compared (aligned).

In addition, in this specification, "mutant" or "modified" in the context of "mutant DNA polymerase" or "modified DNA polymerase" means that the polymerase has an amino acid sequence different from that of conventionally known DNA polymerases, and does not distinguish between artificial mutations and mutations occurring in nature.

In a particular preferred embodiment, the DNA polymerase of the present invention is one in which the amino acid at position 751 in the amino acid sequence of SEQ ID NO: 1 or the like has been modified to a neutral amino acid. Preferably, the amino acid at position 751 has been substituted with tyrosine, cysteine, glutamine, serine, threonine, asparagine, or tryptophan, and more preferably, tyrosine, cysteine, glutamine, serine, threonine, or asparagine. Tyrosine, cysteine, glutamine, serine, threonine, and asparagine are all known as amino acids with polar neutral side chains, and their isoelectric points are close at about 5.0 to about 5.7, and it is expected that they will exert similar effects as amino acids that can exhibit common properties.

In a further preferred embodiment, the modified DNA polymerase of the present invention is an amino acid sequence of SEQ ID NO: 1, etc., in which the amino acid at the position corresponding to position 509 is further modified to a basic amino acid. Preferably, the amino acid at the position corresponding to position 509 is substituted with arginine, lysine, or histidine. For example, the modified DNA polymerase of the present invention may be an amino acid at the position corresponding to position 509 substituted with arginine or lysine. It is known that the isoelectric point of basic amino acids is about 10.8 for arginine, about 9.7 for lysine, and about 7.6 for histidine. In other words, since all of these basic amino acids have high isoelectric points, it can be expected that they will exert the same effects as amino acids that can exhibit common properties.

A conventional method can be used to produce the modified DNA polymerase of the present invention. Preferably, a method is used in which a mutation is introduced into a gene encoding a wild-type DNA polymerase, and a mutant (modified) DNA polymerase having a new function is produced by protein engineering techniques.

As one embodiment of a method for introducing amino acid modifications, a site-specific mutagenesis method based on the inverse PCR method can be used. For example, the KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) is a kit that can obtain a transformant carrying a plasmid with a target mutation by (1) denaturing a plasmid into which a target gene has been inserted, annealing a mutation primer to the plasmid, and then performing an extension reaction using KOD DNA polymerase, (2) repeating the cycle of (1) 15 times, (3) selectively cutting only the template plasmid using the restriction enzyme DpnI, (4) phosphorylating the newly synthesized gene and carrying out ligation to cyclize it, and (5) transforming the cyclized gene into E. coli. This kit can be suitably used for producing the modified DNA polymerase of the present invention.

The DNA polymerase of the present invention may be in the form of a fusion protein of Sso7d or PCNA. It may also be a fusion protein to which a protein tag such as a His tag or a GST tag has been added.

The above DNA polymerase gene is transferred to an expression vector as necessary, and a host such as E. coli is transformed with the expression vector, and then spread onto an agar medium containing a drug such as ampicillin to form colonies. The colonies are inoculated onto a nutrient medium such as LB medium or 2xYT medium, and cultured at 37°C for 12 to 20 hours, after which the cells are disrupted and a crude enzyme solution is extracted. A vector derived from pBluescript is preferred. Any known method may be used to disrupt the cells, but for example, ultrasonic treatment, physical disruption methods such as French press or glass bead disruption, or lytic enzymes such as lysozyme can be used. The crude enzyme solution is heat-treated at 80°C for 30 minutes to inactivate the polymerase derived from the host, and the DNA polymerase activity is measured.

Any method may be used to obtain purified DNA polymerase from the strain selected by the above method, but examples include the following method. After culturing in a nutrient medium and recovering the bacterial cells, the cells are disrupted and extracted by enzymatic or physical disruption to obtain a crude enzyme solution. The crude enzyme extract obtained is heat-treated, for example at 80°C for 30 minutes, and then the DNA polymerase fraction is recovered by ammonium sulfate precipitation. This crude enzyme solution can be desalted by gel filtration using Sephadex G-25 (Amersham Pharmacia Biotech). After this operation, the crude enzyme solution is separated and purified by heparin Sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified to the extent that it shows an almost single band by SDS-PAGE.

Specifically, whether or not the modified DNA polymerase of the present invention has reverse transcription activity and DNA polymerase activity can be measured by the following method.

[Method of evaluating reverse transcription activity (RT activity)]
The DNA polymerase of the present invention has superior reverse transcription activity compared to conventional ones, and may be capable of performing reverse transcription reaction in a short time. Specifically, although not particularly limited, when the total length of the target RNA is, for example, 50 to 300 bp, preferably 150 to 250 bp, the reverse transcription reaction from the target RNA is completed in 5 minutes or less, preferably 3 minutes or less, more preferably 1 minute or less. Here, in the present invention, the completion of the reverse transcription reaction is defined as follows. That is, the condition for the RT-PCR reaction to be established is that the PCR efficiency is within 70 to 130% and the correlation coefficient r2 is 0.97 or more. The PCR efficiency can be calculated according to the following formula (1) from the slope of a calibration curve obtained by plotting the results with the amount of serially diluted nucleic acid on the X-axis and the Ct corresponding to the amount of nucleic acid on the Y-axis.
PCR efficiency (%) = (10 ^−1/Slope −1) × 100 ... Formula (1)
The correlation coefficient r2 represents the linearity of the calibration curve. In this manner, the reverse transcription reaction is considered to be complete when PCR is possible after the reverse transcription reaction.
Since the reverse transcription reaction is completed in such a short time, the time required for the entire nucleic acid amplification method using RNA as a template (e.g., RT-PCR method) can be shortened, which is extremely beneficial when rapid testing or diagnosis is required.

[Method for measuring DNA polymerase activity]
In the present invention, the activity of purified DNA polymerase is measured by the following method. When the enzyme activity is strong, the sample is diluted with a storage buffer (50 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM dithiothreitol, 0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin) and then measured. (1) 25 μl of solution A, 5 μl of solution B, 5 μl of solution C, 10 μl of sterile water, and 5 μl of enzyme solution are added to a microtube and reacted at 75° C. for 10 minutes. (2) After that, the mixture is cooled on ice, 50 μl of solution E and 100 μl of solution D are added, stirred, and cooled on ice for another 10 minutes. (3) This solution is filtered through a glass filter (Whatman GF/C filter) and thoroughly washed with 0.1 N hydrochloric acid and ethanol. (4) The radioactivity of the filter is measured using a liquid scintillation counter (Tri-Carb 2810 TR manufactured by Packard) to measure the incorporation of nucleotides into the template DNA. One unit of enzyme activity is defined as the amount of enzyme that incorporates 10 nmol of nucleotides into the acid-insoluble fraction (i.e., the fraction that becomes insoluble when Solution D is added) per 30 minutes under these conditions.
Solution A: 40 mM Tris-HCl buffer (pH 7.5), 16 mM magnesium chloride, 15 mM dithiothreitol, 100 μg/ml BSA (bovine serum albumin)
Solution B: 1.5μg/μl activated calf thymus DNA
Solution C: 1.5mM dNTP (250cpm/pmol [3H]dTTP)
Solution D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)
Solution E: 1 mg/ml calf thymus DNA

The modified DNA polymerase of the present invention can be used in any nucleic acid amplification method known in the art. The conditions for nucleic acid amplification, such as temperature, time, and reaction cycle, vary depending on the type of nucleic acid to be amplified, the base sequence, the chain length, etc., but can be appropriately set by a person skilled in the art. As an example, when PCR or RT-PCR is performed as a nucleic acid amplification method using the modified DNA polymerase of the present invention, the extension time may be 30 seconds or less per 1 kb. The modified DNA polymerase of the present invention may be capable of performing a sufficient nucleic acid amplification reaction even with such a short extension time. Usually, in PCR or RT-PCR, one cycle consists of three steps: (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) extension of the primer using a DNA polymerase, and this cycle is repeated. The extension time in the present invention refers to the time required for one cycle of the reaction (3) to extend the primer. In addition, in PCR and RT-PCR, the above (2) annealing and (3) primer extension may be performed in two steps at the same temperature. In this case, for convenience, the extension time of the present invention refers to the time during which (2) annealing and (3) extension are performed in parallel.

In a particular embodiment, the modified DNA polymerase of the present invention preferably has a level of heat resistance that allows it to function sufficiently even at high temperatures in a PCR reaction cycle. For example, it is preferable that the modified DNA polymerase has a level of heat resistance that does not reduce the enzyme activity by more than half even when heat treatment is performed at 85°C for 1 minute or more. For example, the reaction temperature used in RT-PCR is not particularly limited, but examples include PCR reaction cycle conditions in which a reverse transcription reaction is performed at 40 to 80°C, followed by thermal denaturation at 90 to 100°C and association and extension reaction at 40 to 80°C, and preferably PCR reaction cycle conditions in which a reverse transcription reaction is performed at 50 to 65°C, followed by thermal denaturation at 94 to 98°C and association and extension reaction at 55 to 65°C (for example, PCR reaction cycle conditions in which a reverse transcription reaction is performed at 60°C, followed by thermal denaturation at 95°C and association and extension reaction at 60°C) can be exemplified. The modified DNA polymerase of the present invention maintains good activity in such a temperature range and can effectively amplify nucleic acids.

The modified DNA polymerase of the present invention can be applied not only to nucleic acid amplification methods from RNA that require reverse transcription (e.g., RT-PCR), but also to nucleic acid amplification methods that use DNA as a template (e.g., PCR). In such RT-PCR and/or PCR methods, for example, at least one type of primer, dNTP (deoxyribonucleotide triphosphate), is reacted to extend the primer, synthesizing a DNA primer extension product. Specifically, it can be applied to primer extension methods, sequencing methods, conventional methods that do not involve temperature cycling, cycle sequencing methods, etc.

As a further embodiment, the present invention provides a nucleic acid amplification reagent containing the modified DNA polymerase as described above. This nucleic acid amplification reagent can be used in any nucleic acid amplification reaction, but since it contains a DNA polymerase that has not only DNA polymerase activity but also reverse transcription activity, it can be used, for example, for amplifying nucleic acid from RNA, and preferably can be used in the RT-PCR method. Examples of RT-PCR include, but are not limited to, RT-PCR and qRT-PCR. In other words, the nucleic acid amplification reagent of the present invention can amplify nucleic acid regardless of whether the target nucleic acid (nucleic acid to be detected) used as a template is DNA or RNA, making it a highly versatile nucleic acid amplification reagent. The amount of the mutant DNA polymerase in the nucleic acid amplification reagent of the present invention is not limited as long as the effects of the present invention are achieved, but an example of an amount that results in a final concentration in the nucleic acid amplification reaction of 0.1 to 20 U/20 μl can be given.

An example of a nucleic acid amplification reagent includes two types of primers, one of which is complementary to the DNA extension product of the other primer, dNTP, and the DNA polymerase of the present invention as described above, divalent ions, monovalent ions, and a buffer solution, and more specifically, it can include two types of primers, one of which is complementary to the DNA extension product of the other primer, dNTP, and the above-mentioned DNA polymerase, magnesium ions and/or manganese ions, ammonium ions and/or potassium ions, BSA, the above-mentioned nonionic surfactant, and a buffer solution. When used as a reagent for RT-PCR, it is preferable to include, but is not limited to, a salt such as manganese salt and/or magnesium salt having a final concentration of 1 mM or more in the reaction solution. Examples of manganese salts include manganese chloride, manganese sulfate, manganese acetate, etc. Examples of magnesium salts include magnesium chloride, magnesium sulfate, magnesium acetate, and other magnesium salts.

Another embodiment of the nucleic acid amplification reagent is a nucleic acid amplification reagent that includes two types of primers, one of which is complementary to the DNA extension product of the other primer, dNTPs, the DNA polymerase of the present invention as described above, divalent ions, monovalent ions, a buffer solution, and, if necessary, an antibody having activity to inhibit the polymerase activity and/or 3'-5' exonuclease activity of the heat-stable DNA polymerase. Examples of the antibody include monoclonal antibodies and polyclonal antibodies. This nucleic acid amplification reagent is particularly effective in increasing the sensitivity of PCR and reducing non-specific amplification.

Yet another aspect of the present invention may be a kit containing the above-mentioned nucleic acid amplification reagent. Specifically, this kit may be used for nucleic acid amplification using DNA and/or RNA as a template. Since the kit contains a DNA polymerase with improved reverse transcription activity, it may also be used for nucleic acid amplification reactions using RNA as a template, and may be suitably used, for example, in RT-PCR methods. The kit may also include an attached document or the like that describes the procedure of the nucleic acid amplification method of the present invention.

In a further embodiment, the present invention provides a method for amplifying nucleic acid using the modified DNA polymerase of the present invention as described above, a nucleic acid amplification reagent containing the DNA polymerase, or a nucleic acid amplification kit containing the nucleic acid amplification reagent. These DNA polymerases, nucleic acid amplification reagents, and kits can exhibit excellent reverse transcription activity and high amplification efficiency, and therefore can be suitably used not only in a method for amplifying nucleic acid from DNA, but also in a method for amplifying nucleic acid using RNA as a template. Thus, the nucleic acid amplification method of the present invention can include, for example, a step of converting RNA to cDNA (also referred to as a reverse transcription reaction step), and can further include a step of performing an RT-PCR reaction. The reverse transcription reaction step can be performed by coexisting the modified DNA polymerase of the present invention with template RNA under predetermined conditions (for example, at 50 to 65°C for 1 to 30 minutes).

The present invention will be described in more detail below based on examples. Note that the present invention is not particularly limited to the examples.

Example 1
Preparation of DNA polymerase plasmid The artificially synthesized DNA polymerase gene (SEQ ID NO: 2) derived from Thermus thermophilus HB8 was cloned into pBluescript to prepare a plasmid incorporating wild-type Tth DNA polymerase (pTth). The plasmid with the mutation was prepared using pTth as a template and KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) according to the instruction manual. The double mutation was prepared by using the prepared mutant plasmid as a template and further introducing a mutation using the same kit. The template and primers used for the preparation of the plasmid are shown in Table 1. The obtained plasmid was transformed into Escherichia coli JM109 and used for enzyme preparation.

Example 2
Preparation of DNA polymerase The cells obtained in Example 1 were cultured as follows. First, 80 mL of sterilized TB medium (Molecular cloning 2nd edition, p.A.2) containing 100 μg/ml ampicillin was dispensed into a 500 mL Sakaguchi flask. Escherichia coli JM109 (plasmid transformed strain) (test tube used) previously cultured in 3 ml of LB medium (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride; Gibco) containing 100 μg/ml ampicillin at 37° C. for 16 hours was inoculated into this medium, and aerated culture was performed at 37° C. for 16 hours. The cells were collected from the culture 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 disrupted by sonication to obtain a cell disruption solution. The cell disruption solution was then treated at 80°C for 15 minutes, and the insoluble fraction was removed by centrifugation. Further, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and heparin sepharose chromatography were performed, and finally, the buffer was replaced with a 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 modified DNA polymerase.

The DNA polymerase activity measurement in the above purification process was performed as follows. In addition, if the enzyme activity was high, the sample was diluted and then measured.

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

(method)
25 μl of solution A, 5 μl of solution B, 5 μl of solution C and 10 μl of sterile water were added to a microtube and mixed with stirring, then 5 μl of the above purified enzyme dilution solution was added and reacted at 75 ° C for 10 minutes. After cooling, 50 μl of solution E and 100 μl of solution D were added, stirred and further cooled on ice for 10 minutes. This solution was filtered through a glass filter (Whatman GF/C filter), thoroughly washed with 0.1 N hydrochloric acid and ethanol, and the radioactivity of the filter was measured using a liquid scintillation counter (Packard Tri-Carb 2810 TR) to measure the incorporation of nucleotides into the template DNA. One unit of enzyme activity was defined as the amount of enzyme that incorporates 10 nmol of nucleotides into the acid-insoluble fraction per 30 minutes under these conditions.

The results of the above measurements confirmed that the modified DNA polymerase of the present invention has sufficient DNA polymerase activity.

Example 3
Evaluation of amplification efficiency by RT-PCR method (reverse transcription reaction time: 5 minutes)
One-step RT-PCR was carried out from RNA using the DNA polymerase prepared in Example 2. For RT-PCR, the buffer attached to Tth DNA Polymerase RT-PCR Buffer ( manufactured by Roche) was used, and enterovirus RNA of unknown concentration was added to 20 μl of reaction solution containing 1×PCR Buffer, 0.4 mM dNTPs, 4 pmol of primers (SEQ ID NO: 7 and 8) that amplify enterovirus RNA (approximately 196 bp), 4 pmol of Taqman probe (SEQ ID NO: 9), and 1 U of enzyme, and a pre-reaction was carried out at 90° C. for 30 seconds, followed by a reverse transcription reaction at 60° C. for 5 minutes, followed by PCR using Step One (manufactured by Applied Biosystem) with a schedule of 95° C. for 5 seconds → 60° C. for 10 seconds repeated for 45 cycles. The enzymes used were the conventionally known Tth DNA polymerase (Q509R) that does not have an amino acid modification at the site corresponding to position 751, and the Tth NA polymerase of the present invention (Q509R/F751Y) that has an amino acid modification at the site corresponding to position 751.

Table 2 shows the results of the RT-PCR Cq values. The smaller the Cq value, the higher the PCR amplification efficiency. As shown in the results in Table 2, the Cq value of the modified Tth DNA polymerase of the present invention is clearly smaller than that of the conventionally known Tth DNA polymerase that does not have an amino acid modification at the site corresponding to position 751, confirming that the amplification efficiency is very high. Therefore, it was demonstrated that the modified DNA polymerase of the present invention has high amplification sensitivity in a nucleic acid amplification method including a reverse transcription reaction using RNA as a template by having an amino acid modification at position 751.

Example 4
Evaluation of reverse transcription efficiency in RT-PCR (reverse transcription reaction time: 5 minutes, 1 minute)
Using the modified DNA polymerase of the present invention (Q509R/F751Y) prepared in Example 2, one-step RT-PCR was carried out from RNA. For RT-PCR, the buffer attached to Tth DNA Polymerase RT-PCR Buffer ( manufactured by Roche) was used, and RNA was added to 20 μl of reaction solution containing 1×PCR Buffer, 0.4 mM dNTPs, 4 pmol of primers (SEQ ID NO: 10 and 11) that amplify human β-globin (about 188 bp), 4 pmol of Taqman probe (SEQ ID NO: 12), and 1 U of enzyme so that the amounts were 500 ng, 50 ng, 5 ng, and 0.5 ng. After a pre-reaction at 90° C. for 30 seconds, a reverse transcription reaction was carried out at 60° C. for 1 minute or 5 minutes, followed by PCR using Step-OnePlus (manufactured by Applied Biosystem) with a schedule of 45 cycles of →95° C. for 15 seconds →60° C. for 1 minute. The enzyme used was the modified Tth DNA polymerase of the present invention (Q509R/F751Y) having an amino acid modification at the site corresponding to position 751.

Table 3 (when the reverse transcription reaction time is 5 minutes) and Table 4 (when the reverse transcription reaction time is 1 minute) show the results of summarizing the Cq values and PCR efficiency in RT-PCR. As a result, it was revealed that the modified Tth DNA polymerase of the present invention shows almost no change in Cq value between the reverse transcription reaction of 5 minutes (Table 3) and the reverse transcription reaction of 1 minute (Table 4), and satisfies the PCR efficiency conditions in both cases. Therefore, it was found that by using the modified DNA polymerase of the present invention into which the F751Y mutation has been introduced, sufficient RT-PCR can be performed even with a short reverse transcription reaction time, and nucleic acid amplification from RNA can be efficiently performed.

The present invention provides a modified DNA polymerase and a composition thereof that are useful in the field of molecular biology. In addition, the present invention can significantly reduce the time required for a nucleic acid amplification reaction using RNA as a template. The present invention is particularly useful in gene expression analysis, and can be used not only for research purposes but also for clinical diagnosis, environmental testing, etc.

Claims (18)

A DNA polymerase having reverse transcription activity and consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:1, characterized in that the amino acid at the position corresponding to position 751 is replaced with a neutral amino acid having a polar side chain selected from the group consisting of tyrosine , glutamine , and asparagine. A DNA polymerase having reverse transcription activity, characterized in that in an amino acid sequence in which one or more amino acids are deleted, substituted, and/or added in the amino acid sequence of SEQ ID NO: 1, the amino acid at position 751 is replaced with a neutral amino acid having a polar side chain selected from the group consisting of tyrosine, glutamine , and asparagine. A DNA polymerase having reverse transcription activity and consisting of the amino acid sequence of SEQ ID NO:1, characterized in that the amino acid at the position corresponding to position 751 is replaced with a neutral amino acid having a polar side chain selected from the group consisting of tyrosine , glutamine , and asparagine. The DNA polymerase according to any one of claims 1 to 3, wherein the modification of the amino acid at the site corresponding to position 751 is a substitution with tyrosine. The DNA polymerase according to any one of claims 1 to 4, in which the reverse transcription reaction is completed in 5 minutes or less. The DNA polymerase according to any one of claims 1 to 5, in which the reverse transcription reaction is completed in 1 minute or less. The DNA polymerase according to any one of claims 1 to 6, further comprising an amino acid modification at the site corresponding to position 509. The DNA polymerase according to claim 7, wherein the modification of the amino acid at the site corresponding to position 509 is a substitution with a basic amino acid selected from the group consisting of histidine, lysine, and arginine. The DNA polymerase according to claim 7 or 8, wherein the modification of the amino acid at the site corresponding to position 509 is a substitution with arginine. A nucleic acid amplification reagent comprising the DNA polymerase according to any one of claims 1 to 9. The nucleic acid amplification reagent according to claim 10, which is used for amplifying nucleic acids from RNA. The nucleic acid amplification reagent according to claim 10 or 11, which is used in an RT-PCR method. A nucleic acid amplification kit comprising the nucleic acid amplification reagent according to any one of claims 10 to 12. A method for amplifying nucleic acid using a DNA polymerase according to any one of claims 1 to 9, a reagent for amplifying nucleic acid according to any one of claims 10 to 12, or a kit for amplifying nucleic acid according to claim 13. The nucleic acid amplification method according to claim 14, wherein RNA is the nucleic acid to be detected. The nucleic acid amplification method according to claim 14 or 15, wherein the reverse transcription reaction time is 5 minutes or less. The nucleic acid amplification method according to any one of claims 14 to 16, wherein the reverse transcription reaction time is 1 minute or less. The nucleic acid amplification method according to any one of claims 14 to 17, comprising a step of performing an RT-PCR reaction.