JP2003125767A - In vitro transcription reaction method - Google Patents

In vitro transcription reaction method

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Publication number
JP2003125767A
JP2003125767A JP2001325016A JP2001325016A JP2003125767A JP 2003125767 A JP2003125767 A JP 2003125767A JP 2001325016 A JP2001325016 A JP 2001325016A JP 2001325016 A JP2001325016 A JP 2001325016A JP 2003125767 A JP2003125767 A JP 2003125767A
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JP
Japan
Prior art keywords
transcription reaction
rna polymerase
salt
reaction
vitro transcription
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP2001325016A
Other languages
Japanese (ja)
Inventor
Nobuo Kitamura
Yasuo Oshima
Masanori Watabiki
Sukeyasu Yoneda
暢夫 北村
泰郎 大島
祐康 米田
正則 綿引
Original Assignee
Nippon Genetech Co Ltd
株式会社ニッポンジーンテク
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Priority to JP2001325016A priority Critical patent/JP2003125767A/en
Publication of JP2003125767A publication Critical patent/JP2003125767A/en
Application status is Pending legal-status Critical

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Abstract

(57) [Problem] To provide an improved method of in vitro transcription reaction for improving reaction efficiency. SOLUTION: This is a method for obtaining a nucleic acid transcript by mixing an RNA polymerase and a template DNA having a corresponding promoter sequence, comprising tetrakis (3-aminopropyl) ammonium represented by the following chemical formula 1 or a salt thereof. as well as/
Alternatively, cardo pentamine represented by the chemical formula 2 or a salt thereof is added and a transcription reaction is performed at 50 ° C. or higher. Embedded image Embedded image

Description

Description: TECHNICAL FIELD [0001] The present invention relates to an RNA polymerase.
The present invention relates to an in vitro transcription reaction method using the enzyme. Especially thermophiles
By adding polyamines found from
The present invention relates to a reaction method with improved efficiency. [0002] The in vitro transcription reaction is T7 phage.
T3, SP6, K11 phage
In the field of molecular biology using RNA polymerases of origin
Is one of the widely used technologies. In vitro
The transcription reaction was performed in situ hybridization (Nakamur
a, H., et al .: Biosci Biotechnol Biochem. 61: 256-
262, 1997) or probes such as DNA microarrays
(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 sequencing (Sasa
ki, N., et al .: Proc Natl Acad Sci USA. 95: 3455-3
460, 1998). [0003] Recently, human genome analysis has progressed, and
High speed for diagnosis of offspring, etc. or gene function research
DNA microarray is rapidly becoming a popular technique for analysis.
It is spreading. The importance of probe preparation has increased accordingly.
are doing. Generally complementary DNA-DNA hydrogen bonds
Hydrogen bonding of DNA-RNA is thermodynamically less expensive than that of DNA-RNA.
It is said to be constant. In particular, in vitro transcription reactions
Transcripts used as probes (riboprobes)
Is thermodynamically stable, the
DNA can be probed
To obtain less noisy results than when used as
Widely used because it can be. [0004] In particular, the expression pattern of a large number of genes at once
Microarray technology can analyze RNs from biological samples.
Preparation of probes derived from all mRNA species by extracting A
But only a small amount of tissue
In some cases, the amount of RNA cannot be obtained. What to do about this
Reverse transcriptase, RNA polymerase and RNA
-Enzymes such as RNaseH that degrades only the double-stranded RNA strand of DNA
A linear amplification method combining elementary reactions is performed (Eberwi
ne, J., et al .: Proc Natl Acac Sci USA, 89: 3010-3
014,1992). And this method is based on the original RNA sample.
Can be amplified in large quantities without changing the substantial relative amount of
When performing quantification from the above, a better process than RT-PCR
It is a lobe preparation method. Further, 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).
ing. These methods involve the use of DNA polymerase.
Unlike the conventional PCR method, the reaction can be performed at a constant temperature.
Does not require special equipment such as a thermal cycler
Rather, the target nucleic acid can be amplified in a short time. [0006] In performing gene function analysis, DNA
Nucleotide sequencing is one of the important means. direct
Transcription sequencing is currently the most useful sequencing
(Sasaki, N., et al .: Proc Natl Acad Sci US
A. 95: 3455-3460, 1998). This method uses traditional sequencing
Sanger method (also known as the dideoxy method)
DNA polymerase used in 2) -deoxyribonucleic acid
Nucleoside-5'-triphosphate (2'-dNTPs) and
2 ', 3'-dideoxyribonucleoside-5'-trifosfe
Polymerase (2 ', 3'-ddNTPs)
Bonnucleoside-5'-triphosphate (rNTPs) and 3'-
Deoxynucleoside-5'-triphosphate (3'-dNTP
s). [0007] Sequence reaction of the PCR product as it is
When used as a template for direct transcription
Since the method uses RNA polymerase,
Unlike the conventional method using merase, PCR
Remove unreacted primer and 2'-dNTPs remaining in the solution
Is a great way to sequence without the need to
In principle, amplification and template DNA amplification by PCR
The transcription reaction of the direct sequencing method is performed in the same reaction solution.
It can be performed simultaneously and in parallel (Patent No. 3155279)
). However, RNA polymerase has poor thermostability
Must be added after the PCR reaction.
Technology that automates the sequence reaction from PCR is important
There is a demand for a heat-resistant RNA polymerase
I have. [0008] Furthermore, the cell-free protein synthesis system
Eukaryotes or raw materials to synthesize proteins
Expression vectors required for expression systems in eukaryotic cells
Introduction into host cells, culture, selection of positive clones,
Restriction enzyme mapping, culture of positive clones, etc.
This is an excellent method that does not require complicated identification and confirmation. Cell extraction
Utilizing the endogenous RNA polymerase contained in the effusion
When performing mRNA synthesis, the activity is low and the efficiency is low,
When preparing large quantities of protein, use T7 RNA Polymerase
Is often used. In this case, the T7 promoter
If it has been introduced upstream of the target gene, the template D
Because it can be NA, plasmid or PCR product
It can be used as a template as it is,
It has the feature of easily and efficiently synthesizing proteins in a short time.
I do. And these are wheat germ cell-free extraction systems
Name (TNT Coupled Wheat Germ Extract Systems (Promega
The cell-free extraction system derived from Escherichia coli is a trade name (E.coli T7
S30 Extract System for Circular DNA (Promega
)). [0009] Draft sequence of human genome revealed
In the future, post-sequence analysis
Later, it has shifted to gene function analysis. Among them, i
The in vitro transcription reaction uses a small amount of sample as described above.
Being effective for analysis, it is now one of the important analysis technologies
ing. Therefore, a more efficient in vitro reaction system is expected.
Was desired. Promoting in vitro transcription reaction so far
Some have been reported as additives. In the living body
That polyamines, which are commonly observed in
(Stripe, F., et al .: Eur. J. Bioch
em., 15: 505-512, 1979). Polyamines are thriving
Substances commonly observed in cells, especially spermidine,
Putrescine and spermine are used in urinary polyamines in cancer patients.
Cell levels are reported to be abnormally high,
Research on the relationship between polyamines has become active. [0011] Regarding the in vitro transcription reaction,
Is promoted by polyamines derived from natural products such as
Known and commercially available buffers for RNA polymerase activity
-Also contains spermidine. More spermi
Non-natural polyamines other than gin can achieve the same accelerating effect.
(Frugier, M. et al Nucleic
Acids Res., 22,2784-2790 (1994), Iwata, M., et alBi.
oorg.Med.Chem., 8: 2185-2194 (2000)). About the above
Alternatively, polyamines become ammonium ions in solution
Nuclei such as DNA due to the electrostatic interaction
It is thought to be due to binding and stabilization of the acid. In addition, Oshima et al. In the study of thermostable bacteria
Reported the presence of various polyamines in cells.
Was. In general, polyamines produced by thermostable bacteria
Polya with a longer chain length than that of the representative room-temperature bacteria
Features such as min or branched quaternary amine
(Uzawa, T., et al .: J. Bioc
hem (Tokyo). 114: 478-486 (1993)). In addition, these heat resistance
Polyamines produced by bacteria are proteins in vitro
As an activator of synthesis and as an inhibitor of nucleic acid metabolizing enzymes
(H, Kirino., Et al .: JB
iochem. (tokyo), 107: 661-665 (1990)). However,
Whether these polyamines promote transcriptional activity
Was not reported. [0013] A cell-free tamper using an extract of a thermophilic bacterium is also provided.
A cell synthesis system has also been constructed, and it is cell-free at high temperatures.
(Uzawa, T., et a)
l .: JBiochem (Tokyo) .114: 478-486.1993), protein
Is expected to be applied to research on heat stabilization and heat resistance
However, none has been put to practical use yet. On the other hand, polyamines derived from thermophilic bacteria are
Polyamines, putrescine, spelling
In addition to midines, those with longer chain lengths and branched ones
Is known to exist (Uzawa, T., et al .: J.
Biochem (Tokyo). 114: 478-486 (1993)). In particular, long chain length
Cardo pentamine or branched quaternary amines
About tetrakis (3-aminopropyl) ammonium
Is effective when added to a cell-free protein synthesis system
It is reported to have an inhibitory effect on fruits and restriction enzymes
(H, Kirino., Et al .: J. Biochem. (Tokyo), 107: 661-
665 (1990)), but in this report phenylalanyl-tRNA
Is a reaction to synthesize polypeptides directly using
It does not use RNA polymerase. [0015] Therefore, the present inventors have
Whether polyamines enhance transcriptional activity
I tried to verify. However, it is obtained from thermophilic bacteria cells.
Polyamines are too small to supply enough to withstand experiments
Therefore, polyamines found in thermophilic bacteria are chemically synthesized.
And examined its action spectrum. In particular, these ports
Previous examples of in vitro transcription reactions using lamine
Since there was no such research, the present inventors have conducted intensive research and completed the present invention. [0017] The present invention relates to an invitro having improved reaction efficiency.
The purpose is to obtain a transcription reaction method. [0018] According to the present invention, there is provided an electronic apparatus comprising:
The in vitro transcription reaction method according to Ming
And a template DNA having a corresponding promoter sequence.
A method for obtaining a nucleic acid transcript, comprising:
Tetrakis (3-aminopropyl) ammonium represented by the formula
Or a salt thereof and / or a cardo represented by the chemical formula
Add pentamine or a salt thereof and conduct transcription reaction at 50 ° C or higher.
Is what you do. Embedded image Embedded image [0021] The in-bit according to the invention described in claim 2
The transcription reaction method uses the tetrakis (3-amino
Nopropyl) ammonium or its salt and / or cardo
Pentamine or its salt obtained by chemical synthesis
Is used. [0022] The in-bit according to the third aspect of the present invention.
The transcription reaction method is the method of claim 1, wherein
Merase was mediated by T7, T3, SP6 and K11 phage.
An RNA polymerase derivative comprising at least one
Added, deleted, inserted or substituted R amino acids
This uses NA polymerase. BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, wild-type RN
Mutant enzyme as A polymerase or its derivative
Using these at various concentrations in in vitro transcription reactions
A polyamine was added and the effect was investigated. On this occasion
Therefore, the demand for thermostable RNA polymerase is increasing.
In recent years, mutations have been introduced into wild-type RNA polymerase.
Attempts have been made to increase thermal stability by introducing
1-54387).
Was. However, using the above-mentioned commercially available products, the present inventors
Further research on the transcription reaction in the temperature range
As a result, the optimum reaction temperature was at most about 50 ° C,
Remarkably higher yield of RNA transcripts when reaction is performed in the upper temperature range
Is reduced, and efficient
We found that there were issues that needed to be improved in order to perform
I did. Further, the present inventors have found that heat-resistant
In a high temperature range using T7 RNA polymerase,
Perform in vitro transcription reaction without polyamines
The change in the amount of RNA transcript produced when
went. As a result, in the temperature range of 50 ° C or higher,
It was confirmed that the production amount of the product was significantly reduced. Therefore, the inventor of the present invention has proposed a method other than spermidine.
Cell-free tan at high temperatures as natural polyamines
In a protein synthesis system, the activity of promoting polypeptide synthesis
(Uzawa, T., et al .: J Bioch
em (Tokyo) .114: 478-486.1993) Polyamines derived from thermophilic bacteria
Focusing on the type of
And examined. As a result, the condition of 50 ° C. or more, specifically 60 ° C.
When the in vitro transcription reaction was performed at
The tetrakis shown in the above chemical formula 3, which is the released polyamine
(3-aminopropyl) ammonium (taa)
Adding cardo pentamine (cpa) shown in
Increased the amount of RNA transcripts produced
And its effect is higher than spermidine
This led to the present invention. That is, the present invention relates to an RNA polymerase
Mix with template DNA having corresponding promoter sequence
And a method for obtaining a transcript of a nucleic acid, the method comprising:
Tetrakis (3-aminopropyl) ammonium represented by the formula
Or a salt thereof and / or a cardo represented by the chemical formula
Add pentamine or a salt thereof and conduct transcription reaction at 50 ° C or higher.
Is what you do. This transcription reaction is carried out by using an RNA polymerase,
A template DNA having a promoter sequence and a thermophilic bacterium
Trakis (3-aminopropyl) ammonium or a salt thereof
And / or cardopentamine or a salt thereof, and an aqueous solution
That can give rise to magnesium ions in magnesium
Salts and potassium that can generate potassium ions in aqueous solution
Salt, a promoter substantially recognized by RNA polymerase
In a composition consisting of nucleic acid containing
In vitro transcription reaction is performed. In the present invention, the in vitro transcription reaction is carried out at a high temperature.
Temperature, specifically at 50 ° C or higher, and the transcription enzyme is inactivated
Temperature range up to the maximum temperature. Transcription
Specifically, the temperature at which the enzyme is inactivated is lower than 70 ° C.
More preferably, it is performed at about 53 ° C to 65 ° C. From 53 ° C
Low temperature increases the production of RNA transcripts of the invention
Does not show significance, the enzyme itself is inactivated if it is higher than 65 ° C
And the improvement of the production amount cannot be expected.
You. Polyamide found from the thermophilic bacterium of the present invention
Is a polyamine that has been confirmed to be produced by thermophiles
It is also possible to extract and use those produced by thermophilic bacteria
But enough to withstand the experiment just extracted from the cells
Can not supply. Therefore, preferably use chemically synthesized products.
I have. Specific polyamines are represented by the above formula (3).
Tetrakis (3-aminopropyl) ammonium, and
Cardo pentamine shown in Chemical formula 4 is exemplified. In the present invention, the polyamine salt is an inorganic salt.
It may be either an acid salt or an organic acid salt. Hydrochloric acid as inorganic salt
Salt, bromate, iodate, perchlorate, perbromate,
Periodate, sulfate and the like. As organic acid salt
Include acetate, citrate, glutamic acid and the like.
Preferably, a hydrochloride can be used. But more
However, the present invention is not limited to this. In the in vitro transcription reaction, the RNA poly
DNA fragment having promoter sequence for merase
Enzymatic synthesis of purified RNA transcript using DNA as a template
The method is a method known in principle. The template used for performing the in vitro transcription reaction is
DNA fragments are promoters for RNA polymerase.
There is no restriction other than including a sequencer. For example, Promo
DNA fragment containing the DNA sequence
It can be an NA product. Further amplified DNA
Primers used for PCR reaction and / or 2-
Deoxyribonucleoside-5-triphosphate and
And / or the method of the invention without removing its derivatives
In vitro transcription reaction can be performed. Also,
The DNA fragment containing the promoter sequence is
After ligation of the sequence and the DNA fragment to be amplified,
DNA fragment cloned using an appropriate host
You can also. That is, in the present invention,
Certain DNA sequences, primers, conditions for amplification, etc.
There is no particular limitation. For example, phage-derived RNA polymerase
Primers containing the corresponding promoter sequence
PCR product that has been amplified. Further increase
Primer and 2-deoxyribose from PCR product
Nucleoside-5-triphosphate and its derivatives
In vitro in the method of the present invention without removing
A transcription reaction can be performed. In addition, the promoter sequence
The DNA fragment containing the promoter sequence and the DN
After ligation with fragment A, using an appropriate host
It may be a cloned DNA fragment. Immediately
That is, in the present invention, the DNA sequence to be amplified,
There are no particular restrictions on the primer, amplification conditions, and the like. The RNA polymerase used in the present invention is wild
RNA polymerase and mutant RNA polymerase
Either is acceptable. However, the corresponding wild-type RNA
To improve thermostability compared to the ability of Rase
At least one amino acid of a wild-type RNA polymerase
Is modified (ie, replaced, added, inserted or deleted)
It is desirable that it is done. In the present invention, “wild-type RNA poly
“Merase” refers to all naturally occurring RNA polymers.
Means Rase. Furthermore, "Wild-type RNA polymerase"
Ze "is a wild-type RNA polymerase,
Improved thermostability compared to native RNA polymerase
Substitution, addition,
It may further have a missing part or an inserted part.
That is, the wild-type RNA polymerase is
The RNA polymerase modified for the purpose is also described in the above “wild-type R
NA polymerase ". However, such a net
No acid substitutions, additions, insertions or deletions are RNA polymerase
The activity was performed within the range that maintains the activity as
Is appropriate. As the “wild-type RNA polymerase”,
For example, T3 phage, T7 phage, SP6 phage,
List RNA polymerases derived from K11 phage
be able to. However, these RNA polymerases
It is not limited. In the present invention, “wild-type RNA poly
Merase is a naturally occurring thermostable RNA polymer
Heat-stable and naturally occurring RNA polymerase
Has been artificially modified to have
Replacement, addition, insertion or deletion).
However, the modification for imparting heat resistance is RNA polymer
The activity was performed to the extent that the activity as
Is appropriate. T7 RNA polymerase is very specific
Known as high-specificity promoter-specific RNA polymerase
Have been. T7 RNA polymerase base sequence and raw
For production methods, see Davanloo et al., Proc. Natl. Acad. Sci.
USA., 81: 2035-2039 (1984). Even larger
For mass production, see Zawadzki et al., Nucl.
s., 19: 1948 (1991). This fur
Di-derived RNA polymerase is used in E. coli and higher organisms.
Only a single polypeptide, unlike RNA polymerase
To perform a transcription reaction. (Chamberlin et al., N
ature, 288: 277-231 (1970). Therefore, the transcription mechanism
Is a great material for analyzing DNA, and many mutants are isolated
Has been reported. See Sousa et al., Nature, 3
64: 593-599 (1993) describes the crystal analysis results. Furthermore, other highly specific promoters
Infects Escherichia coli as a protein-specific RNA polymerase
T3 phage, SP6 fur infecting Salmonella
K11 fur that infects Ji and Klebsiella pneumoniae
Three well-known RNA polymerases from di
You. In addition, the above-mentioned four types of RNA polymerases
Extremely similar in primary structure, promoter sequence, etc.
You. [Example] In vitro using T7 RNA polymerase
B) In the transcription reaction, tetrakis-3- (aminopropyl)
Effects of ammonium and cardopentamine [Example 1] In vitro transcription reaction using the following reaction system
Of Polyamines from Thermophilic Bacteria in Rice
The fruit was tested. Specifically, from the viewpoint of two types of thermophiles
The released polyamine, tetrakis-3- (aminopro
Pyr) ammonium and cardopentamine, respectively
40 mM Tris so that the concentration is 0.05, 0.1, 0.2, 0.5 mM.
s-HCl (pH 8.0), 8mM MgCl Two , 25mM NaCl, 5mM DTT, 25μM
ATP, 25μM CTP, 25μM GTP, 16.25μM UTP, 8.75μM
Biotin-UTP, 20U T7 RNA polymerase (F644Y), 4U
RNasin (promega), plasmid p as template DNA
Bluescript (Stratagene) 40ng
Prepare a 20 μl transcription reaction solution and incubate at 37 ° C for 60 minutes.
Was. After the reaction, 2 μl of 500 mM EDTA was added to this solution.
To stop the reaction, and further add 10 μl of 2.5 mM LiCl, H 2 O 18μ
l, EtOH 100μl, add vortex mixer and mix
After standing at 30 ℃ for 30 minutes, centrifuge at 12,000rpm, 4 ℃ for 20 minutes to generate
RNA was precipitated. The precipitated RNA is phosphorylated with 70% EtOH.
Then, ethanol was removed with a vacuum centrifugal dryer. Collected
H in the generated RNA Two Add 40 μl of O (RNase free) to dissolve
2 μl of Hybond N + membrane (Amersham
(Lumacia Biotech). Purple on the membrane
RNA transcripts are exposed to external light to the membrane.
Fix to surface and treat membrane with blocking solution
Followed by streptavidin-alkaline phosphatase
To emit CDP-Star (Roche), which is a luminescent substrate, through Fl
Biotin-UTP uptake by uor-S Multi-imager (Bio-Rad)
The amount of detection was determined. As a control, tetrakis-3- (aminopropyl
L) Ammonium and cardopentamine are added
A similar experiment was performed with no reaction solution, and the results are described below.
The results are shown in Table 1. [Example 2] In vivo reaction was carried out using the following reaction system.
Polyamines found from thermophiles in Toro transcription reaction
Was tested for its reaction promoting effect. Specifically, two types of
Tetrakis-3-, a polyamine found from thermophiles
(Aminopropyl) ammonium and cardopentamine are
Final concentrations should be 0.05, 0.1, 0.2 and 0.4 mM respectively
40mMHepes-KOH (pH 7.5 at 25 ° C), 4mM Mg (OAc) Two ,Ten
0 mM KGlu, 5 mM DTT, 0.5 mM rNTPs, 5 μM Biotin-UTP,
50U Thermo T7 RNA polymerase (Toyobo),
4U RNasin (promega), T7 prom as template DNA
100 ng of 500 bp dsDNA containing oter
Prepare a 20 μl transcription reaction solution and react at 60 ° C for 30 minutes.
I let you. The template DNA is plasmid pBluescript (Strata
Gene7) as a template for the T7 promoter
Primers to contain
Got ready. After the reaction, add 2 μl of 500 mM EDTA to this solution.
To stop the reaction, and further add 10 μl of 2.5 mM LiCl, H 2 O 18μ
l, EtOH 100μl, add vortex mixer and mix
After standing at 30 ° C for 30 minutes, centrifuge at 12,000 rpm, 4 ° C for 20 minutes.
NA was precipitated. Rinse precipitated RNA with 70% EtOH
Then, ethanol was removed with a vacuum centrifugal dryer. Recovered raw
H for adult RNA Two Add 40 μl of O (RNase free) to dissolve
2 μl of this was applied to Hybond N + membrane (Amersham Far
Macia Biotech). By irradiating the membrane with ultraviolet light,
RNA transcript is immobilized on the membrane surface and
After treating the membrane with the solution, streptavidin
-Luminescent group via alkaline phosphatase (Wako Pure Chemical Industries)
Fluor-S Multi-im by emitting CDP-Star (Roche)
Detection and quantification of Biotin-UTP uptake by ager (Bio-Rad)
Was done. As a control, tetrakis-3- (aminopropyl
L) Ammonium and cardo pentamine are added
A similar experiment was performed with no reaction solution, and the results are shown in Table 1 below.
It was shown to. [Table 1] From the results shown in Table 1, it was found that tetrakis-3- (amido
Nopropyl) ammonium or cardo pentamine
At 37 ° C., about 2
Remarkable transcription of about 6.5 to 7.5 times at 60 ° C
It was shown to have a reaction promoting effect. REFERENCE EXAMPLE Polyamine found from a thermophilic bacterium
[Reference Example 1] Tetrakis (3-aminopropyl) ammonium
Synthesis of chloride [Reference Example 1-1] Synthesis of tris (3-aminopropyl) amine Hydrogen was added to 150 mL of THF as shown in the following chemical reaction formula.
5.93 g (156.3 mmol) of lithium aluminum chloride were dissolved
3,3 ', 3''-Nitrilotrispropionamide (1) in solution
6.00 g (26.06 mmol) was added and refluxed for 24 hours under an argon atmosphere
I let it. In addition, H 2 Add O at 25 ° C for 2 hours
Stir, filter the resulting solid and wash with ethanol several times.
After purification, 1M hydrochloric acid was added. What was collected by filtration under reduced pressure
After removing the solvent, the compound (tris (3-aminopropyl) a
Min) (2) (10.62g crude) was obtained as a yellow oil.
Was. Embedded image Reference Example 1-2 Tris- (3-phthalimide
Synthesis of propyl) 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),
45.83 g (309.5 mmol) of phthalic anhydride, 12.70 of sodium acetate
g (154.8 mmol) in an argon atmosphere at 170 ° C. for 2 hours
For a while. Then, cool to room temperature, 200 mL of H 2 O
Was added. The resulting suspension was heated and boiled for 15 minutes
Thereafter, the pH was adjusted to 8 with an aqueous sodium hydrogen carbonate solution. Get
The tan solids removed from the aqueous layer by filtration,
The filtrate was washed and extracted with ethyl acetate, and dried over anhydrous magnesium sulfate.
Then, the solvent was removed under reduced pressure. The residue obtained
Is a silica gel using hexane-ethyl acetate as a developing solvent.
Compounds separated and purified by column chromatography
(Tris- (3-phthalimidopropyl) amine) (3) (5.0
5g, yield 33.5%) as a white powder. The NMR (1H-NMR,
13C-NMR), FAB-MS, and melting point are as follows. 1H-NMR (400 MHz, CDCl3); δ 1.80 (6H, m), 2.5 (6H, m), 3.74 (6
H, m), 7.68 (6H, m), 7.80 (6H, m) 13C-NMR (400 MHz, CDCl3); δ 26.60, 36.78, 51.68, 123.51,1
32.67,134.11,168.71 FAB-MS; m / z579.36 (M ++ H) mp150-151 ° C Reference Example 1-3 N- (3-iodopropyl) phenyl
Synthesis of Talimide As shown in the following chemical reaction formula, dry acetone
2.80 g (18.65 mmol) of sodium iodide was dissolved in 20 mL
1.00 g (3.7 g) of N- (3-bromopropyl) phthalimide (4) was added to the solution.
3 mmol) and refluxed for 2 hours under an argon atmosphere.
Thereafter, the reaction solution was cooled to room temperature, and H 2 O, and add
Extract three times with chill and dry over anhydrous magnesium sulfate
The solvent was removed under reduced pressure. Almost quantitatively, the compound (N- (3-yo
Propyl) phthalimide) (5) as a yellow powder.
Was done. The NMR (1H-NMR,
13C-NMR), FAB-MS, and melting point are as follows. 1H-NMR (400MHz, CDCl3); δ 2.25 (2H, m), 3.17 (2H, t), 3.78
(2H, t), 7.73 (2H, m), 7.85 (2H, m) 13C-NMR (400 MHz, CDCl3); δ 1.12, 32.54, 38.63, 123.31, 13
1.98,134.04,168.21 FAB-MS; m / z316.22 mp88-89 ° C Reference Example 1-4 Tetrakis (3-phthalimiimi)
Synthesis of (propyl) ammonium iodide 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,
The mixture was stirred at 200 ° C. for 3 hours under a nitrogen atmosphere. White solid formed
The substance was collected by filtration and washed with anhydrous ethyl acetate to remove the solvent.
This solid is dissolved again in anhydrous dioxane and refluxed for 1 hour
After that, the precipitate was filtered. Repeat this operation several times
Was. By drying the obtained precipitate, the compound (tetra
Kis (3-phthalimidopropyl) ammonium iodide
(6) (3.23 g, yield 76.2%) was obtained as a white powder.
Was. The NMR (1H-NMR,
13C-NMR) and FAB-MS are as follows. 1H-NMR (400 MHz, DMSO-d6); δ 1.92 (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 / z 766.49 (M ++ H) Reference Example 1-5 Tetrakis (3-aminopro
Synthesis of pyr) ammonium chloride As shown in the chemical reaction formula shown in the following chemical formula 9, the compound (tetra
Kis (3-phthalimidopropyl) ammonium iodide
(6) 0.30 g (0.34 mmol) of hydrazine 0.33 mL (10.34 mmol)
Add to 6 mL of anhydrous ethanol solution containing under argon atmosphere
Refluxed for 2 hours. After cooling the reaction solution to room temperature, 1M salt
The acid was added. The resulting precipitate solid is filtered, and the filtrate is depressurized.
It was concentrated below. The resulting residue is washed with Dowex 50W-X4 (H + form)
After subjecting to ram, elution was carried out with 6M hydrochloric acid. Fractionated image
The fraction was analyzed by HPLC, and the fraction containing the quaternary amine was
The compound (tetrakis) was concentrated at 40 ° C under reduced pressure and dried to dryness.
(3-aminopropyl) ammonium chloride) (7) (0.01
8g, 12.4% yield) as a yellow powder. Embedded image REFERENCE EXAMPLE 2 Synthesis of cardo pentamine As shown in the chemical reaction formula shown in the following chemical formula 10, N, N-bis (3-
(Aminopropyl) -1,3-propanediamine (8) 2.50 mL (12.
(21 mmol) was dissolved in 120 mL of 2-propanol.
4.00 g of N- (3-bromopropyl) phthalimide (9)
(14.92 mmol) was added to the mixture for 20 hours under an argon atmosphere.
Reflux for a while. After cooling the reaction solution to room temperature, 12M hydrochloric acid was added.
Added and refluxed for another 10 hours. Thereafter, the solvent was removed from the mixture under reduced pressure.
Was. H is added to the obtained residue. 2 10 mL of O was added and the precipitate was filtered.
Was. The filtrate was concentrated under reduced pressure, and the obtained residue was Dowex 50W-X4 (H
+ form) After applying to the column, elution was carried out with 3M hydrochloric acid.
The fraction collected is analyzed by HPLC and contains cardopentamine.
The fraction was concentrated under reduced pressure at about 40 ° C. and dried. The residue obtained is
By recrystallizing with ethanol: methanol (= 1: 1)
Compound (cardopentamine) (10) (0.079 g, yield 15.1%)
Was obtained as a white powder. Embedded image The present invention relates to an in vitro transcription reaction.
To add polyamines found from thermophiles.
This makes it possible to increase the amount of RNA synthesis.
Therefore, RNA transcripts in in vitro transcription
By increasing the amount of genetic material, only a small amount of sample can be obtained.
It is effective for analysis of offspring and preparation of probes. Also high
It also promotes the transcription reaction in the temperature range, thus stabilizing the reaction.
Gene analysis for long reaction and automation
It is particularly effective in fields related to genetic diagnosis
Has the effect.

   ────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Yuyasu Yoneda             1-5, Kandanishikicho, Chiyoda-ku, Tokyo Fri             Gonishikicho Building Nippon Genetec Co., Ltd.             Inside (72) Inventor Yasuo Oshima             1826-2 Takagasaka, Machida City, Tokyo (72) Inventor Masanori Wataniki             1-5, Kandanishikicho, Chiyoda-ku, Tokyo Fri             Gonishikicho Building Nippon Genetec Co., Ltd.             Inside F term (reference) 4B024 AA20 BA80 CA01 CA04 CA11                       HA01

Claims (1)

  1. Claims: 1. A method for obtaining a nucleic acid transcript by mixing an RNA polymerase and a template DNA having a corresponding promoter sequence, comprising the steps of: Aminopropyl) ammonium or a salt thereof and / or cardopentamine or a salt thereof represented by the chemical formula 2
    An in vitro transcription reaction method comprising performing a transcription reaction as described above. Embedded image Embedded image 2. The in vitro transcription reaction according to claim 1, wherein said tetrakis (3-aminopropyl) ammonium or a salt thereof and / or cardopentamine or a salt thereof are obtained by chemical synthesis. 3. The method of claim 3, wherein 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.
JP2001325016A 2001-10-23 2001-10-23 In vitro transcription reaction method Pending JP2003125767A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006197255A (en) * 2005-01-13 2006-07-27 Nec Corp Local content connection system, mobile terminal, method for connecting local content, and client program
US10034951B1 (en) 2017-06-21 2018-07-31 New England Biolabs, Inc. Use of thermostable RNA polymerases to produce RNAs having reduced immunogenicity

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006197255A (en) * 2005-01-13 2006-07-27 Nec Corp Local content connection system, mobile terminal, method for connecting local content, and client program
JP4561983B2 (en) * 2005-01-13 2010-10-13 日本電気株式会社 Local content connection system, mobile terminal, local content connection method, and client program
US10034951B1 (en) 2017-06-21 2018-07-31 New England Biolabs, Inc. Use of thermostable RNA polymerases to produce RNAs having reduced immunogenicity

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