JP2003125767A - In vitro transcription reaction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a method for improving an in vitro transcription reaction, by which the efficiency of the reaction is improved. SOLUTION: This in vitro transcription reaction method for obtaining a nucleic acid transcript, comprising mixing an RNA polymerase with a template DNA having the corresponding promoter sequence, is characterized by adding tetrakis(3-aminopropyl)ammonium represented by chemical formula 1 or its salt and/or caldopentamine represented by chemical formula 2 or its salt and performing the transcription reaction at >=50 deg.C.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an in vitro transcription reaction method using RNA polymerase. In particular, it relates to a reaction method in which the reaction efficiency is improved by adding polyamines found from thermophilic bacteria.

[0002]

The in vitro transcription reaction is one of the techniques widely used in the field of molecular biology using RNA polymerase derived from T7 phage or related phages T3, SP6 and K11 phages. The in vitro transcription reaction is performed in situ hybridization (Nakamur
a, H., et al .: Biosci Biotechnol Biochem. 61: 256-
262, 1997) or preparation of probes such as DNA microarray (Luo, L., et al .: Nature Med. 5: 117-122,199).
9), Synthesis of template RNA in cell-free protein synthesis system (Kawarasaki, Y., et al .: Biotechnol Prog. 16:
517-521, 2000), direct transcription sequence method (Sasa
ki, N., et al .: Proc Natl Acad Sci USA. 95: 3455-3
460, 1998) and so on.

Recently, the human genome analysis has advanced, and DNA microarrays are rapidly becoming widespread as a technique capable of high-speed analysis in diagnosing a disease gene or conducting a functional study of a gene. Accompanying this, the importance of probe preparation methods is increasing. Generally, it is considered that the hydrogen bond of DNA-RNA is thermodynamically stable rather than the hydrogen bond of complementary DNA-DNA. In particular, when a transcript obtained by an in vitro transcription reaction is used as a probe (riboprobe), it is thermodynamically stable, so that hybridization can be performed at a high temperature, which causes less noise than when DNA is used as a probe. It is widely used because it can obtain few results.

[0004] In particular, a microarray technique capable of analyzing the expression pattern of a large number of genes at once is used for RN from a biological sample.
Although A is extracted to prepare probes derived from all mRNA species, there are cases where a sufficient amount of RNA cannot be obtained due to only a very small amount of tissue. Reverse transcriptase, RNA polymerase and RNA
-A linear amplification method that combines enzymatic reactions such as RNase H that decomposes only RNA strands of DNA double strands is performed (Eberwi
ne, J., et al .: Proc Natl Acac Sci USA, 89: 3010-3
014, 1992). In addition, this method is a probe preparation method superior to RT-PCR in the case of quantification because it can be amplified in a large amount without changing the substantial relative amount of the original RNA sample.

Furthermore, the NASBA method (Sooknanan, R., et a
l.:Biotechniques.17:1077-1080,1083-1085,1994), T
MA method (Pasternack, J., et al .: Clin Microbiol. 35: 6
76-678, 1997) and the like, and a nucleic acid amplification method based on transcription is known. Since these methods can perform the reaction at a constant temperature unlike the conventional PCR method using DNA polymerase,
Not only special equipment such as a thermal cycler is not required, but the target nucleic acid can be amplified in a short time.

[0006] The method for determining the nucleotide sequence of DNA is one of the important means for analyzing the function of genes. The direct transcription sequence method is currently the most useful nucleotide sequencing method (Sasaki, N., et al .: Proc Natl Acad Sci US
A. 95: 3455-3460, 1998). This method uses the DNA polymerase, 2'-deoxyribonucleoside-5'-triphosphate (2'-dNTPs) and the DNA polymerase used in the Sanger method (also called the dideoxy method) which is a conventional sequencing method.
RNA polymerase, ribonucleoside-5'-triphosphate (rNTPs) and 3'- instead of 2 ', 3'-dideoxyribonucleoside-5'-triphosphate (2', 3'-ddNTPs)
Deoxynucleoside-5'-triphosphate (3'-dNTP
s) is used.

When the PCR product is used as it is as a template for a sequencing reaction, RNA polymerase is used in the direct transcription sequence method. Therefore, unlike the conventional method using DNA polymerase, unreacted primers remaining in the PCR reaction solution are left unreacted. And is an excellent method that can perform sequencing without the need to remove 2'-dNTPs. In principle, amplification of template DNA by PCR reaction and transcription reaction of direct sequencing method are performed simultaneously in parallel in the same reaction solution. It is possible (Japanese Patent No. 3155279). However, since RNA polymerase has weak thermal stability, it is necessary to add it after the PCR reaction. For large-scale analysis, an automated technique for the sequencing reaction from PCR is important, and a heat-resistant RNA polymerase is demanded. .

Further, since the cell-free protein synthesis system synthesizes a protein in vitro, the expression vector required for the expression system in eukaryotic or prokaryotic cells is introduced into a host cell, cultivated, or positive cloned. Choice,
It is an excellent method that does not require complicated identification and confirmation such as restriction enzyme mapping and culture of positive clones. When mRNA synthesis is performed using the endogenous RNA polymerase contained in the cell extract, the activity is low and the efficiency is low.
T7 RNA polymerase is often used for large-scale preparation of proteins. In this case, if the T7 promoter is introduced upstream of the target gene, template D
Since it can be NA, a plasmid or PCR product can be used as a template as it is, and it has a characteristic that a protein can be synthesized very easily and in a short time with high efficiency. And these are the product name of the wheat germ cell-free extraction system (TNT Coupled Wheat Germ Extract Systems (manufactured by Promega)), the cell-free extraction system derived from Escherichia coli (E.coli T7
S30 Extract System for Circular DNA (manufactured by Promega)) is commercially available.

[0009] While the human genome draft sequence has been clarified, it will be shifted to post sequence analysis, that is, gene function analysis in the future. Among them, the in vitro transcription reaction is currently one of the important analysis techniques because it is effective for the analysis using a small amount of sample as described above. Therefore, a more efficient in vitro reaction system has been desired.

There have been several reports as additives that accelerate the in vitro transcription reaction. Among them, polyamines that are universally observed in the living body have been reported to promote transcription (Stripe, F., et al .: Eur.J.Bioch
em., 15: 505-512, 1979). Polyamines are substances that are universally observed in proliferating cells, especially spermidine,
As putrescine and spermine were reported to have abnormally high urinary polyamine levels in cancer patients, research on the relationship between cell proliferation and polyamines became active.

It is known that the in vitro transcription reaction is promoted by a polyamine derived from a natural product such as spermidine, and spermidine is added to a commercially available buffer for RNA polymerase activity. Furthermore, it has been reported that a similar accelerating effect can be obtained with non-natural polyamines other than spermidine (Frugier, M. et al Nucleic.
Acids Res., 22,2784-2790 (1994), Iwata, M., et alBi
oorg.Med.Chem., 8: 2185-2194 (2000)). Regarding the above matters, it is considered that the polyamine is ammonium ion in the solution, and therefore it is considered to be bound to nucleic acid such as DNA and stabilized by its electrostatic interaction.

[0012] Oshima et al. Reported that various polyamines exist in cells from the research on thermostable bacteria. Generally, polyamines produced by thermostable bacteria are known to have characteristics such as polyamines having a long chain length or branched quaternary amines as compared with those of room temperature bacteria represented by E. coli (Uzawa , T., et al .: J. Bioc
hem (Tokyo). 114: 478-486 (1993)). Further, it has been reported that polyamines produced by these thermostable bacteria act as activators of in vitro protein synthesis and also as inhibitors of nucleic acid metabolizing enzymes (H, Kirino., Et al .: JB.
iochem. (tokyo), 107: 661-665 (1990)). However,
It was not reported whether these polyamines promote transcriptional activity.

Since a cell-free protein synthesis system using a thermophilic extract has also been constructed, and cell-free protein synthesis in a high temperature range is possible (Uzawa, T., et al.
l.:JBiochem (Tokyo). 114: 478-486.1993), although it is expected to be applied to research such as heat stabilization and heat resistance of proteins, none have been put to practical use yet.

On the other hand, polyamines derived from thermophiles are known to exist in addition to putrescine and spermidine, which are polyamines also present in room temperature bacteria, with longer chain lengths and branched ones ( Uzawa, T., et al .: J.
Biochem (Tokyo). 114: 478-486 (1993)). In particular, cardiopentamine, which has a long chain length, or tetrakis (3-aminopropyl) ammonium, which has a branched quaternary amine, can be added to the cell-free protein synthesis system to have a promoting effect and an inhibitory effect on restriction enzymes. Reported (H, Kirino., Et al .: J. Biochem. (Tokyo), 107: 661-
665 (1990)), in this report, is a reaction for directly synthesizing a polypeptide using phenylalanyl-tRNA, and does not use RNA polymerase.

[0015]

Therefore, the present inventors have found that
Attempts were made to verify whether polyamines promote transcriptional activity.

However, since the amount of polyamine obtained from the thermophilic bacterium cells cannot be supplied in an amount that can withstand the experiment due to a small amount, the polyamine found in the thermophilic bacterium was chemically synthesized and its action spectrum was investigated. In particular, since there was no precedent for the in vitro transcription reaction using these polyamines, the present invention was completed by earnest research.

An object of the present invention is to obtain an in vitro transcription reaction method with improved reaction efficiency.

[0018]

The in vitro transcription reaction method according to the invention described in claim 1 is a method for obtaining a nucleic acid transcript by mixing RNA polymerase with a template DNA having a corresponding promoter sequence. , Tetrakis (3-aminopropyl) ammonium represented by the following chemical formula 3 or a salt thereof and / or cardopentamine represented by the chemical formula 4 or a salt thereof is added, and a transcription reaction is performed at 50 ° C. or higher. is there.

[0019]

[Chemical 3]

[0020]

[Chemical 4]

In the in vitro transcription reaction method according to the invention described in claim 2, the tetrakis (3-aminopropyl) ammonium or salt thereof and / or cardopentamine or salt thereof according to claim 1 can be obtained by chemical synthesis. The one that is used is used.

In the in vitro transcription reaction method according to the invention described in claim 3, at least one RNA polymerase derivative derived from the T7, T3, SP6 and K11 phages is used as the corresponding RNA polymerase according to claim 1. R of amino acid addition, deletion, insertion or substitution of
NA polymerase is used.

[0023]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a wild-type RN is used.
Using a mutant enzyme as A polymerase or its derivative, these polyamines were added to the in vitro transcription reaction at various concentrations, and the effect was examined. At this time, while the demand for thermostable RNA polymerases has been increasing, attempts have been made in recent years to increase the thermostability by introducing mutations into wild-type RNA polymerases (JP-A-200
1-54387), and there are commercial products, and it is now easily available.

However, when the present inventor conducted further diligent research on the transcription reaction in the high temperature range using the above commercial products, the optimum reaction temperature was about 50 ° C. at most, and the reaction was carried out in the temperature range higher than that. It was confirmed that the yield of RNA transcript was remarkably reduced, and it was found that there is a problem to be further improved in order to efficiently perform the transcription reaction in the high temperature range.

Furthermore, the present inventors have used a commercially available thermostable T7 RNA polymerase in the high temperature range,
The change in the amount of RNA transcript produced when the in vitro transcription reaction was carried out in the absence of polyamines was examined. As a result, it was confirmed that in the temperature range of 50 ° C. or higher, the amount of RNA transcript produced was significantly reduced.

Therefore, the present inventor has reported that polyamines derived from natural products other than spermidine have an activity of promoting polypeptide synthesis in a cell-free protein synthesis system in a high temperature range (Uzawa, T. et al. , et al.:J Bioch
em (Tokyo). 114: 478-486.1993) Focusing on polyamines derived from thermophiles, the promoting effect on the in vitro transcription reaction was examined.

As a result, conditions of 50 ° C. or higher, specifically 60
When an in vitro transcription reaction was carried out at 0 ° C., tetrakis (3-aminopropyl) ammonium (taa) shown in the above chemical formula 3 and chemical formula 4
It was confirmed that the addition of cardopentamine (cpa) shown in 1) produced a larger amount of RNA transcript as compared with the case without addition, and that the effect was higher than that of spermidine, leading to the present invention.

That is, the present invention relates to a method for obtaining a nucleic acid transcript by mixing RNA polymerase with a template DNA having a corresponding promoter sequence, which comprises tetrakis (3-aminopropyl) represented by the chemical formula (3). ) Ammonium or a salt thereof and / or cardopentamine represented by the chemical formula (4) or a salt thereof is added, and a transcription reaction is performed at 50 ° C. or higher.

This transcription reaction involves RNA polymerase and
In addition to the template DNA having a promoter sequence and tetrakis (3-aminopropyl) ammonium or a salt thereof and / or cardopentamine or a salt thereof derived from a thermophile, a magnesium salt capable of generating magnesium ion in an aqueous solution, The in vitro transcription reaction in a high temperature range is carried out in a composition consisting of a potassium salt capable of generating potassium ions in an aqueous solution and a nucleic acid substantially containing a promoter sequence recognized by RNA polymerase.

In the present invention, the in vitro transcription reaction is carried out in a high temperature range, specifically, in a temperature range of 50 ° C. or higher and a maximum temperature until the transcription reaction enzyme is deactivated. The temperature at which the transcription reaction enzyme is inactivated is specifically lower than 70 ° C, and therefore, it is more preferably performed at about 53 ° C to 65 ° C. This is because at a temperature lower than 53 ° C., the significance of the improvement of the production amount of the RNA transcript of the present invention is not shown, and at a temperature higher than 65 ° C., the enzyme itself may be inactivated and the improvement of the production amount cannot be expected. .

The polyamine found from the thermophilic bacterium in the present invention is a polyamine confirmed to be produced by the thermophilic bacterium, and one produced by the thermophilic bacterium may be extracted and used. , It is not possible to supply an amount that can withstand the experiment simply by extracting from cells. Therefore, a chemically synthesized product is preferably used. Specific polyamines include tetrakis (3-aminopropyl) ammonium represented by the above chemical formula 3, and
The cardopentamine shown in Chemical formula 4 can be mentioned.

The polyamine salt in the present invention may be either an inorganic acid salt or an organic acid salt. As the inorganic salt, hydrochloride, bromate, iodate, perchlorate, perbromate,
Examples thereof include periodate and sulfate. Examples of the organic acid salt include acetate, citrate, glutamic acid and the like.
Preferably, the hydrochloride salt can be used. However, it is not limited to the above.

In the in vitro transcription reaction, a method of enzymatically synthesizing a purified RNA transcription product using a DNA fragment having a promoter sequence for RNA polymerase as a template is a known method in principle.

There is no limitation other than that the DNA fragment serving as the template for the in vitro transcription reaction contains a promoter sequence for RNA polymerase. For example, a DNA fragment containing a promoter sequence amplified by PCR
It can be a NA product. Further amplified DNA
From the product, the primer used in the PCR reaction and / or 2-
The in vitro transcription reaction in the method of the present invention can be carried out without removing deoxyribonucleoside-5-triphosphate and / or its derivative. Also,
The DNA fragment containing the promoter sequence is ligated to the DNA fragment to be amplified after ligating the promoter sequence with
It may be a DNA fragment cloned using an appropriate host. That is, in the present invention, the DNA sequence to be amplified, the primer, the conditions for amplification, etc. are not particularly limited.

For example, it can be a PCR product amplified by a primer containing a promoter sequence corresponding to phage-derived RNA polymerase. Furthermore, the in vitro transcription reaction in the method of the present invention can be performed without removing the primer and 2-deoxyribonucleoside-5-triphosphate and its derivative from the amplified PCR product. In addition, a DNA fragment containing a promoter sequence is a promoter sequence and a DN to be amplified.
It may also be a DNA fragment cloned using a suitable host after ligation with the A fragment. That is, in the present invention, the DNA sequence to be amplified, the primer, the conditions for amplification, etc. are not particularly limited.

The RNA polymerase used in the present invention may be either wild type RNA polymerase or mutant RNA polymerase. However, to improve thermostability compared to the ability of the corresponding wild-type RNA polymerase,
At least one amino acid in the wild-type RNA polymerase has been modified (ie, substituted, added, inserted or deleted)
It is desirable that the

In the present invention, the "wild type RNA polymerase" means all naturally occurring RNA polymerases. Furthermore, the “wild-type RNA polymerase” is a wild-type RNA polymerase, and has amino acid substitutions or additions other than modifications intended to improve thermostability as compared with the corresponding wild-type RNA polymerase.
It is possible to further have a missing part and an inserted part.
That is, the RNA polymerase obtained by artificially modifying the wild-type RNA polymerase for the purpose other than the above also has the above-mentioned "wild-type R polymerase".
NA polymerase ”. However, it is appropriate that such substitution, addition, insertion or deletion of amino acids is carried out within a range that maintains the activity as RNA polymerase.

As the "wild type RNA polymerase",
For example, T3 phage, T7 phage, SP6 phage,
An example is RNA polymerase derived from K11 phage. However, it is not limited to these RNA polymerases.

In the present invention, the “wild-type RNA polymerase” is a naturally occurring thermostable RNA polymerase, and a naturally occurring RNA polymerase is artificially modified to have thermostability (ie, amino acid Substitutions, additions, insertions or deletions) are also included.
However, it is appropriate that the modification for imparting heat resistance is carried out within the range in which the activity as the RNA polymerase is maintained.

T7 RNA polymerase is known as a promoter-specific RNA polymerase with extremely high specificity. Regarding the nucleotide sequence of T7 RNA polymerase and the production method, Davanloo et al., Proc. Natl. Acad. Sci.
USA., 81: 2035-2039 (1984). For mass production, see Zawadzki et al., Nucl.Acids Re
s., 19: 1948 (1991). Unlike the RNA polymerases of Escherichia coli and higher organisms, this phage-derived RNA polymerase can perform a transcription reaction with only a single polypeptide. (Chamberlin et al., N
ature, 288: 277-231 (1970). Therefore, it became an excellent material for analyzing the transcription mechanism, and many mutants were isolated and reported. Sousa et al., Nature, 3
64: 593-599 (1993) describes the crystal analysis results.

Further, three other well-known promoter-specific RNA polymerases having extremely high specificity are RNA polymerases derived from T3 phage infecting E. coli, SP6 phage infecting Salmonella and K11 phage infecting Klebsiella pneumoniae. . The above four RNA polymerases are very similar in amino acid primary structure, promoter sequence and the like.

[0042]

[Example] Effect of tetrakis-3- (aminopropyl) ammonium and cardopentamine on in vitro transcription reaction using T7 RNA polymerase [Example 1] From thermophilic bacteria in in vitro transcription reaction using the following reaction system The reaction accelerating effect of the found polyamine was tested. Specifically, tetrakis-3- (aminopropyl) ammonium and cardopentamine, which are polyamines found in two types of thermophiles, have final concentrations of 0.05, 0.1, 0.2, and 0.5 mM, respectively. 40mM Tri
s-HCl (pH8.0), 8mM MgCl ₂ , 25mM NaCl, 5mM DTT, 25μM
ATP, 25 μM CTP, 25 μM GTP, 16.25 μM UTP, 8.75 μM
Biotin-UTP, 20U T7 RNA polymerase (F644Y), 4U
RNasin (Promega), plasmid p as template DNA
20 μl of a transcription reaction solution containing 40 ng of Bluescript (Stratagene) and the above was prepared and reacted at 37 ° C. for 60 minutes.

After the reaction, 2 μl of 500 mM EDTA was added to this solution to stop the reaction, and further 10 μl of 2.5 mM LiCl and 18 μm of H ₂ O were added.
l, EtOH 100 μl were added and stirred with a vortex mixer −20
After standing still at 30 ° C. for 30 minutes, it was centrifuged at 12,000 rpm, 4 ° C. for 20 minutes to precipitate the produced RNA. The precipitated RNA was rinsed with 70% EtOH and ethanol was removed by a vacuum centrifugal dryer. 40 μl of H ₂ O (RNase free) was added to and dissolved in the recovered generated RNA, and ₂ μl of the solution was spotted on a Hybond N + membrane (Amersham Pharmacia Biotech). The RNA transcript is immobilized on the surface of the membrane by irradiating the membrane with ultraviolet light, and after treating the membrane with a blocking solution, CDP-Star (Roche), which is a luminescent substrate, is caused to emit light via streptavidin-alkaline phosphatase and Fl.
The amount of Biotin-UTP incorporated was detected and quantified by uor-S Multi-imager (Bio-Rad).

As a control, the same experiment was conducted with a reaction solution containing no tetrakis-3- (aminopropyl) ammonium and cardopentamine, and the results are shown in Table 1 below.

Example 2 Using the following reaction system, the reaction promoting effect of polyamine found in thermophilic bacteria in the in vitro transcription reaction was assayed. Specifically, tetrakis-3-, a polyamine found in two thermophiles
(Aminopropyl) ammonium and cardopentamine are 40 mM Hepes-KOH (pH 7.5 at 25 ° C), 4 mM Mg (OAc) ₂ , 10 so that the final concentrations are 0.05, 0.1, 0.2 and 0.4 mM, respectively.
0 mM KGlu, 5 mM DTT, 0.5 mM rNTPs, 5 μM Biotin-UTP,
50U Thermo T7 RNA polymerase (manufactured by Toyobo Co., Ltd.),
4U RNasin (Promega), T7 prom as template DNA
20 μl of a transcription reaction solution containing 100 ng of 500 bp dsDNA containing oter and the above was prepared and reacted at 60 ° C. for 30 minutes. The template DNA was prepared by PCR by designing primers using the plasmid pBluescript (Stratagene) as a template and containing the T7 promoter present in the plasmid.

After the reaction, 2 μl of 500 mM EDTA was added to this solution to stop the reaction, and further 10 μl of 2.5 mM LiCl and 18 μm of H ₂ O were added.
l, EtOH 100 μl were added and stirred with a vortex mixer −20
After standing at ℃ for 30 minutes, centrifuge at 12,000 rpm, 4 ℃ for 20 minutes to generate R
NA was precipitated. The precipitated RNA was rinsed with 70% EtOH and ethanol was removed by a vacuum centrifugal dryer. 40 μl of H ₂ O (RNase free) was added to and dissolved in the recovered generated RNA, and ₂ μl of the solution was spotted on a Hybond N + membrane (Amersham Pharmacia Biotech).

The RNA transcript was immobilized on the surface of the membrane by irradiating the membrane with ultraviolet rays, and after treating the membrane with a blocking solution, CDP-Star, which is a luminescent substrate, was mediated by streptavidin-alkaline phosphatase (Wako Pure Chemical Industries). Fluor-S Multi-im
The amount of Biotin-UTP incorporated was detected and quantified by ager (Bio-Rad).

As a control, a similar experiment was conducted with a reaction solution containing no tetrakis-3- (aminopropyl) ammonium and cardopentamine, and the results are shown in Table 1 below.
It was shown to.

[0049]

[Table 1]

From the results shown in Table 1, addition of tetrakis-3- (aminopropyl) ammonium or cardopentamine results in about 2% at 37 ° C. in each case.
It was shown to bring about a remarkable transcription reaction promoting effect of about 6.5 times to 7.5 times at 60 ° C.

[Reference Example] Synthesis of polyamines found from thermophile [Reference Example 1] Synthesis of tetrakis (3-aminopropyl) ammonium chloride [Reference Example 1-1] Tris (3-aminopropyl) amine Synthesis of 3,3 ', 3''-nitrilotrispropionamide (1) was added to a solution prepared by dissolving 5.93 g (156.3 mmol) of lithium aluminum hydride in 150 mL of THF as shown in the chemical reaction formula shown below.
6.00 g (26.06 mmol) was added and the mixture was refluxed for 24 hours under an argon atmosphere. Furthermore, H ₂ O was added to this reaction solution, and the mixture was stirred at 25 ° C. for 2 hours. The obtained solid substance was collected by filtration, washed several times with ethanol, and 1M hydrochloric acid was further added. What was collected by filtration under reduced pressure,
The solvent was removed to obtain the compound (tris (3-aminopropyl) amine) (2) (10.62g crude) as a yellow oil.

[0052]

[Chemical 5]

[Reference Example 1-2] Synthesis of tris- (3-phthalimidopropyl) amine As shown in the chemical reaction formula shown in the following Chemical formula 6, the compound (tris
(3-aminopropyl) amine) (2) 10.62 g (30.95 mmol),
Phthalic anhydride 45.83 g (309.5 mmol), sodium acetate 12.70
A mixture of g (154.8 mmol) was heated at 170 ° C. for 2 hours under an argon atmosphere. Then it was cooled to room temperature and 200 mL of H ₂ O was added. The obtained suspension was heated and boiled for 15 minutes, and then adjusted to pH 8 with an aqueous sodium hydrogen carbonate solution. The obtained yellowish brown solid substance was removed from the aqueous layer by filtration, the filtrate was washed and extracted with ethyl acetate, dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. The obtained residue was separated and purified by a silica gel column chromatography method using hexane-ethyl acetate as a developing solvent to give a compound (tris- (3-phthalimidopropyl) amine) (3) (5.0
5 g, yield 33.5%) was obtained as a white powder.

NMR of the obtained white powder (1H-NMR,
13C-NMR), FAB-MS, and melting point are as follows. 1H-NMR (400MHz, CDCl3); δ1.80 (6H, m), 2.5 (6H, m), 3.74 (6
H, m), 7.68 (6H, m), 7.80 (6H, m) 13C-NMR (400MHz, CDCl3); δ26.60,36.78,51.68,123.51,1
32.67,134.11,168.71 FAB-MS; m / z 579.36 (M ++ H) mp 150-151 ℃

[0055]

[Chemical 6]

[Reference Example 1-3] Synthesis of N- (3-iodopropyl) phthalimide As shown in the following chemical reaction formula, dry acetone was used.
To a solution prepared by dissolving 2.80 g (18.65 mmol) of sodium iodide in 20 mL of N- (3-bromopropyl) phthalimide (4), 1.00 g (3.7
(3 mmol) was added and the mixture was refluxed for 2 hours under an argon atmosphere.
After that, the reaction solution was cooled to room temperature, H ₂ O was added, the mixture was extracted 3 times with ethyl acetate, dried over anhydrous magnesium sulfate, and then the solvent was removed under reduced pressure. The compound (N- (3-iodopropyl) phthalimide) (5) was obtained almost quantitatively as a yellow powder.

NMR of the obtained yellow powder (1H-NMR,
13C-NMR), FAB-MS, and melting point are as follows. 1H-NMR (400MHz, CDCl3); δ2.25 (2H, m), 3.17 (2H, t), 3.78
(2H, t), 7.73 (2H, m), 7.85 (2H, m) 13 C-NMR (400MHz, CDCl3); δ1.12,32.54,38.63,123.31,13
1.98,134.04,168.21 FAB-MS; m / z316.22 mp 88-89 ℃

[0058]

[Chemical 7]

[Reference Example 1-4] Synthesis of tetrakis (3-phthalimidopropyl) ammonium iodide As shown in the chemical reaction formula shown in the following Chemical formula 8, the compound (tris
-(3-phthalimidopropyl) amine) (3) 2.74g (4.74mmo
l) and the compound (N- (3-iodopropyl) phthalimide) (5) 1.
79 g (5.68 mmol) was dissolved in 2.5 mL of anhydrous dioxane and stirred at 200 ° C. for 3 hours under an argon atmosphere. The produced white solid substance was collected by filtration and washed with anhydrous ethyl acetate to remove the solvent.
This solid substance was again dissolved in anhydrous dioxane and refluxed for 1 hour, and then the precipitate was filtered. This operation was repeated several times. The compound obtained by drying the obtained precipitate (tetrakis (3-phthalimidopropyl) ammonium iodide)
(6) (3.23 g, yield 76.2%) was obtained as a white powder.

NMR of the obtained white powder (1H-NMR,
13C-NMR) and FAB-MS are as follows. 1H-NMR (400MHz, DMSO-d6); δ1.92 (8H, m), 3.32 (8H, m), 3.5
8 (8H, t), 7.84 (16H, m) 13C-NMR (400MHz, DMSO-d6); δ20.96,34.46,56.07,123.0
4,131.64,134.30,167.84 FAB-MS; m / z766.49 (M ++ H)

[0061]

[Chemical 8]

[Reference Example 1-5] Synthesis of tetrakis (3-aminopropyl) ammonium chloride As shown in the chemical reaction formula shown in Chemical Formula 9 below, a compound (tetrakis (3-phthalimidopropyl) ammonium iodide)
(6) 0.30 g (0.34 mmol) was added to 6 mL of anhydrous ethanol solution containing 0.33 mL (10.34 mmol) of hydrazine, and the mixture was refluxed for 2 hours under an argon atmosphere. After the reaction solution was cooled to room temperature, 1M hydrochloric acid was added. The formed solid precipitate was filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was applied to a Dowex 50W-X4 (H + form) column and then eluted with 6M hydrochloric acid. The collected fractions are analyzed by HPLC and the fractions containing quaternary amine
The compound (tetrakis) was concentrated by vacuum concentration at 40 ° C and dried to dryness.
(3-aminopropyl) ammonium chloride) (7) (0.01
8 g, yield 12.4%) was obtained as a yellow powder.

[0063]

[Chemical 9]

Reference Example 2 Synthesis of Cardopentamine As shown in the chemical reaction formula shown in Chemical formula 10 below, N, N-bis (3-
Aminopropyl) -1,3-propanediamine (8) 2.50 mL (12.
21 mmol) was dissolved in 120 mL of 2-propanol, and N- (3-bromopropyl) phthalimide (9) 4.00 g was added to the solution.
The mixture to which (14.92 mmol) was added was refluxed for 20 hours under an argon atmosphere. The reaction solution was cooled to room temperature, 12M hydrochloric acid was added, and the mixture was refluxed for 10 hours.

Then, the solvent was removed from this mixture under reduced pressure. 10 mL of H ₂ O was added to the obtained residue, and the precipitate was filtered. The filtrate was concentrated under reduced pressure and the resulting residue was filtered using Dowex 50W-X4 (H
+ form) column and then eluted with 3M hydrochloric acid.
The collected fractions were analyzed by HPLC, and the fraction containing cardopentamine was concentrated under reduced pressure at about 40 ° C. and dried. The obtained residue was recrystallized from ethanol: methanol (= 1: 1) to give the compound (cardopentamine) (10) (0.079g, yield 15.1%).
Was obtained as a white powder.

[0066]

[Chemical 10]

[0067]

INDUSTRIAL APPLICABILITY The present invention makes it possible to increase the amount of RNA synthesis by adding polyamines found from thermophiles in an in vitro transcription reaction.
Therefore, by increasing the RNA transcription product in the in vitro transcription reaction, it exerts an effect in the analysis of genes and the preparation of probes in which only a small amount of sample can be obtained. Further, since it promotes the transcription reaction in the high temperature range, it leads to long-term reaction and automation due to the stabilizing action of the reaction, so that it is particularly effective in the field of gene analysis and fields related to gene diagnosis.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yasuyasu Yoneda             5-5 Kandanishikicho, Chiyoda-ku, Tokyo             Gonishikicho Building Nippon Genetech Co., Ltd.             Within (72) Inventor Yasuo Oshima             1826-2 Takagasaka, Machida City, Tokyo (72) Inventor Masanori Watabiki             5-5 Kandanishikicho, Chiyoda-ku, Tokyo             Gonishikicho Building Nippon Genetech Co., Ltd.             Within F-term (reference) 4B024 AA20 BA80 CA01 CA04 CA11                       HA01

Claims (3)

[Claims] 1. A method for mixing a RNA polymerase and a template DNA having a corresponding promoter sequence to obtain a nucleic acid transcript, which comprises tetrakis (3-aminopropyl) ammonium represented by the following chemical formula: Add a salt and / or cardopentamine represented by the chemical formula of Chemical formula 2 or a salt thereof, and
An in vitro transcription reaction method characterized by carrying out a transcription reaction as described above. [Chemical 1] [Chemical 2] 2. The in vitro transcription reaction according to claim 1, wherein the tetrakis (3-aminopropyl) ammonium or a salt thereof and / or cardopentamine or a salt thereof is obtained by chemical synthesis. Law 3. The RNA polymerase is T7, T
3, an RNA polymerase derivative derived from SP6 and K11 phage, wherein at least one amino acid is added,
The in vitro transcription reaction method according to claim 1, wherein a deleted, inserted or substituted RNA polymerase is used.