Modified DNA polymerase

Abstract

To provide DNA polymerase having reverse transcription activity and improved amplification efficiency.SOLUTION: The present invention relates to a DNA polymerase with reverse transcription activity having a specific amino acid sequence, to a nucleic acid amplification reagent containing the DNA polymerase, to a nucleic acid amplification kit containing the nucleic acid amplification reagent, and to a nucleic acid amplification method using the DNA polymerase, the nucleic acid amplification reagent, or the nucleic acid amplification kit.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a variant of a DNA polymerase having reverse transcriptase 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 inspection, and the like.

Background Art

[0002] Nucleic acid amplification methods are techniques for amplifying several copies of a target nucleic acid to a visible level, that is, more than several hundred million copies, and are widely used not only in the field of life science research but also in medical fields such as gene diagnosis and clinical tests, or in the inspection of microorganisms in foods and the environment.

[0003] A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR uses (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to the template single-stranded DNA, and (3) extension of the primer using DNA polymerase as one cycle, and by repeating this cycle, a method for amplifying the target nucleic acid in a sample.

[0004] 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 quantification of mRNA, RT-PCR, which performs a reaction (reverse transcription reaction) for converting RNA into cDNA by reverse transcriptase before PCR, is also widely used.

[0005] In RT-PCR, it is common to use two types of enzymes: reverse transcriptase and DNA polymerase. However, some DNA polymerases also have reverse transcription activity, and in recent years, such DNA polymerases with reverse transcription activity may be used. Examples of DNA polymerases with reverse transcription activity include DNA polymerase (Tth) derived from Thermus thermophilus HB8 (Thermus thermophilus HB8), DNA polymerase (Z05) derived from Themus sp Z05, and DNA polymerase (Tma) derived from Thermotoga maritima. However, it is difficult to say that the reverse transcription activity of these DNA polymerases is high, and further improvement has been demanded. Also, due to the low reverse transcription activity, it is common for RT-PCR using these DNA polymerases to require a reaction time of about 20 minutes for reverse transcription. In situations where rapid testing and diagnosis are required, it is desired to shorten the reaction time, and improvement of reverse transcription activity has been demanded.

[0006] Previously, various mutants of DNA polymerases with reverse transcription activity have been studied (Patent Documents 1, 2, 3). However, even such mutants may not be sufficient to efficiently carry out the reaction, and the development of a polymerase with even higher performance has been demanded.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present invention has been made in view of the above prior art, and an object thereof is to provide a DNA polymerase having reverse transcriptase activity and high amplification efficiency. [Means for Solving the Problems]

[0009] In view of the above problems, the present inventors have conducted intensive studies and as a result, have found that by modifying amino acids at specific sites of a DNA polymerase derived from Thermus thermophilus, efficient amplification can be achieved, and thus the present invention has been reached.

[0010] That is, the present invention mainly has the following configurations. [Item 1] A DNA polymerase having reverse transcriptase activity and comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at the position corresponding to position 751 has been modified. [Item 2] A DNA polymerase having reverse transcriptase activity and comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at the position corresponding to position 751 has been modified. [Item 3] A DNA polymerase having reverse transcriptase activity and comprising the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at the position corresponding to position 751 has been modified. [Item 4] The DNA polymerase according to any one of Items 1 to 3, wherein the modification of the amino acid at the position 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 modification of the amino acid at the position 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 a modification of the amino acid at the site corresponding to position 509. [Item 9] The DNA polymerase according to Item 8, 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. [Item 10] The DNA polymerase according to Item 8 or 9, wherein the modification of the amino acid at the site corresponding to position 509 is a substitution with arginine. [Item 11] A reagent for nucleic acid amplification containing the DNA polymerase according to any one of Items 1 to 10. [Item 12] The reagent for nucleic acid amplification according to Item 11, which is used for nucleic acid amplification from RNA. [Item 13] The reagent for nucleic acid amplification according to Item 11 or 12, which is used in the RT-PCR method. [Item 14] A kit for nucleic acid amplification containing the reagent for nucleic acid amplification according to any one of Items 11 to 13. [Item 15] A nucleic acid amplification method 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 the RNA is a 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 nucleic acid amplification method according to any one of Items 15 to 18, including the step of performing an RT-PCR reaction.

Advantages of the Invention

[0011] According to the present invention, a novel DNA polymerase having reverse transcription activity and improved amplification efficiency is provided. By using the DNA polymerase of the present invention, it becomes possible to efficiently perform nucleic acid amplification from RNA.

Modes for Carrying Out the Invention

[0012] The present invention will be described in detail below, but the present invention is not limited thereto.

[0013] The present invention relates to a modified DNA polymerase having reverse transcriptase activity. A DNA polymerase having reverse transcriptase activity is a DNA polymerase that has the ability to convert RNA into cDNA (also referred to as "reverse transcriptase activity" (RT activity)) and the ability to amplify DNA (also referred to as "DNA polymerase activity"). DNA polymerase means an enzyme that synthesizes a DNA strand having a base sequence complementary to a single-stranded nucleic acid using the single-stranded nucleic acid as a template. The presence or absence of reverse transcriptase 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, it can be measured by the procedure described in the method for evaluating reverse transcriptase activity (RT activity) described below. The presence or absence of DNA polymerase activity can be measured according to the method described in the DNA polymerase activity measurement method described below.

[0014] In a specific embodiment, the present invention provides a mutant DNA polymerase having an amino acid modification at a site corresponding to position 751 in a DNA polymerase derived from Thermus thermophilus. The full-length amino acid sequence of this DNA polymerase derived from Thermus thermophilus is shown in SEQ ID NO: 1. Further, the gene sequence derived from Thermus thermophilus encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2.

[0015] In one embodiment, the DNA polymerase of the present invention is a modified DNA polymerase comprising a mutation (preferably substitution with another amino acid) at the amino acid 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 preferably composed of an amino acid sequence having an identity of 90% or more, preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, even more preferably 98% or more, and particularly 99% or more with the amino acid sequence described in SEQ ID NO: 1. Further, 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. For example, it is 1 to 20, preferably 1 to 10, more preferably 1 to 5. The amino acid sequence before modification as described above may be artificially prepared by genetic engineering techniques, for example, or may be the amino acid sequence of a naturally occurring protein. Such a naturally occurring amino acid sequence is not particularly limited. For example, in addition to the DNA polymerase (Tth) derived from Thermus thermophilus HB8, the DNA polymerase (Z05) derived from Themus sp Z05, the DNA polymerase (Tma) derived from Thermotoga maritima, and the like can be mentioned. Preferably, it is an amino acid sequence derived from Tth or Z05, and among them, the amino acid sequence derived from Tth is particularly suitable.

[0016] In certain embodiments, the modified DNA polymerase of the present invention has an amino acid modification at a site corresponding to phenylalanine (F751) at position 751 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 addition to the amino acid modification at the site corresponding to position F751, the modified DNA polymerase of the present invention may contain any amino acid modification at other amino acid sites as long as the effects of the present invention are achieved. From the viewpoint of more reliably and efficiently performing nucleic acid amplification from RNA, it is preferable that the modified DNA polymerase of the present invention 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.

[0017] In this specification, for nucleotide sequences, amino acid sequences, and their individual components, simplified symbols using alphabetical notation may be used, all of which follow the common practice 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 the amino acid residue before substitution, its position, and the type of the amino acid residue after substitution. In addition, the SEQ ID NOs correspond to the SEQ ID NOs described in the sequence listing unless otherwise specified. In the case of multiple mutants, the above notations are connected with " / " to represent. 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 corresponding to a certain position (order) on SEQ ID NO: 1 in an amino acid sequence that is not completely identical to the amino acid sequence shown in SEQ ID NO: 1 refers to the position corresponding to the said position of SEQ ID NO: 1 when comparing (aligning) the primary structures of the sequences.

[0018] In the present specification, the term "mutant DNA polymerase" or "modified DNA polymerase" means having an amino acid sequence different from that of a conventionally known DNA polymerase, and does not distinguish whether it is due to an artificial mutation or a mutation in nature.

[0019] In a specific preferred embodiment, the DNA polymerase of the present invention is one in which the amino acid at the site corresponding to position 751 in the amino acid sequence of SEQ ID NO: 1 etc. is modified to a neutral amino acid. Preferably, the amino acid at the site corresponding to position 751 is substituted with tyrosine, cysteine, glutamine, serine, threonine, asparagine, or tryptophan, and more preferably, it is substituted with tyrosine, cysteine, glutamine, serine, threonine, or asparagine. Tyrosine, cysteine, glutamine, serine, threonine, and asparagine are all known as amino acids having polar neutral side chains, and their isoelectric points are also close to about 5.0 to about 5.7, and it can be expected that they will exhibit equivalent effects as amino acids showing common properties.

[0020] In a further preferred embodiment, the modified DNA polymerase of the present invention is one in which the amino acid at the site corresponding to position 509 in the amino acid sequence of SEQ ID NO: 1 etc. is further modified to a basic amino acid. Preferably, the amino acid at the site corresponding to position 509 is substituted with arginine, lysine, or histidine. For example, the modified DNA polymerase of the present invention may be one in which the amino acid at the site corresponding to position 509 is substituted with arginine or lysine. It is known that the isoelectric point of arginine as a basic amino acid is about 10.8, the isoelectric point of lysine is about 9.7, and the isoelectric point of histidine is about 7.6. That is, since these basic amino acids all have a high isoelectric point, it can be expected that they will exhibit equivalent effects as amino acids showing common properties.

[0021] As a method for producing a modified DNA polymerase in the present invention, known methods can be used. Preferably, a method of introducing mutations into a gene encoding a wild-type DNA polymerase to produce a mutant (modified) DNA polymerase having a new function by protein engineering techniques is used.

[0022] As one embodiment of the method for introducing amino acid modifications, a site-directed 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: (1) denature a plasmid inserted with a target gene, anneal a mutant primer to the plasmid, and then perform an extension reaction using KOD DNA polymerase; (2) repeat the cycle of (1) 15 times; (3) selectively cleave only the plasmid used as a template using the restriction enzyme DpnI; (4) phosphorylate and ligate the newly synthesized gene to cyclize it; and (5) transform Escherichia coli with the cyclized gene to obtain a transformant carrying the plasmid into which the target mutation has been introduced, and can be suitably used for the production of the modified DNA polymerase of the present invention.

[0023] The DNA polymerase of the present invention may be in the form of a fusion protein with Sso7d or PCNA. It may also be a fusion protein to which a protein tag such as a His tag or a GST tag is added.

[0024] Transfer the above DNA polymerase gene to an expression vector as needed. After transforming, for example, Escherichia coli as a host using the expression vector, spread it on an agar medium containing a drug such as ampicillin to form colonies. Inoculate the colonies into a nutrient medium, such as LB medium or 2×YT medium, culture at 37°C for 12 to 20 hours, then disrupt the cells to extract a crude enzyme solution. As the vector, those derived from pBluescript are preferred. As a method for disrupting the cells, any known method may be used, for example, physical disruption methods such as ultrasonic treatment, French press, or glass bead disruption, or lytic enzymes such as lysozyme can be used. Heat-treat this crude enzyme solution at 80°C for 30 minutes to inactivate the host-derived polymerase and measure the DNA polymerase activity.

[0025] Any method may be used to obtain the purified DNA polymerase from the strain selected by the above method. For example, there are the following methods. After collecting the cells obtained by culturing in a nutrient medium, disrupt and extract them by an enzymatic or physical disruption method to obtain a crude enzyme solution. Heat-treat the obtained crude enzyme extract, for example, at 80°C for 30 minutes, and then recover the DNA polymerase fraction by ammonium sulfate precipitation. This crude enzyme solution can be desalted by methods such as gel filtration using Sephadex G-25 (manufactured by Amersham Pharmacia Biotech). After this operation, it can be separated and purified by heparin sepharose column chromatography to obtain a purified enzyme preparation. The purified enzyme preparation is purified to such an extent that it shows almost a single band by SDS-PAGE.

[0026] Whether the modified DNA polymerase of the present invention has reverse transcriptase activity and DNA polymerase activity can be specifically measured by the following methods.

[0027] [Evaluation method for reverse transcriptase activity (RT activity)] The DNA polymerase of the present invention is excellent in reverse transcription activity compared to conventional ones and may be capable of performing a reverse transcription reaction in a short time. Specifically, although not particularly limited, when the full 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 within 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, as conditions for the RT-PCR reaction to be established, the PCR efficiency is within 70 to 130%, and the correlation coefficient r2 is 0.97 or more. The PCR efficiency can be determined according to the following formula (1) from the slope of the calibration curve obtained by plotting the results with the serially diluted nucleic acid amount on the X-axis and the Ct corresponding to the nucleic acid amount 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 such a form, when PCR after the reverse transcription reaction is possible, it is considered that the reverse transcription reaction is completed. Since the reverse transcription reaction is completed in a short time in this way, the total time required for a nucleic acid amplification method using RNA as a template (for example, the RT-PCR method) can be shortened, which is very beneficial when rapid inspection and diagnosis are required.

[0028] [DNA Polymerase Activity Measurement Method] In the present invention, the activity of the purified DNA polymerase is measured by the method shown below. 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% glycerol) and then measured. (1) Add 25 μl of Solution A below, 5 μl of Solution B, 5 μl of Solution C, 10 μl of sterilized water, and 5 μl of the enzyme solution to a microtube, and react at 75°C for 10 minutes. (2) Then cool on ice, add 50 μl of Solution E and 100 μl of Solution D, stir, and then cool on ice for another 10 minutes. (3) Filter this solution through a glass filter (Whatman GF / C filter), and wash thoroughly with 0.1 N hydrochloric acid and ethanol. (4) Measure the radioactivity of the filter with a liquid scintillation counter (Packard Tri-Carb 2810 TR) to measure the incorporation of nucleotides of the template DNA. One unit of enzyme activity is defined as the amount of enzyme that incorporates 10 nmol of nucleotides per 30 minutes into the acid-insoluble fraction (i.e., the fraction that precipitates when Solution D is added) 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: 1.5 μg / μl activated calf thymus DNA Solution C: 1.5 mM dNTP (250 cpm / pmol [3H]dTTP) Solution D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) Solution E: 1 mg / ml calf thymus DNA

[0029] The modified DNA polymerase of the present invention can be used in any nucleic acid amplification method known in the art. Conditions such as temperature, time, and reaction cycles for nucleic acid amplification vary depending on the type of nucleic acid to be amplified, the base sequence, the strand length, etc., but can be appropriately set by those skilled in the art. As an example, when performing PCR or RT-PCR 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, three steps: (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of primers to the template single-stranded DNA, and (3) extension of the primers using DNA polymerase, are regarded as one cycle, and this cycle is repeated. The extension time in the present invention indicates the time required for one cycle of the reaction to extend the primers in (3). Also, in PCR or RT-PCR, (2) annealing and (3) primer extension may be performed in two steps at the same temperature. In this case, for the sake of convenience, the extension time in the present invention refers to the time when (2) annealing and (3) extension are performed in parallel.

[0030] In certain embodiments, it is preferred that the modified DNA polymerase of the present invention has heat resistance such that it can function sufficiently even under the high temperature of a PCR reaction cycle. For example, it is preferred to have heat resistance at a level where the enzyme activity does not decrease by more than half even when heat treatment at 85 °C for 1 minute or longer is carried out. For example, the reaction temperature used in RT-PCR is not particularly limited, but examples include PCR reaction cycle conditions in which reverse transcription is carried out at 40 to 80 °C followed by heat denaturation at 90 to 100 °C and association / elongation reaction at 40 to 80 °C. Preferably, PCR reaction cycle conditions in which reverse transcription is carried out at 50 to 65 °C followed by heat denaturation at 94 to 98 °C and association / elongation reaction at 55 to 65 °C (for example, PCR reaction cycle conditions in which reverse transcription is carried out at 60 °C followed by heat denaturation at 95 °C and association / elongation reaction at 60 °C) can be exemplified. The modified DNA polymerase of the present invention maintains good activity within such a temperature range and can effectively amplify nucleic acids.

[0031] The modified DNA polymerase of the present invention can be applied not only to nucleic acid amplification methods from RNA that require reverse transcription (for example, RT-PCR), but also to nucleic acid amplification methods using DNA as a template (for example, PCR). In such RT-PCR methods and / or PCR methods, for example, at least one kind of primer and dNTP (deoxyribonucleotide triphosphate) are reacted to extend the primer and synthesize a DNA primer extension product. Specifically, it can be applied to primer extension methods, sequencing methods, methods without performing conventional temperature cycles, cycle sequencing methods, and the like.

[0032] 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. Since it contains a DNA polymerase having not only DNA polymerase activity but also reverse transcription activity, it can be used, for example, for nucleic acid amplification 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. That is, since the nucleic acid amplification reagent of the present invention can amplify nucleic acids regardless of whether the target nucleic acid (nucleic acid to be detected) serving as a template is DNA or RNA, it can be 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. For example, an amount such that the final concentration in the nucleic acid amplification reaction is 0.1 to 20 U / 20 μl can be exemplified.

[0033] As an example of the nucleic acid amplification reagent, it can contain two types of primers in which one primer 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. More specifically, it can contain two types of primers in which one primer is complementary to the other primer DNA extension product, dNTP, and the above DNA polymerase, magnesium ions and / or manganese ions, ammonium ions and / or potassium ions, BSA, a nonionic surfactant as described above, and a buffer. When used as a reagent for RT-PCR, although not limited, for example, it preferably contains salts such as manganese salts and / or magnesium salts having a final concentration of 1 mM or more in the reaction solution. Examples of the manganese salt include manganese chloride, manganese sulfate, manganese acetate, etc. Examples of the magnesium salt include magnesium salts such as magnesium chloride, magnesium sulfate, magnesium acetate, etc.

[0034] As another embodiment of the nucleic acid amplification reagent, there is a nucleic acid amplification reagent containing two kinds of primers in which one primer is complementary to the DNA extension product of the other primer, dNTP, and the DNA polymerase, divalent ions, monovalent ions, buffer solution, and, if necessary, an antibody having an activity of suppressing the polymerase activity and / or 3'-5' exonuclease activity of the thermostable DNA polymerase as described above in the present invention. 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.

[0035] Still another aspect of the present invention can be a kit containing the nucleic acid amplification reagent as described above. Specifically, this kit can 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 can also be used for nucleic acid amplification reactions using RNA as a template, and can be suitably used, for example, in RT-PCR methods and the like. The kit may include an attached document or the like describing the procedure of the nucleic acid amplification method of the present invention.

[0036] As a further embodiment, the present invention provides a nucleic acid amplification method using the modified DNA polymerase of the present invention as described above, the nucleic acid amplification reagent containing the DNA polymerase, or the nucleic acid amplification kit containing the nucleic acid amplification reagent. Since these DNA polymerases, nucleic acid amplification reagents, and kits can exhibit excellent reverse transcription activity and high amplification efficiency, they can be suitably used not only for nucleic acid amplification methods from DNA but also for nucleic acid amplification methods using RNA as a template. Therefore, 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 further can be a method including a step of performing an RT-PCR reaction. The reverse transcription reaction step can be carried out by co-existing the modified DNA polymerase of the present invention with the template RNA under predetermined conditions (for example, at 50 to 65°C for 1 to 30 minutes).

[0037] Hereinafter, the present invention will be described in more detail based on examples. It should be noted that the present invention is not particularly limited to the examples.

Example

[0038] Example 1 Preparation of DNA polymerase plasmid The DNA polymerase gene derived from Thermus thermophilus HB8 (SEQ ID NO: 2) prepared by artificial synthesis was cloned into pBluescript to prepare a plasmid incorporating wild-type Tth DNA polymerase (pTth). The plasmid with mutations was prepared using the KOD-Plus-Mutagenesis Kit (manufactured by Toyobo) according to the instruction manual with pTth as a template. For double mutations, the prepared mutant plasmid was used as a template, and further mutations were introduced using the same kit. The templates and primers used for plasmid preparation are shown in Table 1. The obtained plasmids were used to transform Escherichia coli JM109 and used for enzyme preparation.

[0039]

Table 1

[0040] Example 2 Preparation of DNA polymerase The cultivation of the bacterial cells obtained in Example 1 was carried out as follows. First, 80 mL of TB medium (Molecular cloning 2nd edition, p.A.2) containing 100 μg / mL of ampicillin that had been sterilized was dispensed into a 500 mL Sakaguchi flask. 3 mL of LB medium (1% Bacto-tryptone, 0.5% yeast extract, 0.5% sodium chloride; manufactured by Gibco) containing 100 μg / mL of ampicillin in advance was inoculated with Escherichia coli JM109 (plasmid-transformed strain) (using a test tube) that had been cultured at 37 °C for 16 hours, and aerated culture was carried out at 37 °C for 16 hours. The bacterial cells were recovered 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 the bacterial cells were disrupted by sonication treatment to obtain a cell disruption solution. Next, the cell disruption solution was treated at 80 °C for 15 minutes, and then the insoluble fraction was removed by centrifugation. Furthermore, nucleic acid removal treatment using polyethyleneimine, ammonium sulfate salting out, and heparin sepharose chromatography were carried out, and finally, it was replaced with 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% glycerol) to obtain a modified DNA polymerase.

[0041] The measurement of the DNA polymerase activity in the above purification process was carried out by the following operation. Also, when the enzyme activity was high, the sample was diluted for measurement.

[0042] (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) Solution D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) Solution E: 1 mg / mL calf thymus DNA

[0043] (Method) 25 μl of Solution A, 5 μl of Solution B, 5 μl of Solution C, and 10 μl of sterilized water were added to a microtube, stirred and mixed, then 5 μl of the above purified enzyme dilution was added, and the mixture was reacted at 75°C for 10 minutes. Then it was cooled, 50 μl of Solution E and 100 μl of Solution D were added, stirred, and further ice-cooled for 10 minutes. This solution was filtered through a glass filter (Whatman GF / C filter), thoroughly washed with 0.1N 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 incorporated 10 nmol of nucleotides into the acid-insoluble fraction per 30 minutes under these conditions.

[0044] As a result of the above measurement, it was confirmed that the modified DNA polymerase of the present invention has sufficient DNA polymerase activity.

[0045] Example 3 Evaluation of amplification efficiency in RT-PCR method (reverse transcription reaction time: 5 minutes) Using the DNA polymerase prepared in Example 2, RT-PCR in one step from RNA was carried out. For RT-PCR, Tth DNA Polymerase RT-PCR Buffer (Roche Manufacture) Using the attached buffer, 1×PCR Buffer, 0.4 mM dNTPs, 4 pmol each of primers (SEQ ID NO: 7 and 8) for amplifying enterovirus RNA (about 196 bp), 4 pmol of Taqman probe (SEQ ID NO: 9), and 1 U of enzyme were included in a 20 μl reaction solution. Enterovirus RNA with an unknown concentration was added, and after a pre-reaction at 90°C for 30 seconds, a reverse transcription reaction at 60°C for 5 minutes was performed, followed by PCR using Step One (Applied Biosystem) with a schedule of repeating 95°C for 5 seconds → 60°C for 10 seconds 45 cycles. As the enzyme, a conventionally known Tth DNA polymerase (Q509R) without an amino acid modification at the site corresponding to position 751 and the Tth NA polymerase (Q509R / F751Y) of the present invention with an amino acid modification at the site corresponding to position 751 were used.

[0046]

Table 2

[0047] Table 2 shows the results of summarizing the Cq values of RT-PCR. The smaller the Cq value, the higher the amplification efficiency of PCR. As shown in the results of Table 2, compared with the conventionally known Tth DNA polymerase that does not have an amino acid modification at the site corresponding to position 751, the Cq value of the modified Tth DNA polymerase of the present invention is clearly smaller, and it was confirmed that the amplification efficiency is very high. Therefore, by having an amino acid modification at position 751, it was shown 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.

[0048] Example 4 Evaluation of reverse transcription reaction efficiency in RT-PCR method (reverse transcription reaction time 5 minutes, 1 minute) Using the modified DNA polymerase (Q509R / F751Y) of the present invention prepared in Example 2, one-step RT-PCR was performed from RNA. For RT-PCR, Tth DNA Polymerase RT-PCR Buffer (Roche Manufacture) Using the attached buffer, 500 ng, 50 ng, 5 ng, and 0.5 ng of RNA were added to a 20 μl reaction solution containing 1×PCR Buffer, 0.4 mM dNTPs, 4 pmol of primers (SEQ ID NOs: 10 and 11) for amplifying human β-globin (about 188 bp), 4 pmol of Taqman probe (SEQ ID NO: 12), and 1 U of the enzyme. After a pre-reaction at 90°C for 30 seconds, a reverse transcription reaction was performed at 60°C for 1 minute or 5 minutes, followed by PCR using Step-OnePlus (manufactured by Applied Biosystem) according to a schedule of repeating 45 cycles of →95°C for 15 seconds →60°C for 1 minute. As the enzyme, the modified Tth DNA polymerase (Q509R / F751Y) of the present invention having an amino acid modification at the site corresponding to position 751 was used.

[0049]

Table 3

[0050]

Table 4

[0051] 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 respective Cq values and PCR efficiencies in RT-PCR. As a result, it was revealed that the modified Tth DNA polymerase of the present invention showed almost no change in the Cq value between the case of a 5-minute reverse transcription reaction (Table 3) and the case of a 1-minute reverse transcription reaction (Table 4), and in any case, it satisfied the conditions of PCR efficiency. Therefore, by using the modified DNA polymerase of the present invention into which the F751Y mutation has been introduced, it was found that RT-PCR can be sufficiently performed even with a short reverse transcription reaction time, and nucleic acid amplification can be efficiently performed from RNA.

Industrial Applicability

[0052] The present invention provides a modified DNA polymerase useful in the field of molecular biology and a composition thereof. Further, according to the present invention, the time required for a nucleic acid amplification reaction using RNA as a template can be significantly shortened. 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 inspection, and the like.

Claims

1. A DNA polymerase having reverse transcriptase activity and consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at the position corresponding to position 751 is substituted with a neutral amino acid having a polar side chain selected from the group consisting of tyrosine, glutamine, and asparagine.

2. A DNA polymerase having reverse transcriptase activity and consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at the position corresponding to position 751 is substituted with a neutral amino acid having a polar side chain selected from the group consisting of tyrosine, glutamine, and asparagine.

3. A DNA polymerase having reverse transcriptase activity and consisting of the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at the position corresponding to position 751 is substituted with a neutral amino acid having a polar side chain selected from the group consisting of tyrosine, glutamine, and asparagine.

4. The DNA polymerase according to any one of claims 1 to 3, wherein the modification of the amino acid at the position corresponding to position 751 is substitution with tyrosine.

5. The DNA polymerase according to any one of claims 1 to 4, wherein the reverse transcription reaction is completed within 5 minutes.

6. The DNA polymerase according to any one of claims 1 to 5, wherein the reverse transcription reaction is completed within 1 minute.

7. The DNA polymerase according to any one of claims 1 to 6, further comprising modification of the amino acid at the position corresponding to position 509.

8. The DNA polymerase according to claim 7, wherein the modification of the amino acid at the position corresponding to position 509 is substitution with a basic amino acid selected from the group consisting of histidine, lysine, and arginine.

9. The DNA polymerase according to claim 7 or 8, wherein the modification of the amino acid at the position corresponding to position 509 is substitution with arginine.

10. A reagent for nucleic acid amplification containing the DNA polymerase according to any one of claims 1 to 9.

11. The reagent for nucleic acid amplification according to claim 10, which is used for nucleic acid amplification from RNA.

12. The reagent for nucleic acid amplification according to claim 10 or 11, which is used in the RT-PCR method.

13. A kit for nucleic acid amplification containing the reagent for nucleic acid amplification according to any one of claims 10 to 12.

14. A nucleic acid amplification method using the DNA polymerase according to any one of claims 1 to 9, the nucleic acid amplification reagent according to any one of claims 10 to 12, or the nucleic acid amplification kit according to claim 13.

15. The nucleic acid amplification method according to claim 14, wherein the RNA is a nucleic acid to be detected.

16. The nucleic acid amplification method according to claim 14 or 15, wherein the reverse transcription reaction time is 5 minutes or less.

17. 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.

18. The nucleic acid amplification method according to any one of claims 14 to 17, comprising a step of performing an RT-PCR reaction.

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