JP4329365B2 - Single component avian osteoblastosis virus reverse transcriptase and its uses - Google Patents

Description

【図面の簡単な説明】
【図１】図１は、実施例１の電気泳動の結果を示す図であり、電気泳動用ゲルを銀染色した様子を示すものである。フラクション２〜６０の偶数フラクションを泳動した。レーンＭは分子量マーカーであり、ＡＭＶ ８Ｕは精製前のＡＭＶＲＴである。
【図２】図２は、実施例２のＲＴ活性測定結果である。フラクション１０〜５０の偶数フラクションの活性を測定した。
【図３】図３は、実施例３のＴＲＣ反応の結果である。フラクション２０〜４８の偶数フラクションの活性を測定した。
【図４】図４は、実施例４に示す方法で得られたαβヘテロダイマー濃縮物のＴＲＣ反応の結果である。図中、αβ体濃縮物１は濃縮物液１を、αβ体濃縮物２は濃縮液２をそれぞれ示す。陰性対照はＲＮＡを添加せずにＴＲＣ反応を行ったものである。なお初期ＲＮＡ量１０³コピー／試験である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an avian osteoblastosis virus reverse transcriptase composition and the like.
[0002]
[Prior art]
Avian myeloblastosis virus reverse transcriptase (hereinafter referred to as “AMVRT”) is an enzyme that can be purified from individuals infected with avian myeloblastosis virus (AMV), and is used for molecular biological studies and gene amplification reactions. Widely used. AMVRT purified from a solid infected with AMV is composed of an α monomer consisting of one molecule of α subunit having a molecular weight of 68000 (α subunit is generated by fragmentation of β subunit), α subunit and β subunit having a molecular weight of 92,000. It is known to contain three components: a heterodimer consisting of one molecule (αβ heterodimer) and a homodimer consisting of two β subunit molecules (ββ homodimer).
[0003]
Since the main enzyme activity of AMVRT is reverse transcription activity, that is, RNA-dependent DNA polymerase activity, the activity of AMVRT has been conventionally used for 10 minutes at 37 ° C. using poly (A) / Oligo (dT) as a template primer. Is expressed using RNA-dependent DNA polymerase activity, where 1 unit is the amount of enzyme that incorporates 1 nmol of deoxyribonucleotide into the acid-precipitated fraction.
[0004]
[Problems to be solved by the invention]
In recent years, genetic diagnosis that can qualitatively and quantitatively analyze a small amount of nucleic acid in a test sample is particularly useful for early detection of diseases such as infectious diseases and monitoring of therapeutic effects, screening of blood for transfusion, monitoring of food and environment, etc. Although widely used, AMVRT is also used in gene amplification reactions that are the premise of such genetic diagnosis.
[0005]
An example of the gene amplification reaction using AMVRT is, for example, the so-called NASBA (Nucleic Acid Sequence Based Amplification) method described in Japanese Patent No. 2650159 or the so-called Japanese National Patent Publication No. 4-5000759. There is a TMA (Transcribion-Medialation Amplification) method, and a TRC (Transcribion-Reverse transcription Interaction) method described in Japanese Patent Application Laid-Open No. 2000-14400 reported by the present applicants.
[0006]
The amplification methods described above generate RNA copies that are homologous or complementary to the nucleic acid (target RNA) to be detected via a transcription reaction under conditions of relatively low temperature and constant temperature. Specifically, for example, a first DNA primer (including a promoter site) is attached to and extended from the target RNA to generate a DNA-RNA heteroduplex nucleic acid, and the target in the heteroduplex nucleic acid RNA that degrades RNA, attaches and extends a second DNA primer to the DNA remaining after the degradation, generates double-stranded DNA containing a promoter site, and RNA complementary to the target RNA from the double-stranded DNA A method of generating a copy (the second DNA primer is attached to and extended from the RNA copy generated here to generate a DNA-RNA heteroduplex nucleic acid, and the target in the heteroduplex nucleic acid The first DNA primer is attached to and extends from the DNA remaining after the RNA is decomposed to generate a double-stranded DNA containing a promoter site, from which the target R RNA complementary copies A is generated, amplification cycles is established that). Also, specifically, for example, a first DNA primer is attached to and extended from the target RNA to generate a DNA-RNA heteroduplex nucleic acid, the target RNA in the heteroduplex nucleic acid is degraded, and the degradation is performed. A second DNA primer (including a promoter site) is attached to the remaining DNA and extended to generate a double-stranded DNA containing the promoter site, and an RNA copy homologous to the target RNA is produced from the double-stranded DNA. (The first DNA primer is attached and extended to the RNA copy generated here to generate a DNA-RNA heteroduplex nucleic acid, and the target RNA in the heteroduplex nucleic acid is The second DNA primer is attached to and extended from the DNA remaining after being decomposed to generate a double-stranded DNA including a promoter site, and the target RN is generated from the double-stranded DNA. And homologous RNA copies are produced, amplification cycles is established that). The target RNA in these amplification methods is not limited even if it is RNA produced by a reaction system mediated by transcription as described above.
[0007]
AMVRT has not only RNA-dependent DNA polymerase activity but also DNA-dependent DNA polymerase activity and ribonuclease H activity. In the above amplification method, not only RNA-dependent DNA polymerase activity, which is the main activity of AMVRT, but also DNA-dependent DNA polymerase activity and ribonuclease H activity are important. In particular, the ribonuclease H activity is important in order to eliminate the operation of separating the heteroduplex due to the temperature increase and the temperature decrease for the subsequent reaction from the amplification method, and in the amplification method that does not additionally use RNaseH or the like. Is particularly important.
[0008]
However, as is clear from the fact that the enzyme activity of AMVRT has been conventionally expressed as reverse transcription activity (RNA-dependent DNA polymerase activity), for each component contained in AMVRT, only the reverse transcription activity has attracted attention, No other studies have been made from the viewpoint of other activities. That is, it is known that the reverse transcription activity of AMVRT is expressed by any of α monomer, αβ heterodimer, and ββ homodimer, but no other activity has been studied.
[0009]
In view of this situation, the inventors of the present invention have conducted extensive studies on each component of AMVRT. As a result, the reverse transcription activity is surely expressed in all of α monomer, αβ heterodimer and ββ homodimer. When used alone as a reagent for an amplification method as described above, it has surprisingly been found that only αβ heterodimer is effective, and the amplification reaction does not proceed with the α monomer component or ββ heterodimer.
[0010]
Accordingly, a first object of the present invention is to provide an AMVRT effective for the RNA amplification method as described above. A second object of the present invention is to provide a method for manufacturing such AMVRT. A third object of the present invention is to provide a reagent for a nucleic acid amplification method as described above, which contains such AMVRT.
[0011]
[Means for Solving the Problems]
The invention of claim 1 made to achieve the first object is an AMVRT in which 80% or more of the components are αβ heterodimers composed of an α subunit having a molecular weight of 68000 and a β subunit having a molecular weight of 92000. It is a composition. The invention of claim 2 of the present application relates to the invention of claim 1, and is an AMVRT composition characterized by consisting essentially of an αβ heterodimer consisting essentially of an α subunit having a molecular weight of 68000 and a β subunit having a molecular weight of 92000. is there.
[0012]
In order to achieve the second object, the invention according to claim 3 of the present invention includes an αβ heterodimer comprising an α subunit having a molecular weight of 68000 and a β subunit having a molecular weight of 92000, and a monomer of the α subunit (α monomer). ) And / or the β subunit homodimer (ββ homodimer), and a method comprising producing the αβ heterodimer by subjecting the solution to ion exchange chromatography. The invention of claim 4 relates to the invention of claim 3, wherein the ion exchange chromatography is ion exchange chromatography using an ion exchanger having a DEAE group as an ion exchange group.
[0013]
The invention of claim 5 made to achieve the third object is the attachment and extension of the first DNA primer to the target RNA, and the target RNA in the DNA-RNA heteroduplex nucleic acid generated by the extension. The target RNA comprising the following reactions: degradation, attachment and extension of a second DNA primer to DNA remaining after the degradation, transcription of target RNA or target RNA and complementary RNA based on double-stranded DNA generated by the extension A reagent used for an amplification reaction of a target RNA and a complementary RNA (wherein either the first or second DNA primer has a promoter site for RNA transcription or a complementary site of the site), A reagent comprising the AMVRT composition of claim 1.
[0014]
Hereinafter, the present invention will be described in detail. The AMVRT composition of the present invention can be produced from AMVRT obtained from an individual infected with AMV or a commercially available naturally-derived AMVRT reagent (for example, CHIMERx, trade name: AMV Reverse Transscriptase, 40 U / μl). Since the present AMVRT produced by gene recombination consists of only α monomer, it is not suitable as a raw material for producing the AMVRT composition of the present invention.
[0015]
The AMVRT composition of the present invention can be produced by performing liquid chromatography using the above-mentioned commercially available AMVRT reagent or the like as a raw material. As for liquid chromatography, it is particularly preferable to employ so-called high performance liquid chromatography as long as the mechanical strength of the column (packing agent) to be used can be sufficiently secured.
[0016]
Specific examples of the liquid chromatography include those using an ether column, but ion exchange chromatography using, for example, an ion exchanger having a DEAE group as a filler is particularly preferable. There are no particular limitations on the base material of the filler, and silica-based, polymer-based, etc. can be used. On the other hand, there are no particular limitations on the column packed with the packing material, and ordinary stainless steel, polyetheretherketone, glass or the like can be used. As such a suitable commercial packed column, for example, TSKgel DEAE-5PW (trade name, manufactured by Tosoh Corporation) can be exemplified.
[0017]
The raw material for producing the AMVRT composition of the present invention is an AMVRT solution in which commercially available AMVRT is dissolved in an appropriate buffer or the like. There are no particular limitations on the buffer used for dissolution, for example, 20 mM calcium phosphate (pH 7.2), Triton X-100 (0.2%), dithiothreitol (hereinafter referred to as DTT) (2 mM), and glycerol (10% ). DTT is added for protection of AMVRT. According to the findings of the present inventors, the αβ heterodimer of AMVRT is not monomerized with a DTT of about 2 mM. Triton X-100 is added to prevent adsorption of AMVRT to the column. According to the knowledge of the present inventors, the αβ heterodimer of AMVRT is not monomerized with about 0.2% Triton X-100.
[0018]
After AMVRT dissolved in the above buffer solution is applied to the column, AMVRT retained in the packing material is eluted. The elution of AMVRT can be performed, for example, by increasing the salt concentration of the buffer solution supplied to the column stepwise or continuously. For example, when a buffer as described above (hereinafter referred to as buffer A) is used for dissolving AMVRT and retaining it in a filler, for example, 20 mM calcium phosphate (pH 7.2), Triton X-100 (0.2 %), DTT (2 mM), glycerol (10%) and NaCl (1M) (hereinafter referred to as buffer solution B) are mixed at a fixed ratio. More specifically, at a flow rate of about 1 ml / min, first, buffer A is provided for about 15 minutes to elute components not retained in the filler, and then the ratio of buffer B is 50% over 20 minutes. It can be exemplified that the ratio of buffer B is gradually increased to 100% over 10 minutes, and then the column is washed with only buffer B over 15 minutes. Here, Triton X-100 is added to prevent adsorption of AMVRT to the column.
[0019]
By performing gradient elution as described above, each component of AMVRT is eluted in the order of α monomer, αβ heterodimer and ββ homodimer. These all have reverse transcription activity, which is the main enzyme activity of AMVRT, but when used for gene amplification reaction using each component, only αβ heterodimer can proceed, and α monomer and ββ homodimer The amplification reaction does not proceed. Therefore, the eluate is fractionated as a certain amount, and the fraction of the desired αβ heterodimer, that is, both are present approximately 1: 1 by confirming the amount ratio of α subunit and β subunit in each fraction. What is necessary is just to acquire the fraction to perform. In order to confirm the quantitative ratio of each subunit in each fraction, for example, an enzyme immunoassay (EIA) using an antibody capable of specifically recognizing each subunit or SDS-PAGE under reducing conditions is performed. It ’s fine. The reducing condition here refers to obtaining a test solution from each fraction and adding a strong reducing agent such as mercaptoethanol to about 5%, or heating at 95 ° C. for 5 minutes, for example. As long as the dimer can be forcibly monomerized. In confirming the quantitative ratio of α subunit and β subunit by SDS-PAGE, the density of each band may be quantified using a densitometer or the like after staining the gel after electrophoresis with a silver staining reagent or the like. It can be illustrated.
[0020]
Another AMVRT composition of the present invention, that is, an AMVRT composition consisting essentially only of αβ heterodimer, for example, an AMVRT composition produced as described above is an affinity filler in which an antibody against the α subunit is immobilized. And a column packed with an affinity packing material in which an antibody against the β subunit is immobilized.
[0021]
By the above operation, an AMVRT composition in which 80% or more of the components are αβ heterodimers and an AMVRT composition substantially consisting only of αβ heterodimers can be produced. The AMVRT composition of the present invention thus produced can be concentrated by a conventional method such as dialysis, and can be stored according to a general enzyme storage method.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
Examples are shown below to describe the present invention in more detail, but the present invention is not limited to these Examples.
[0023]
Example 1 Production of AMVRT Composition
500 μl of a commercially available AMVRT reagent (trade name; native, 40 U / μl, manufactured by CHIMERx) was diluted with the following buffer A (4.5 ml).
[0024]
Buffer A (all concentrations are final)
20 mM potassium phosphate (pH 7.2)
2 mM dithiothreitol
0.2% Triton X-100
10% glycerol
After supplying the diluted AMVRT solution to a commercially available column for ion-exchange chromatography (trade name; DEAE-5PW, 7.5 mm ID × 7.5 cm, manufactured by Tosoh Corporation), the following buffer B was added at 4 ° C. 60 fractions were obtained, 1 ml per minute according to the elution time. The gradient conditions are as follows, and the buffer flow rate is 1 ml / min.
[0025]
Buffer B (all concentrations are final)
20 mM potassium phosphate (pH 7.2)
2 mM dithiothreitol
0.2% Triton X-100
10% glycerol
1M NaCl
Gradient condition
0 to 15 minutes: Buffer A 100%
15-35 minutes: gradient from buffer A 100% to buffer A 50% + buffer B 50%
From 35 to 45 minutes: Buffer A 50% + Buffer B 50% to Buffer B 100% gradient
45 to 60 minutes: Buffer B 100%
Next, in order to confirm the quantitative ratio of α subunit and β subunit in each fraction, SDS-PAGE was performed under reducing conditions.
[0026]
First, 5 μl of a test solution is obtained from each fraction, 5 μl of the following SDS-PAGE sample buffer solution is added, heated at 95 ° C. for 5 minutes, and then a commercially available 10% electrophoresis gel (manufactured by ATTO Corporation) Name: e-Pagel, model: E-R10L, 90 mm (W) × 73 mm (H) × 1 mm (t)) 7 μl each was subjected to electrophoresis at 30 mA. The electrophoresis buffer is as follows.
[0027]
Sample buffer
0.25M Tris-HCl buffer (pH 6.8)
20% SDS
50% glycerol
0.05% bromophenol blue
Electrophoresis buffer (adjusted to 1 liter with distilled water)
3.0g Tris-hydrochloric acid
14.4 g glycine
1.0g SDS
The gel after the electrophoresis was stained with a commercially available silver staining reagent (Daiichi Chemical Co., Ltd., trade name: No. 1). The state of the gel after dyeing is shown in FIG. Moreover, the gel after dyeing | staining was used for the densitometer (Ato Co., Ltd. make, brand name; Densitograph Lane Analyzer), and the quantitative ratio of (alpha) subunit and (beta) subunit was confirmed. As a result, α monomer was eluted in fractions 4-16 and 26-28, αβ heterodimer was eluted in fractions 30-34, and ββ homodimer was eluted in fractions 36-58. The measurement result of each fraction with a densitometer (alpha subunit band density: β subunit band density) is 1: 0 for fractions 4-16, 1: 0 for fractions 26-28, and fractions. It was 55:45 for 30-34 and 16:84 for fractions 36-58. As described above, α monomer, αβ heterodimer, and ββ homodimer could be produced by this example. Fraction 30 to 34 (αβ heterodimer fraction) had a densitometer measurement of 55:45. The result of analysis with a densitometer shows that 80% or more of the fraction of this fraction is αβ heterodimer, and the total of α monomer and ββ homodimer is 20% or less.
Example 2 Measurement of RT activity
The AMVRT reagent used in Example 1 was diluted with water to prepare AMVRT solutions having various concentrations (30 U / μl, 10 U / μl, 3 U / μl, 1 U / μl, 0.1 U / μl). Each concentration of AMVRT solution (each 5 μl) was incubated with a reaction solution (20 μl) having the following composition at 37 ° C. for 10 minutes.
[0028]
Reaction solution (all concentrations are final)
50 mM Tris-HCl buffer (pH 8.3)
40 mM KCl
8.75 mM MgCl₂
10 mM dithiothreitol
0.1% bovine serum albumin
250 mM Poly (A) (Pharmacia)
5 mM Oligo (dT) 30
1 mM Thymidine 5'-triphosphate (TTP) sodium salt [methyl-3H] (Daiichi Chemical Co., Ltd.)
Next, 133 μl of a mixed solution (a mixed solution of TE 80 μl, Glycogen 3 μl, 7.5 M ammonium acetate 50 μl) was added, 375 μl of 100% ethanol was added, and the mixture was allowed to stand at −20 ° C. for 30 minutes. After centrifugation at 4 ° C. and 15000 rpm (rotation radius 7 cm) for 10 minutes, carefully remove the supernatant, add 500 μl of 70% ethanol, and again after centrifugation at 4 ° C. and 15000 rpm (rotation radius 7 cm) for 10 minutes, carefully remove the supernatant. Removed. This was suspended in 100 μl of TE, and the entire amount was measured with a scintillation counter to prepare a calibration curve. TE is 10 mM Tris-hydrochloric acid buffer (pH 8.0) and 1 mM EDTA.
[0029]
Subsequently, after 5 μl of each fraction produced in Example 1 as an AMVRT solution was subjected to the same treatment as above, the total amount was measured with a scintillation counter, and reverse transcription of each fraction was performed from the calibration curve prepared as described above. Activity was sought. The results are shown in FIG. As is clear from FIG. 2, reverse transcription activity was observed in all fractions 26 to 44, that is, any fraction of α monomer, αβ heterodimer and ββ homodimer.
[0030]
Example 3 Measurement of RNA amplification activity
According to the method described in Japanese Patent Application Laid-Open No. 2001-353000, amplification targeting RNA derived from mecA gene (hereinafter referred to as mecA-RNA) of methicillin-resistant Stephyrococcus Aureus (hereinafter referred to as MRSA) Reaction was performed. The mecA-RNA referred to in the present example is RNA synthesized and purified by in vitro transcription using a double-stranded DNA containing the mecA base sequence as a template.
(1) Base number 1 to 2013 of mecA-RNA encoding cell wall synthetic protein PBP-2 ′ produced by MRSA (base number of RNA is according to Matsuhashi et al. “FEBS Lett. 221, 167 to 171 (1987)”) ) Containing standard RNA (2016mer) as a sample, quantified by UV absorption at 260 nm, and then diluted with RNA (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0.5 U / μl RNase Inhibitor, 5. 1.0 mM with 0 mM DTT)^ThreeDilute to copy / 2.5 μl. Only the diluted solution was used in the control test group (Nega). The initial RNA amount is 10^ThreeCopy / test.
(2) 23.3 μl of the reaction solution having the following composition was dispensed into a 0.5 ml PCR tube (Gene Amp Thin-Walled Reaction Tubes, manufactured by PerkinElmer), and 2.5 μl of the RNA sample was added thereto.
[0031]
Composition of reaction solution (concentration is final concentration of reaction system after addition of enzyme solution)
60.0 mM Tris-HCl buffer (pH 8.6)
13.0 mM magnesium chloride
90.0 mM potassium chloride
1.0 mM DTT
0.25 mM dATP, dCTP, dGTP, dTTP each
Each 3.0 mM ATP, CTP, UTP
2.25 mM GTP
3.6 mM ITP
1.0 μM of each first primer (SEQ ID NO: 1) and second primer (SEQ ID NO: 2)
0.16 μM oligonucleotide probe for cleavage (SEQ ID NO: 3, oligonucleotide for cleaving the target RNA at a position where the first primer can bind, 3 ′ end is aminated)
Oligonucleotide labeled with 25 nM intercalating fluorescent dye (SEQ ID NO: 4, where the intercalating fluorescent dye is composed of the 6th “G” and the 7th “A” at the 5 ′ end of the oligonucleotide of SEQ ID NO: 4). (In addition, the hydroxyl group at the 3 'end is modified with a glycol group. See Japanese Patent No. 31899000.)
39U Ribonuclease inhibitor (manufactured by Takara Shuzo Co., Ltd.)
15.0% DMSO
Volumetric distilled water
(3) The above reaction solution was incubated at 41 ° C. for 5 minutes, and then 5 μl of the AMVRT solution of each fraction produced in Example 1 per 30 μl of the solution, the following composition was preliminarily incubated at 41 ° C. for 2 minutes. 2 μl was added.
[0032]
Composition of enzyme solution (reconcentration at the time of reaction)
1.7% sorbitol
142 units T7 RNA polymerase (manufactured by GIBCO)
3μg bovine serum albumin
Volumetric distilled water
Excitation wavelength while keeping the reaction solution at 41 ° C. using an apparatus (see Japanese Patent Application Laid-Open No. 2000-214092) configured to measure the fluorescence intensity of the reaction solution in the tube while controlling the temperature of the PCR tube. With respect to the fluorescence measurement at 470 nm and the fluorescence wavelength of 510 nm, the time when the enzyme solution was added was 0 minute, and the change over time in the fluorescence increase ratio from the reaction solution (fluorescence intensity value at a predetermined time ÷ background fluorescence intensity value) was measured. The results are shown in FIG.
[0033]
From FIG. 3, an increase in fluorescence intensity is observed in fractions 30 to 34, and only the αβ heterodimer contained in the fraction can effectively proceed with the above RNA amplification reaction, that is, not only reverse transcription activity, but also at least ribonuclease. It was confirmed to have H activity. On the other hand, RNA amplification reaction did not proceed in other fractions.
[0034]
Example 4 Measurement of concentration and RNA amplification activity
The fraction containing αβ heterodimer produced in Example 1 (fractions 30 to 34) was concentrated by the following two methods.
[0035]
First, as a first method, 1.3 ml of the mixed solution of fractions 30 to 34 is placed in a dialysis tube (SPECTRA / POR, manufactured by Spectrum Medical Industries), and an external solution (20 mM potassium phosphate (pH 7.2), Dialyzed against 500 ml of 2 mM dithiothreitol, 0.2% Triton X-100) for 3 hours. After exchanging the external solution with a new solution, dialysis was further performed for 10 hours. The fraction mixture remaining after dialysis was concentrated to 25 μl with a concentrator (trade name; Centricon 30, manufactured by Millipore). After concentration, glycerol was added to a final concentration of 50% for storage. Hereinafter, the solution containing the αβ heterodimer thus produced is referred to as a concentrated solution 1.
[0036]
Next, as a second method, 1.3 ml of the mixed solution is put into a dialysis tube (SPECTRA / POR, manufactured by Spectrum Medical Industries) at 4 ° C. with an external solution (2 mM potassium phosphate (pH 7.2), 0 Dialyzed against 500 ml of 2 mM dithiothreitol, 0.02% Triton X-100, 0.01 M trehalose) for 3 hours. After exchanging the external solution with a new solution, dialysis was further performed for 10 hours. The fraction mixture remaining after dialysis was concentrated to 130 μl with a concentrator (trade name; Centricon 30, manufactured by Millipore). After concentration, glycerol was added to a final concentration of 50% for storage. Hereinafter, the solution containing the αβ heterodimer thus produced is referred to as a concentrated solution 2.
[0037]
Concentrated solution 1 and concentrated solution 2 produced as described above were used for the amplification of mecA-RNA as in Example 3. However, instead of AMVRT used in Example 3, 0.25 μl of concentrated solution 1 or concentrated solution 2 was used per 30 μl of reaction solution. The results are shown in FIG. As can be seen from FIG. 4, RNA could be amplified using either concentrated solution 1 or concentrated solution 2.
[0038]
【The invention's effect】
In the conventional AMVRT, the amount of enzyme (unit) is expressed based on its main activity, reverse transcription activity, that is, RNA-dependent DNA polymerase activity. Therefore, even if a predetermined amount of AMVRT containing three components of α monomer, αβ heterodimer and ββ homodimer is used based on the unit as in the past, AMVRT that truly contributes to the gene amplification reaction as described above It is impossible to keep the amount of In addition, the content ratio of the three components of α monomer, αβ heterodimer, and ββ homodimer is likely to vary depending on the production lot and purchaser of AMVRT, but RNA-dependent DNA polymerase activity and DNA dependency depend on the variation of these content ratios. If the ratio of DNA polymerase activity or ribonuclease H activity varies, the efficiency of the amplification reaction will vary even if the same amount of enzyme is used, and the qualitative / quantitative determination of the gene using such amplification reaction may be affected. Easily expected.
[0039]
Therefore, by using the AMVRT composition provided in the present invention, the amplification efficiency per added AMVRT composition is improved, and by using a smaller amount of AMVRT, equivalent gene amplification is achieved as compared with the conventional case. At the same time, in a more strict sense, it becomes possible to make the comparison of qualitative / quantitative results easier when AMVRTs of different lots are used by homogenizing gene amplification reaction conditions.
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing the results of electrophoresis of Example 1, and shows a state in which an electrophoresis gel is stained with silver. Even fractions of fractions 2-60 were run. Lane M is a molecular weight marker and AMV 8U is AMVRT before purification.
FIG. 2 shows the RT activity measurement results of Example 2. The activity of even fractions of fractions 10-50 was measured.
FIG. 3 shows the results of TRC reaction in Example 3. The activity of the even fractions of fractions 20-48 was measured.
4 is a result of TRC reaction of αβ heterodimer concentrate obtained by the method shown in Example 4. FIG. In the figure, αβ-form concentrate 1 represents the concentrate liquid 1, and αβ-form concentrate 2 represents the concentrate 2. The negative control is a TRC reaction without addition of RNA. The initial RNA amount is 10^ThreeCopy / test.

Claims (1)

(1) Attachment and extension of the first DNA primer to the target RNA,
(2) degradation of the target RNA in the DNA-RNA heteroduplex nucleic acid produced by the extension,
(3) Attachment and extension of the second DNA primer to the DNA remaining after the degradation,
(4) transcription of target RNA based on double-stranded DNA generated by the extension or RNA complementary to target RNA,
A method for amplifying the target RNA or a complementary RNA to the target RNA, wherein either of the first or second DNA primer has a promoter site for RNA transcription,
The avian bone myeloblastosis virus reverse transcriptase enzyme used in the reaction of (1) to (3), .alpha..beta hetero more than 80% of the component, and a β-subunit of α-subunit having a molecular weight 92000 of molecular weight 68000 Said method, characterized in that it is a dimer.

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