JP3391629B2 - Method for synthesizing polydeoxyribonucleotide - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for synthesizing polydeoxyribonucleotide (DNA) in a template-independent and primer-independent manner, in which genetic information is created depending only on the information possessed by a protein. To enable.

[0002]

PRIOR ART AND PROBLEM TO BE SOLVED BY THE INVENTION Presently, D
There are at least two types of methods for synthesizing NA. It is a method of using a DNA synthase or a method of chemically synthesizing it. A method for synthesizing DNA using a DNA synthase is, for example, a template DN using DNA polymerase.
There is a so-called “template-dependent DNA synthesis method” such as a method of synthesizing DNA from A and a method of synthesizing DNA from template RNA by a reverse transcriptase. However, in any of these methods, a primer composed of a short single-stranded DNA or RNA and a single-stranded template DNA were required to synthesize DNA. Recently, the polymerase chain reaction method or the PCR method [Saiki et al., Science (Sc
ience), vol. 239, pp. 487-491, (1
988)], a method for easily amplifying a template DNA has been developed, but the template DNA and the primer are still required. Also, since this primer can only be made by chemical synthesis, currently D
A chemical synthesis method is mainly used for synthesizing NA. As a method of chemically synthesizing the phosphonate, there is a method of synthesizing by the phosphoric acid diester method, the phosphoric acid triester method or the like, but these are complicated in the synthetic process, take time, and can synthesize only about several tens of bases. D synthesized above
NA is difficult to purify. In addition, although a device is commercially available for this synthesis, there is a drawback in that an expensive and special reagent must be used. In addition, although it has not been attempted at present, even if one relies on a chemical synthesis method to create DNA, this method can produce thousands of bp (base pair,
It is virtually impossible to synthesize a DNA having a base pair), and even if it can be synthesized, it can be expected to be very expensive. Therefore, it can be expected that it is impossible to create a protein by expressing the created DNA.

Next, as a cell transformation technique, a method of inserting a gene for transformation into a vector derived from a plasmid or a virus and inserting the gene into a host cell has been mainly used at present. . In order to carry out this method, it is necessary to first isolate the gene from the chromosome or various gene libraries and then insert the gene into a vector. This operation is a very cumbersome, time-consuming and laborious task, and has often plagued scientists.

Recombinant DNA that supports the current genetic engineering
The technology is, for example, by incorporating an existing DNA (gene) into a vector capable of carrying a gene, introducing it into a host cell that does not have the gene, and expressing it in a protein, for example, in a host cell. It refers to technologies such as imparting resistance to environmental stress and producing a large amount of proteins that are naturally present in trace amounts. Using this technology, it is possible to use a naturally occurring protein in another host or mass-produce it in a host that is easy to culture, but to create a new protein that does not exist in nature ( In other words, you cannot create).

With the recent chemical DNA synthesis technology, it is almost impossible to synthesize a long DNA in a single reaction. Even if it is possible to synthesize a long DNA, many complicated steps and great cost are required. There is a drawback that the purity of the obtained DNA is extremely low. In addition, there is a possibility that a DNA having a long sequence can be synthesized by several times of reaction, but in this case, there is a disadvantage that some person's will is inevitably introduced into the sequence.

[0006] However, considering the next generation of current genetic engineering, the present inventors consider that the emergence of more useful and effective proteins over existing genetic material which is a product of evolution is indispensable. . Therefore, the template-independent DNA synthesis method disclosed in the present invention is different from mere template DNA replication in that DNA is created depending only on the information that the protein has (that is, its amino acid sequence or three-dimensional structure). Was intended to create freely non-naturally occurring genes and to create more useful non-naturally occurring proteins from this gene.

[0007]

The present inventors have made various discoveries in the study of DNA synthesis. For example, Thermococcus litoralis DNA polymerase (New England Bio-Raves) and buffer A [10 mM
KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris / HCl (pH 8.8), 6 mM MgCl ₂ ,
0.1% Triton X-100 (all final concentrations)] and deoxyribonucleotides [dATP, dCTP, dGT
P and dTTP (all final concentration 200 μM)] was reacted at 74 ° C. for 3 hours, and a part of the reaction mixture was electrophoresed on agarose (1%) gel, stained with ethidium bromide, and then irradiated with ultraviolet rays. Observed under the lamp. Then, about 1 kbp (1000 base pairs) to 30 k as shown in the photograph in Figure 1.
A DNA band stained in a wide range of bp was confirmed.

Next, a reaction solution containing no deoxyribonucleotide triphosphate was prepared, and deoxyribonuclease I or ribonuclease A was added to the reaction solution, and the mixture was kept at 37 ° C. for 2 hours.
The reaction was allowed to proceed for a period of time to completely decompose DNA or RNA (which may be a primer or template) that may be present as an impurity in the reaction solution and the enzyme. Then, deoxyribonucleotide triphosphate was added and the same reaction as above was performed. If the reaction is template / primer dependent, then DNA synthesis should no longer occur. However, if it is template / primer-independent, a DNA synthesis reaction occurs, and a DNA band should be confirmed as a result of agarose gel electrophoresis. As a result, by agarose gel electrophoresis, a band stained in a wide range of about 1 kbp to 30 kbp was confirmed, and it was confirmed that this reaction was independent of template / primer. In other words, this reaction is independent of DNA or RNA that can serve as a template or primer
It turned out that it was done according to the information that creates A.

Next, this Thermococcus litoralis D
Template / primer independent DN by NA polymerase
In the A synthesis reaction, it was examined by changing the reaction conditions whether or not a DNA having a size other than about 1 kbp to 30 kbp can be synthesized. That is, when deoxyribonuclease I (DNaseI) was added to a normal reaction solution and reacted at 37 ° C. for 30 minutes and then at normal 74 ° C. for 3 hours, brominated around 10 kbp as shown in FIG. A DNA band that was strongly stained by ethidium appeared. From this result, it was confirmed that the length of the generated DNA can be arbitrarily changed depending on the reaction conditions.

Next, the DNA of Thermococcus litoralis
The time course of template / primer independent synthesis reaction by polymerase was confirmed. As shown in FIG. 6, the reaction did not occur until 1 hour after the reaction, and a so-called lag period was observed.
The reaction started after 2 hours. After 2 hours, 3
The maximum reaction rate was observed by the hour. The reaction rate at this time was 0.88 bases / second / one molecule of enzyme, which is 76 times the normal template-primer-dependent DNA polymerization reaction rate of Thermococcus litoralis DNA polymerase.
1 [Kong et al., The Journal of Biological Chemistry (J. Biol. Chem.), 268, 1965-1975, (1993)], 1 / 57th of E. coli DNA polymerase I. [Cornberg, DNA Replication Second Edition (DNA Rep
license 2nd ed), W. H. Freeman & Company, San Francisco, CA,
(1992)], which is considered to be a completely different reaction from the ordinary template / primer-dependent DNA synthesis reaction.

Next, the D independent of this template / primer
In the NA synthesis reaction, the specificity for the substrate deoxyribonucleotide triphosphate (ie, the substrate specificity of this reaction) was examined. Prepare 4 kinds (A, T, G, C) of deoxyribonucleotide triphosphates individually or in combination with 2, 3 and 4 kinds of deoxyribonucleotide triphosphates, and subject them to ordinary reaction. A portion of this was electrophoresed on an agarose (1%) gel and then stained with ethidium bromide. As shown in FIG. 2, in addition to the addition of four types of deoxyribonucleotides, three types of dATP, dCTP and dTTP, or d
A DNA band was confirmed in the reaction solution containing three kinds of ATP, dGTP and dTTP added. However, there was a difference in the distribution of DNA bands from the reaction in which all four types were added and the latter two in which three types of deoxyribonucleotide triphosphates were added. In other words, the size of the former DNA is about 1k
Whereas the latter is distributed in the range of bp to 30 kbp, the latter is distributed in the range of about 10 kbp or more, and the size larger than that is hardly recognized. Therefore, it was suggested that these template / primer-independent DNA synthesis products may be of different types. That is, it is suggested that this template / primer-independent DNA synthesis reaction may include a plurality of different reactions.

Next, in order to prepare a material for investigating the characteristics of the template / primer-independent synthetic DNA (hereinafter abbreviated as ntp-DNA) of the present invention, a large amount of the DNA was prepared. Also, as a method for purifying DNA, cesium chloride-ethidium bromide equilibrium density gradient centrifugation [Samrock et al.
Molecular Cloning (A Laboratory Manual) (Molecular Cloning (A
Laboratory Manual)), Cold Spring Harbor Laboratory Press, 19
1989 issue] was used. As a result, 9.5 mg of DNA could be recovered from 475 ml of reaction solution. Using the sample prepared in this manner, S1 nuclease or mang which is a single-strand-specific DNA nuclease is then used to examine whether the template-primer-independent DNA synthesis product is single-stranded or double-stranded. Cleavage with bean nuclease was performed. As a result, as shown in FIG. 7, ntp-DNA could not be cleaved by these single-strand-specific nucleases. From this, it was suggested that this template / primer-independent DNA may be double-stranded DNA.

Next, in order to investigate the structure of ntp-DNA, circular dichroism spectrum analysis and observation with a transmission electron microscope were carried out. First, regarding circular dichroism spectrum analysis, as shown in FIG. 8, it was almost the same as the typical B-type double-helix structure DNA in which the plasmid DNA used as a target was linearized by a restriction enzyme. Is similar,
Therefore, this ntp-DNA is a DN of B type double helix structure.
It was confirmed to be A. Next, as a result of observation with a transmission electron microscope, as shown in FIG. 9, it was confirmed that the DNA was linear DNA and the diameter thereof was double-stranded.
From these analyzes, the present inventors determined that the template-primer-independent synthetic DNA was a double-stranded linear DNA.

Next, the base composition of ntp-DNA was analyzed. First, ntp-DNA was cleaved (hydrolyzed) by deoxyribonuclease I, phosphodiesterase I, and alkaline phosphatase to decompose various deoxyribonucleosides. This is Cosmosil 5C
₁₈ Chromatograph (high performance liquid chromatography, manufactured by JASCO Corporation) on a reverse phase column (manufactured by Nacalai Tesque),
Various deoxyribonucleosides were quantified. as a result,
As shown in Table 1, the base composition is 3 for deoxyadenosine.
4.4%, deoxycytosine 15.6%, deoxyguanine 17.4% and deoxythymidine 32.6%, thus the GC content was 33%.

Next, the frequency of adjacent bases of ntp-DNA (n
(earest neighbor frequency)
Was analyzed. [Α- ³² P] -labeled deoxyribonucleotide triphosphate was added to a usual reaction solution to carry out a usual reaction.
This reaction solution was applied to a Superose 12 gel filtration column (manufactured by Pharmacia), and a DNA solution was separated from the DNA peak. This solution was purified by ethanol precipitation and dissolved in TE buffer. This labeled DNA was cleaved with micrococcal nuclease and pancreatic phosphodiesterase and decomposed into deoxyribonucleoside-3'-monophosphate. This was subjected to a Cosmocil 5C ₁₈ reverse phase column, and deoxyadenosine-3'-monophosphate (dAp), deoxycytidine-3'-monophosphate (dCp), deoxyguanosine-3'-monophosphate (dGp), deoxythymidine. Each peak of -3'-monophosphate (dTp) was collected. Then, the radioactivity of each ³² P was measured with a liquid scintillation counter (manufactured by Beckman), and the adjacent base frequency was analyzed. As a result, as shown in Table 2, an array having a certain regularity was obtained. That is, d before dA
T and dG frequently appeared, dT frequently appeared before dC, dA frequently appeared before dG, and dA and dC often appeared before dT. . From this,
It was confirmed that there was a bias in the analyzed ntp-DNA sequence.

Next, molecular cloning of this ntp-DNA was carried out. As a cloning vector, pUC
19 was cleaved with a restriction enzyme Sma I (manufactured by Toyobo Co., Ltd.). As the insert DNA, ntp-DNA
Was blunt ended with DNase I (flush end,
Blunt end), dephosphorylated with alkaline phosphatase (Takara Shuzo), and then again phosphorylated with T4 polynucleotide kinase (Toyobo), and finally into Klenow fragment. It was used after being repaired. This inserted DNA was ligated to the above-mentioned cloning vector, and the cloned plasmid was confirmed by cutting with a restriction enzyme and the presence or absence of the inserted DNA was confirmed by electrophoresis. As a result, a clone in which the DNA fragment of interest was integrated can be selected, and the present inventors have found that the plasmid pTL34,
It was named pTL57 and pTL85.

Next, plasmids pTL34 and pTL57
And the nucleotide sequence of the inserted DNA portion of pTL85 was determined. The nucleotide sequence was determined by the dideoxy terminator method [Sanger et al., Proceedings of the National
Academy of Sciences of the USA
(Proc. Natl. Acad. Sci. USA),
Vol. 74, 5463-5467, (1977)]. The determined nucleotide sequences are shown in SEQ ID NOs: 1, 2 and 3 in the sequence listing (Table 3). Next, this DNA
The sequence was searched for homology to existing DNA. The results are disclosed in Table 4, Table 5 and Table 6 (same order). This indicates that this DNA sequence is conserved in the chromosomes of many organisms including humans. Also, it is interesting to note that this sequence is also contained in gene regulatory sites such as microsatellite DNA existing near the centromere.

Next, in order to confirm whether or not this ntp-DNA can be used as a probe for searching a tomato chromosome library having a sequence homologous to or similar to the nucleotide sequence of this DNA, for example, , This nt
The phosphate residue at the 5'end of p-DNA was replaced with [γ- ³² P] AT.
A probe for searching was prepared by labeling with P. As the labeled probe, various fluorescent labeled probes may be used. Tomato chromosome c using this probe
As a result of screening the DNA λ phage library by plaque hybridization, many positive signals were detected. As a result of isolating λ phage from the plaque in which this positive signal was detected and determining the nucleotide sequence, it was confirmed that the sequence contained ntp-DNA as expected. From this, this ntp-DN
It was confirmed that A can be a probe that can easily screen homologous or similar sequences existing in various cDNA libraries.

Next, the following experiment was carried out in order to confirm that ntp-DNA has a high frequency recombination activity. In addition, the gene manipulation method of yeast used in the experiment is described by Rose et al. [Methods in East Genetics (A Laboratory Course Manual) (Method).
s in Yeast Genetics (A Lab
oratory Course Manual)),
Cold Spring Harbor Laboratory Press, (1990)]. First, the β-galactosidase gene is amplified from the plasmid pCH110 (Pharmacia) by the PCR method. However,
On the 5'end side of the upstream primer, baker's yeast ( Sacch
GAL1 of Aromyces cerevisiae )
A promoter sequence is added, an antisense sequence of the transcription termination signal CYC1 of baker's yeast is added to the 5'end of the downstream primer, and both are aligned with the codon of the β-galactosidase gene in frame. By this operation, β
-The galactosidase gene will be expressible in yeast. Next, ntp-DN was added to the 3'OH end of the double-stranded DNA containing the β-galactosidase gene by, for example, Thermococcus litoralis DNA polymerase.
A is synthesized. This operation uses DNA ligase to add ntp-DN at both ends of the β-galactosidase gene.
It is also possible to substitute by connecting A. Thereafter, this is transfected into baker's yeast, and the yeast cells are cultured in a plate medium. From this plate, yeasts were separately cultured and the β-galactosidase activity was measured. If β-
When the galactosidase activity is confirmed, it means that β-galactosidase, which yeast originally does not have, is expressed. The transfected DNA does not have a yeast origin of replication and therefore cannot replicate in yeast cells. Therefore, β-galactosidase in yeast cells is considered to have been expressed by the gene being integrated into the yeast chromosome. As a result of measuring the β-galactosidase activity of yeast, it was confirmed that the β-galactosidase activity was indeed detectable, and that the gene was integrated into the yeast chromosome (Table 7). From these things, this ntp-D
NA has a high frequency of recombination,
It was demonstrated that the protein can be expressed from the inserted gene without the need for a special expression vector. According to the present invention, it becomes possible to express a target protein without the need for the complicated work of constructing an expression vector. This method can also be applied to organisms other than yeast (for example, E. coli and other prokaryotes as well as eukaryotes).

The present invention is based on many new discoveries including the above findings, and is a method for synthesizing polydeoxyribonucleotide characterized by polymerizing deoxyribonucleotide in the absence of template and primer, and in the presence of protein. , A polydeoxyribonucleotide synthesized by the method, and a novel D by culturing cells transfected with the polydeoxyribonucleotide
It relates to a method of creating NA, its DNA and its use.

The npt-DNA synthesis method of the present invention is carried out in the absence of a template and a primer, and in the presence of a protein. The polymerization reaction proceeds with deoxyribonucleotide polymerase, but the polymerase itself may be used as the protein, or a protein different from the polymerase may be used. DNA polymerase is about 60 ° C
As described above, those which are not inactivated at a temperature of preferably about 70 ° C. or higher are preferable, and examples thereof include Thermococcus litoraris .
Examples thereof include deoxyribonucleotide polymerases of bacteria belonging to the genus Sermococcus, such as Cermococcus , Thermus aquaticus , and T. thermophilus .

As a deoxyribonucleotide, dAT
P, dTTP, dGTP and dCTP are used, and the reaction proceeds if four or three of them are present in the reaction system. The reaction between deoxyribonucleotide and deoxyribonucleotide polymerase has a pH of about 7 or 1.
It is preferably carried out with a weak alkaline of 0 or less. The reaction temperature and time can be selected within a range in which the polymerase is not inactivated, and the reaction can be rapidly advanced by conducting the reaction at a temperature rise of about 60 ° C. or higher at which the polymerase activity is exhibited. For example, the reaction may be carried out at 74 ° C. for several hours, or conditions for ordinary PCR reaction, for example, (1) 9
Hold at 5 ° C for 1 minute, (2) Hold at 45 ° C for 2 minutes, (3)
The cycle of holding at 74 ° C for 3 minutes may be repeated. The reaction is initially initiated after some lag time and eventually reaches a maximum rate. Nt synthesized by the method of the present invention
p-DNA is DNA or RN that should serve as a template or primer.
It is considered that it is synthesized without depending on A, depending on the information of the protein (polymerase) present in the reaction system.

The size of the synthesized DNA is generally 1 kb.
It is distributed from p to 30 kbp, but its size can be changed depending on the reaction conditions. For example, substrate D
If NA of dATP, dGTP, dCTP, dTTP is used, a DNA of 1 to 30 kbp can be obtained, and one of the four types lacks three types, for example, dA.
If TP, dCTP and dTTP are used as substrates, about 10 kb
p DNA can be obtained, and deoxyribonuclease I can be added to a reaction system using 4 kinds of DNA as substrates to obtain about 10 kbp DNA.

The ntp-DNA product thus obtained is a double-stranded DNA, and its base composition is deoxyadenine (dA) 34.4 according to the example (Example 12).
%, Deoxycytidine (dC) 15.6%, deoxyguanine (dG) 17.4%, deoxythymidine 32.6
%, The sum of dG and dC is 33%. Further, in the nucleotide sequence of DNA in one example (Example 14), dTd
The frequencies of A, dTdC, dAdG, dAdT, and dCdT sequences are high (Example 13, Table 2).

Since the synthesized npt-DNA has a high frequency recombination activity, it is possible to express a protein by npt-DNA by transfecting, for example, yeast, Escherichia coli or other prokaryote or eukaryote without using an expression vector. You can

Further, the ntp-DNA is labeled with, for example, a radioisotope, a fluorescent dye or the like, and the human chromosome library, tomato chromosome library or other cDNA is labeled.
It can be used as a probe for screening a library having a sequence homologous to or similar to ntp-DNA. The screening can be performed by a known method using, for example, plug hybridization and Southern blotting.

[0027]

The present invention will be further described below in the form of examples, but the present invention is not limited thereto.

Example 1 Synthesis buffer ntp-DNA by Thermococcus litoralis DNA polymerase A [10 mM KCl, 10 mM (NH ₄ ) ₂
SO ₄ , 20 mM Tris / HCl (pH 8.8), 6
mM MgCl ₂ , 0.1% Triton X-100 (all final concentrations)], deoxyribonucleotide [dAT
P, dCTP, dGTP, and dTTP (all final concentration 200 μM)], 20 units / ml to 100 μl reaction solution containing Thermococcus litoralis DNA polymerase, an equal amount of mineral oil (manufactured by Sigma) was added, and 7
Incubate for 3 hours at 4 ° C. Then, add 20 μl of reaction solution to 1
% Agarose, applied to Tris / acetic acid / EDTA gel [Samrock T, etc., Molecular Cloning (A Laboratory Manual) (Molecular Cl)
oning (A Laboratory Manual
l)), Cold Spring Harbor Laboratory Press, 1989], and 0.5 μ after electrophoresis.
It was stained with g / ml ethidium bromide and photographed. As a result, about 1 kbp to 30 kbp as shown in the photograph in Figure 1
, A DNA band stained in a wide range was confirmed.

Example 2 Synthesis of ntp-RNA by Thermococcus litoralis DNA polymerase using ribonucleotides Buffer A, ribonucleotides [ATP, CTP, GT]
P and TTP (all final concentrations of 200 μM)], 20 units / ml To 100 μl of reaction solution containing Thermococcus litoralis DNA polymerase, an equal amount of mineral oil is added, and the mixture is reacted at 74 ° C. for 3 hours. Then, 20 μl of the reaction solution was added to 1% agarose, Tris / acetic acid / EDTA.
It was applied to a gel, and after electrophoresis, it was stained with 0.5 μg / ml ethidium bromide and photographed. As a result, it was confirmed that the stained RNA band did not appear, and therefore, in this template / primer-independent synthesis reaction, ribonucleotides cannot serve as substrates.

Example 3 Effect of Deoxyribonucleotide on Synthesis of ntp-DNA by Thermococcus litoralis DNA Polymerase Buffer A, Deoxyribonucleotide [dATP, dC
Any one of TP, dGTP and dTTP 1, 2, 3 and 4 (final concentration 200 μM)], 20 units / ml
An equal amount of mineral oil is added to 100 μl of the reaction solution containing Thermococcus litoralis DNA polymerase, and the mixture is reacted at 74 ° C. for 3 hours. Then, 10 μl of each reaction solution was applied to 1% agarose, Tris / acetic acid / EDTA gel, and after electrophoresis, stained with 0.5 μg / ml ethidium bromide and photographed. As a result, as shown in FIG. 2, when all 4 types, 3 types of dATP, dGTP, and dTTP and 3 types of dATP, dCTP, and dTTP were used, a template / primer-independent synthetic DNA band was confirmed. However, no ntp-DNA band could be confirmed when 1, 2, or 3 types of deoxyribonucleotides other than the above were used. Therefore, it was confirmed that in this ntp-DNA synthesis reaction, there is specificity for the substrate deoxyribonucleotide triphosphate (that is, the substrate specificity of this reaction). Moreover, in the reaction in which all four kinds of deoxyribonucleotide triphosphates were added, the size of the synthesized DNA was distributed in the range of about 1 kbp to 30 kbp, whereas in the reaction in which three kinds of deoxyribonucleotide triphosphates were added, It was distributed in the range of about 10 kbp or more, and almost no size larger than that was recognized. The difference in the distribution of these DNA bands suggested that these ntp-DNA synthesis products may be of different types. That is, it was suggested that this template / primer-independent DNA synthesis reaction may include a plurality of different reactions.

Example 4 Ntp-DNA synthesis buffer A by various DNA polymerases, deoxyribonucleotides [dATP, dC]
TP, dGTP, and dTTP (all final concentration 200μ
M)], various DNA polymerases [20 units / ml Thermococcus litoralis DNA polymerase, or
25 units / ml Natural Pyrococcus friosas DNA
Polymerase or 25 units / ml Recombinant Pyrococcus friosas DNA polymerase or 50 units / ml Selmus aquaticus DNA polymerase or 40 units / ml Selmus thermophilus D
NA polymerase (all final concentrations)]
An equal amount of mineral oil is added to μl of the reaction solution, and the mixture is reacted at 74 ° C. for 3 hours. Separately, buffer B [60 mM phosphate buffer (pH 7.4), 6 mM MgCl ₂ ], deoxyribonucleotides [300 μM dATP and dTT]
P and 1.5 mM dGTP and dCTP (all final concentrations)] and 2.5 units / ml of E. coli DNA polymerase I to 100 μl of reaction solution, an equal amount of mineral oil was added, and the mixture was reacted at 37 ° C. for 3 hours. Then, 10 μl of each reaction mixture was added to 1% agarose, Tris / acetic acid / ED.
It was applied to a TA gel, and after electrophoresis, it was stained with 0.5 μg / ml ethidium bromide and photographed. As a result, as shown in FIG. 3, in the natural DNA polymerase and the recombinant DNA polymerase from Thermococcus litoralis and the closely related archaebacterium Pyrococcus friosas, no band of the template / primer-independent synthetic DNA was confirmed. On the other hand, DNA bands of several bp to 10 kbp and several bp to 7 kbp (in the same order) were confirmed in the DNA polymerases from the prokaryotic thermophilic bacteria Cermouth aquaticus and Selmus thermophilus, respectively. However, no DNA band was confirmed in the DNA polymerase from Escherichia coli, which is the same prokaryotic bacterium. From this,
It was confirmed that this template / primer-independent DNA synthesis reaction greatly differs depending on the type of polymerase. Moreover, in DNA polymerases other than the Thermococcus litoralis DNA polymerase, ntp-DN
It was proved that the synthetic reaction of A could occur, and it was shown that this reaction is a fairly universal phenomenon.

Example 5 Confirmation of Presence / Absence of DNA or RNA that May Be Mixed in Enzyme in ntp-DNA Synthesis Reaction Buffer A, 1 μg / ml Ribonuclease A (RNa
seA Washington) or 1.25 μg / ml
Deoxyribonuclease I (DNaseI, manufactured by Boehringer Mannheim Biochemical), 20 units /
An equal amount of mineral oil is added to 92 μl of a reaction solution containing ml Thermococcus litoralis DNA polymerase, and the mixture is reacted at 37 ° C. for 2 hours. Continued 2.5m
8 μl of M deoxyribonucleotide [dATP, dC
TP, dGTP, and dTTP (all final concentration 200μ
M)] is added and reacted at 74 ° C. for 3 hours. afterwards,
1 μl of 500 mM EDTA was added to 20 μl of the reaction solution,
This was applied to a 1% agarose, Tris / acetic acid / EDTA gel, and after electrophoresis, stained with 0.5 μg / ml ethidium bromide and photographed. Separately, as a target experiment to see the positive reaction of each nuclease, buffer A, 1 μg
/ Ml RNase A or 1.25 μg / ml DN
case I, 0.3 mg / ml transfer RNA (tRNA,
(Manufactured by Sigma) or 92 μl of a reaction solution containing 30 μg / ml Hin dIII-cut λDNA (λ / Hin dIII) manufactured by Toyobo) is prepared, and the same reaction and electrophoresis as described above are performed. As a result, as shown in FIG.
It was confirmed that aseA and DNaseI can certainly cleave RNA and DNA (in the same order) in the buffer. Therefore, the reaction solution was treated with RNase A and DNas.
It was confirmed that RNA and DNA primers were certainly unnecessary for this reaction, since template / primer-independent synthesis also occurs for those that have been treated with eI and decomposed RNA and DNA that may coexist.

Example 6 Synthesis buffer 10 Kbp ntp-DNA by Thermococcus litoralis DNA polymerase A, 1.25 μg / ml DNase I, deoxyribonucleotide [dATP, dCTP, dGTP,
And dTTP (all final concentration 200 μM)], 20 units / ml to 100 μl of reaction solution containing Thermococcus litoralis DNA polymerase, an equal amount of mineral oil is added, and the mixture is allowed to react at 37 ° C. for 30 minutes. Then 74
After reacting at 0 ° C. for 3 hours, 20 μl of this was applied to a 1% agarose, Tris / acetic acid / EDTA gel, and 0.5% after electrophoresis.
It was stained with μg / ml ethidium bromide and photographed.
As a result, as shown in FIG. 5, a dark band appeared around 10 kbp. From this result, it was confirmed that the length of the generated DNA can be arbitrarily changed depending on the reaction conditions.

Example 7 Time transition of ntp-DNA synthesis reaction Buffer A, deoxyribonucleotide [dATP, d
CTP, dGTP, and dTTP (all at a final concentration of 200
μM)], [α- ³² P] -labeled deoxyribonucleotides [dATP, dCTP, dGTP, and dTTP (all final concentrations 100 nM, 3.3 × 10 ⁶ Bq / pmo).
l)], 20 units / ml to 50 μl of a reaction solution containing Thermococcus litoralis DNA polymerase, an equal amount of mineral oil was added, and each of them was adjusted to 0, 1,
React for 2, 3 and 4 hours. Then 500 mM ED
Stop the reaction by adding 2 μl of TA, and add 4 μl of this reaction solution.
l was added to 96 μl of calf thymus DNA and the acid insoluble fraction
The radioactivity of ³² P was measured with a liquid scintillation counter. As a result, as shown in FIG. 6, up to 1 hour after the reaction, there was no uptake of deoxyribonucleotides and 2
The capture appeared only after an hour. By 4 hours thereafter, about 9% of the added deoxyribonucleotide was incorporated. When the maximum reaction rate was calculated from the reaction from 1 hour to 2 hours, 0.88 bases were incorporated per protein molecule per second. This is Thermococcus
One-76th of the normal template-primer-dependent DNA polymerization rate of litoralis DNA polymerase, D of E. coli
1/57 of NA polymerase I
It is considered to be a completely different reaction different from the primer-dependent DNA synthesis reaction.

Example 8 Purification of ntp-DNA synthesis product Buffer A, deoxyribonucleotides [dATP, dC
TP, dGTP, and dTTP (all final concentration 200μ
M)], 20 units / ml An equivalent amount of mineral oil is added to 475 ml of a reaction solution containing Thermococcus litoralis DNA polymerase, and the mixture is reacted at 74 ° C. for 3 hours. Next, the same amount of chloroform as the mineral oil is added to the reaction solution, and the mixture is centrifuged at 17700 × g for 2 minutes, and the upper layer solution is transferred to another container. By this operation, the mineral oil was removed from the reaction solution, and the 3M sodium acetate solution was added to
Add 1 volume and 2.5 volumes of ethanol, and add 2 at -85 ° C.
Save time. This is centrifuged to collect the DNA, and 5
ml TE buffer [10 mM Tris / HCl (pH 8.
0) Dissolve in 1 mM EDTA]. This crude DN
Solution A was cesium chloride-ethidium bromide equilibrium density gradient centrifugation [Samrock et al., Molecular Cloning (A Laboratory Manual) (Molecular).
Cloning (A Laboratory Manu
al)), Cold Spring Harbor Laboratory Press, published in 1989]. As a result, 9.5 mg of ntp-DNA was obtained.

Example 9 Cleavage of ntp-DNA by single-stranded DNA-specific nuclease S1 buffer [50 mM acetic acid / NaOH (pH 5.
0), 30 mM NaCl, 1 mM ZnSO ₄ , 26
Unit / ml S1 nuclease (manufactured by Takara Shuzo)] or MB buffer [30 mM acetic acid / NaOH (pH 4.
6) 280 mM NaCl, 1 mM ZnSO ₄ , 4,
3 units / ml Mung bean nuclease (manufactured by Toyobo Co., Ltd.)], 58.5 μg / ml ntp-DNA, or
10 containing 20 μg / ml M13mp18 single-stranded DNA
The reaction solution (1 μl) is reacted at 37 ° C. for 5 minutes. Then, add 1 μm of 500 mM EDTA solution to stop the reaction.
1 was added to 1% agarose, Tris / acetic acid / EDTA gel, and after electrophoresis, it was stained with 0.5 μg / ml ethidium bromide and photographed. As a result, as shown in FIG. 7, the single-stranded DNA was cleaved and the band disappeared, but the ntp-DNA did not change before and after the reaction and was not cleaved by these nucleases. From this, n
tp-DNA is DNA other than single-stranded, for example, double-stranded D
The possibility of NA was suggested.

Example 10 Analysis of DNA double helix structure by circular dichroism of ntp-DNA Template / primer independent synthesis D prepared in Example 8
NA 700 μg in TE buffer [10 mM Tris / HC
1 (pH 8.0), 1 mM EDTA] 0.5 ml was dissolved and a circular dichroism spectrum was measured at room temperature using a circular dichroism dispersion meter J-500A (JASCO Corporation). As a result of this measurement, ntp-DN shown by the solid line in FIG.
A shows a spectrum almost similar to the circular dichroism spectrum of the linear pPT1 plasmid DNA that is typical of the typical double-stranded B-shaped DNA shown by the broken line. That is,
This means that the same "density" per unit concentration of this DNA
Suggests that there is a right-handed helix of
It was concluded that tp-DNA is double-stranded B-type DNA.

Example 11 Observation of ntp-DNA by an electron microscope Template-primer independent synthesis D prepared in Example 7
Using 0.05 μg NA, Sassenbach [Journal of Virology, J. Virol.
Vol., 1131-1138 (1973)], and observed using a transmission electron microscope. As a result, it was confirmed to be linear DNA as shown in FIG.

Example 12 Ntp-DNA base composition buffer C [25 mM Tris / HCl (pH 8.0),
25 mM MgCl ₂ ], 1.5 mg / ml ntp-
DNA, 50 units / ml DNaseI, 5 units / ml
Phosphodiesterase I, 2.3 units / ml 20 μl of reaction solution containing alkaline phosphatase (manufactured by Takara Shuzo) is reacted at 37 ° C. for 2 hours. After that, the total amount is P
4 ml of Cosmocil 5 equilibrated with solvent A (10 mM sodium dihydrogen phosphate: acetonitrile = 98: 2)
It was applied to a C ₁₈ (manufactured by Nacalai Tesque, Inc.) column and eluted with a PA solvent, and from each peak area of deoxyadenine (dA), deoxycytidine (dC), deoxyguanine (dG), and deoxythymidine (dT), The weight is quantified and the ratio of base composition is calculated. As a result, as shown in Table 1, dA was 34.4%, dC was 15.6%, dG was 17.4%, and dT was 32.6%, so that the GC content was 33%.

Example 13 Ntp-DNA adjacent base frequency analysis buffer A, deoxyribonucleotides [dATP, dC]
TP, dGTP, and dTTP (all final concentration 200μ
M)], [α- ³² P] -labeled deoxyribonucleotides [dATP, dCTP, dGTP, and dTTP (all final concentration 100 nM) 3.3 × 10 ⁶ Bq / pmo.
l)], 20 units / ml To 50 μl of reaction solution containing Thermococcus litoralis DNA polymerase, an equal amount of mineral oil is added and reacted at 74 ° C. for 3 hours.
Then, to stop the reaction, final concentration of 20 mM EDTA
Add. To this, phenol: chloroform (1:
1) was added, and the mixture was centrifuged at 17700 × g for 2 minutes, and 40 μl of this upper layer solution was applied to a 24 ml Superose 12 gel filtration column (Pharmacia) and eluted with TE buffer. To this, 550 μg / ml calf thorax DNA
3 μl of 3M sodium acetate solution and 0.1 times volume of 3M sodium acetate solution and 2.5 times volume of ethanol, and after mixing, the mixture is left to stand at −85 ° C. overnight. After centrifuging at 21600 × g for 40 minutes at 4 ° C., the precipitate is washed with 70% ethanol, dried and dissolved in 20 μl of TE buffer. Buffer MP for this sample
[20 mM succinic acid / NaOH (pH 6.0), 10
840 μl of a reaction solution containing mM CaCl ₂ , 19 units / ml micrococcal nuclease, 0.5 units / ml pancreatic phosphodiesterase (all final concentrations)]
React at 3 ° C for 3 hours. After which the total amount was subjected to Cosmosil _{5C 18} (Nacalai Tesque) column 4 ml, eluted with 50mM calcium formate, deoxyadenosine 3'monophosphate (dAp), deoxycytidine 3'-monophosphate (dCp ), Deoxyguanosine-
Peaks of 3'-monophosphate (dGp) and deoxythymidine-3'-monophosphate (dTp) are collected. The volume of this solution was adjusted to 1.5 ml, 200 μl of each was transferred to a ready cap (manufactured by Beckman), and after drying, the radioactivity of ³² P was measured with a liquid scintillation counter.
As a result, as shown in Table 2, there was a bias in the base sequence.

Example 14 Template / primer-independent synthetic DNA cloning buffer D [50 mM Tris / HCl (pH 7.6),
10 mM MnCl ₂ , 0.1 mg / ml bovine serum albumin (BSA)], 400 μg / ml ntp-D
NA, 0.5 containing 0.08 units / ml DNase I
The reaction solution of ml is reacted at 15 ° C. for 20 minutes. afterwards,
Add 500 mM EDTA solution to stop the reaction.
Add 0 μl, then add equal volume of phenol: chloroform (1: 1) and mix. Centrifuge at 9600 x g for 5 minutes and transfer the upper layer to another container. (The above is abbreviated as phenol treatment.) An equal amount of chloroform is added to this upper layer and mixed, and then the above-mentioned centrifugation is performed and the upper layer is transferred to another container. This upper layer was purified by a cesium chloride-ethidium bromide equilibrium density gradient centrifugation method, and the DNA was converted to TE buffer [10 mM Tris /
Dissolve in 40 μl of HCl (pH 8.0), 1 mM EDTA (designated as ntp-DNA / DNaseI). The above-mentioned dephosphorylation of DNA was performed. Dephosphorylation buffer [5
0 mM Tris / HCl (pH 9.0), 10 mM M
gCl ₂ ], 60 μg / ml ntp-DNA / DNa
seI, 100 units / ml Alkaline phosphatase (manufactured by Takara Shuzo Co., Ltd.), 100 μl of reaction solution at 65 ° C., 1
React for hours. After phenol treatment, add 0.1 volume of 3M sodium acidate solution to the reaction mixture and mix it to 2.5.
Add double volume of ethanol. Leave at -20 ° C overnight, 2
Centrifuge at 1600 × g for 30 minutes at 4 ° C. (hereinafter abbreviated as ethanol precipitation). The precipitate is washed with 70% ethanol, dried, and then dissolved in 10 μl of TE buffer (dephosphorylated ntp-DNA / DNaseI). D above
NA was phosphorylated. Phosphorylation buffer [50m
M imidazole / HCl (pH 6.4), 18 mM
MgCl ₂ , 5 mM DTT], 6% polyethylene glycol # 8000, 150 μM ATP, 120 μg /
40 μl of a reaction solution containing ml dephosphorylated ntp-DNA / DNaseI, 1.5 units / μl T4 polynucleotide kinase (manufactured by Toyobo Co., Ltd.) is reacted at 37 ° C. for 1 hour. Then DN by phenol treatment and ethanol precipitation
A is purified and dissolved in 10 μl of TE buffer (phosphorylated n
tp-DNA / DNaseI). Phosphorylated ntp-
Since there is a possibility that a double-stranded DNA has a single-stranded gap structure in some places in DNA / DNaseI, repair was carried out by DNA polymerase. Repair buffer [50 mM Tris / HCl (pH 7.5), 1
0 mM MgSO ₄ , 0.1 mM dithiothreitol, 50 μg / ml BSA], deoxyribonucleotides [dATP, dCTP, dGTP, and dTTP
(All final concentration 25 μM)], 120 μg / ml phosphorylated ntp-DNA / DNaseI, 166 units / ml Klenow fragment (manufactured by Toyobo Co., Ltd.), 50 μl of reaction solution is reacted at 25 ° C. for 15 minutes. Subsequently, the DNA was purified by phenol treatment and ethanol precipitation, and TE buffer 10
Dissolve in μl (named repair ntp-DNA / DNaseI). The above-mentioned phosphorylation of DNA was performed. Phosphorylation buffer [50 mM imidazole / HCI (pH 6.
4), 18 mM MgCl ₂ , 5 mM DTT], 6%
Polyethylene glycol # 8000, 150 μM AT
P, 120 μg / ml dephosphorylated ntp-DNA / DNa
40 μl of a reaction solution containing seI, 1.5 units / μl T4 polynucleotide kinase (manufactured by Tsukayo Boseki Co., Ltd.),
Let react for 1 hour. Subsequently, the DNA is purified by phenol treatment and ethanol precipitation and dissolved in 10 μl of TE buffer (phosphorylated ntp-DNA / DNaseI).
Double-stranded DNA is included in phosphate ntp-DNA / DNaseI.
Since there is a possibility that there is a gap structure in which a single strand is present in some of the sites, repair was carried out by DNA polymerase. Repair buffer [50 mM Tris / HCl (pH
7.5) 10 mM MgSO ₄ , 0.1 mM dithiothreitol, 50 μg / ml BSA], deoxyribonucleotides [dATP, dCTP, dGTP, and dTTP (all final concentration 25 μM)], 120 pg / m
1 phosphorylated ntp-DNA / DNaseI, 166 units /
50 μl of reaction solution containing ml Klenow fragment (manufactured by Toyobo Co., Ltd.) is reacted at 25 ° C. for 15 minutes. Continued DNA
Is purified by phenol precipitation and dissolved in 10 μl of TE buffer. (Repair ntp-DNA / DN
It is set to aseI. )

Subsequently, the cloning vector was prepared. Sma I buffer [10 mM Tris / HCl
(PH 7.5), 7 mM MgCl ₂ , 20 mM KC
1, 7 mM 2-mercaptoethanol], 125 μg
/ Ml pUC19, 1.6 units / μl Sma I (manufactured by Toyobo Co., Ltd.), 200 μl of reaction solution is reacted at 30 ° C. for 2 hours. Then, the DNA is purified by phenol treatment and ethanol precipitation, and dissolved in 80 μl of distilled water.
To this, 10 μl of 10 times dephosphorylation buffer, 0.4 unit /
μl Add 10 μl of alkaline phosphatase for a total volume of 1
The volume was adjusted to 00 μl, and the reaction was performed at 65 ° C. for 30 minutes. After that, the DNA was purified by phenol treatment and ethanol precipitation and dissolved in 20 μl of TE buffer (pUC19 / Sm).
a I). Next, a ligation reaction of DNA was performed. Ligation buffer [30 mM Tris / H
Cl (pH 7.8), 10 mM DTT, 1 mM AT
P], 10 μg / ml pUC19 / Sma I, 60 μ
g / ml repair ntp-DNA / DNaseI, 600
10 μl of reaction solution containing unit / ml T4 DNA ligase (manufactured by Promega) is reacted at 16 ° C. for 24 hours. Then, the enzyme was inactivated at 65 ° C. for 10 minutes. A part of this ligation reaction solution was used for Escherichia coli ( E. coli ) JM109.
LB ampicillin plate [1
% Tryptone, 0.5% yeast extract, 1% NaCl, 5
0 μg / ml ampicillin, 40 μg / ml isopropyl-β-D-thiogalactopyranoside (IPTG),
40 μg / ml 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), 1.5%
Agar powder] was used to select transformants. Positive colonies were isolated and plasmids were prepared in the above selection [see Samrock et al., Molecular Cloning (A Laboratory Manual)]. As a result, plasmid DNA in which three types of DNA fragments derived from ntp-DNA were cloned could be isolated.
It was named TL34, pTL57 and pTL85.

Example 15 Determination of nucleotide sequence of template / primer-independent synthetic DNA The nucleotide sequence of the DNA derived from ntpDNA inserted in pTL34, pTL57 and pTL85 was determined by the dideoxy terminator method. The obtained sequences are shown in SEQ ID NOS: 1, 2 and 3 of the sequence listing (Table 3). From this result, ntp-D cloned in pTL34
It can be confirmed that the NA product has a repeating sequence of 8 bases, that cloned into pTL57 has a repeating sequence of 12 bases, and that cloned into pTL85 is a sequence similar to the repeating sequence. It was confirmed. Next, SEQ ID NOS: 1 and 2 in the sequence listing
And homology search for the nucleotide sequences shown in
This was done using the bank database. As a result, as shown in Table 4, Table 5 and Table 6 (in the same order), it can be seen that these sequences are largely conserved from lower to higher organisms. In addition, it was revealed that these sequences were present in the microsatellite DNA existing near the centromere, and it could be inferred that there is a possibility that they are genetically important.

Example 16 Screening of repetitive sequence using ntp-DNA as a probe 20 μg / ml ntp-DNA was added with exchange buffer [50
mM imidazole / HCl (pH 6.4), 18 mM
MgCl ₂ , 5 mM disthritol, 0.1 mM
Spermidine HCl, 0.1 mM EDTA] and 0.1
mM ADP, 1 nM ATP, 1 μM [γ- ³² P]
ATP (111 TBq / mmol), 4.8% polyethylene glycol 8000, 400 units / ml T4 polynucleotide kinase is added, and the mixture is incubated at 37 ° C. for 30 minutes, and EDTA is added to a final concentration of 20 mM to stop the reaction. DNA by phenol treatment and ethanol precipitation
Is collected and used as a hybridization probe. Next, plaque hybridization was performed as a screening for repetitive sequences. First, E. coli ( Es
cherichia coli ) Y1090 (r-) containing 20% maltose in NZCYM medium [1% casein (enzyme hydrolyzate), 0.5% NaCl, 0.5% yeast extract, 0.1% casamino acid, 0.2% MgS
O ⁴ · _7H 2 O, it was inoculated into pH 7.0] 50 ml, 37
Incubate at 5 ° C for 5 hours. The cells are collected by centrifugation at 4000 × g for 10 minutes and suspended in 0.01M MgSO ₄ . This was used as a bacterial suspension, and SM [0.58% NaCl,
_{0.2% MgSO 4 · 7H 2 O} , 50mM Tris / H
Cl (pH 7.5), 0.01% gelatin] diluted tomato cDNA library λ phage solution (CLFL1083b, Clontech), and mixed at 37 ° C, 20
Incubate for minutes. This is added to NZCYM low agar medium, then overlaid on NZCYM agar medium, and cultured overnight at 37 ° C. A nitrocellulose filter (manufactured by Millipore) was placed on the agar of this culture plate for 1 minute, gently peeled off, and dried at room temperature for 20 minutes. Then, denatured solution (0.2N
It was placed on a filter paper (Whatman 3MM, Whatman) soaked in NaOH, 1.5M NaCl) for 5 minutes, and then a neutralization solution [3M NaCl, 0.3M trisodium citrate dihydrate (20 × SSC) was used. 2M Tris / HCl
(PH 7.5)], and place on a 3MM filter paper for 5 minutes.
The filters are soaked in neutralization solution for 1 minute, 2 × SSC for 5 minutes and then dried on 3MM filter paper. Filter 3
Sandwiched between MM filter papers, vacuum oven at 80 ℃ 2
Suction dry for hours. Next, the hybridization solution [50 mM HEPES / NaOH (pH 7.0),
0.2% polyvinylpyrrolidone, 0.2% Ficoll 4
00, 0.2% calf serum albumin, 100 μg / ml
The filter after drying is sufficiently moistened with denatured salmon sperm DNA, 50% formamide, and then 50 ml of the hybridization solution is added, sealed and left at 42 ° C. overnight.
Then, the hybridization solution is removed and
After heating at 5 ° C for 5 minutes, 10 ml of a hybridization solution containing a hybridization probe which has been cooled with ice is added, and the mixture is sealed again and cultured at 42 ° C overnight. After that, the filter is immersed in the washing solution 1 (2 × SSC, 0.1% SDS) for 1 hour, and then the washing solution 2 (0.5 × SSC, 0.1% SDS).
Soak in DS) and wash at 50 ° C. for 2 hours. After the filter is dried on 3MM filter paper, positive plaques are confirmed by autoradiography. As a result, positive plaques were confirmed, and thus, template / primer independent synthetic DNA
Was confirmed to be a probe in the screening of microsatellite DNA, for example. Use a Pasteur pipette to remove 1 ml of S from the positive plaque.
Transfer to M, add 1 drop of chloroform and leave at room temperature for 1 hour. This was appropriately diluted with SM, added to 500 ml of NZCYM medium together with 100 μl of the bacterial suspension, and 37 ° C.
Incubate overnight. Add 10 μl of chloroform to this,
The cells are collected by centrifugation at 7870 × g for 10 minutes, and the supernatant is collected. Buffer L1 (20 mg / ml R
NaseA, 6 mg / ml DNaseI, 0.2 mg
/ Ml calf serum albumin, 10 mM EDTA, 1
00 mM Tris / HCl, 300 mM NaCl, p
30 μl of H7.5) is added and the mixture is incubated at 37 ° C. for 30 minutes. Subsequently, 2 ml of ice-cold buffer L2 (30% polyethylene glycol 6000, 3M NaCl) is added, and the mixture is left to stand in ice for 1 hour. After that, 17700 x g for 15 minutes,
Centrifuge at 4 ° C and collect the precipitate. This precipitate was added to buffer L3 (100 mM Tris / HCl, 100 mM N
aCl, 25 mM EDTA, pH 7.5) 1 ml, followed by addition of solution L4 (4% SDS) 1 ml, 7
Incubate at 0 ° C for 10 minutes. After cooling in ice, solution L5 (2.
After adding 1 ml of 25 M potassium acetate buffer, pH 4.8) and mixing, centrifuge at 21600 × g , 30 minutes, 4 ° C., and collect the supernatant. In advance, the buffer solution QBT (750 mM
NaCl, 50 mM MOPS, 15% ethanol,
The above collected supernatant is added to a QIAGEN TIP-20 column (manufactured by Qiagen) equilibrated with pH 7.0, 0.15% Triton X-100), and column chromatography is performed. Then buffer QC (1M NaCl, 50 mM
The column was washed with 2 ml of MOPS, 15% ethanol, pH 7.0, and buffer QF (1.25 M NaCl,
50 mM Tris / HCl, 15% ethanol, pH
8.5) Elute the DNA with 1.5 ml. 0.8 volume of isopropanol is added to this, it is left at -20 ° C for 20 minutes, and the DNA is recovered by centrifugation at 21600 xg for 30 minutes at 4 ° C. This DNA precipitate was washed with 70% ethanol and then dried, and 10 μl of TE buffer solution [10 mM Tris / HCl (pH 8.0), 1 mM EDTA] was used.
Dissolve in. The present inventors have designated this λ phage DNA as λTL
It was named 2DNA. Next, the base sequence of this λTL2 DNA was determined. The method used was the dideoxy terminator method described above. As a primer, a commercially available λgt1
1 primer was used. As a result, a repeat sequence derived from ntp-DNA was present. Therefore, this ntp-DN
It was confirmed that A can be a probe for screening this repeating sequence.

Example 17 High-frequency recombination method using ntp-DNA 25 μg of plasmid pCH110 was added with buffer BM [5.
0 mM Tris / HCl (pH 7.5), 10 mM M
gCl ₂ , 100 mM NaCl, 1 mM disoitol], 2000 units / ml BamHI (manufactured by Toyobo Co., Ltd.) was added to make the total volume 100 μl, and the reaction solution was added at 37 ° C.
React for hours. Then, the DNA is recovered by phenol treatment and ethanol precipitation. Add this to TE buffer [10 m
M Tris / HCl (pH 8.0), 1 mM EDT
A] Dissolve in 25 μl. To 25 μl of this DNA solution,
PCR buffer [10 mM Tris / HCl (pH 8.
3), 50 mM KCl, 3.5 mM MgCl ₂ ,
0.001% gelatin], deoxyribonucleotides [dATP, dCTP, dGTP, and dTTP (all final concentration 200 μM)], 1 nM G1 primer, 1
nM G2 primer, 25 units / ml AmpliT
Prepare 2.5 ml of reaction solution containing aq DNA polymerase. To this upper layer, an equal amount of mineral oil (manufactured by Sigma) was added, and the mixture was heated at 95 ° C for 1 minute, 50 ° C for 2 minutes, and 7
The temperature cycle of 2 minutes at 3 ° C. is repeated a total of 30 times. Then, mineral oil is removed with an equal amount of chloroform, DNA is recovered by phenol treatment and ethanol precipitation, and dissolved in 20 μl of TE buffer. This crude purified DNA
The solution was purified by the cesium chloride-ethidium bromide equilibrium density gradient centrifugation method described above. As a result, 20 μg of DNA containing the GAL1 promoter sequence of baker's yeast upstream of the β-galactosidase gene and the transcription termination signal CYCl terminator sequence of baker's yeast downstream was obtained. This DN
A is dissolved in 20 μl of TE buffer, and the total amount of buffer A is
[10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ ,
20 mM Tris / HCl (pH 8.8), 6 mM M
gCl ₂ , 0.1% X-100 (all final concentrations)], deoxyribonucleotides [dATP, dCTP, dGT
P, and dTTP (all final concentrations 200 μM)], 20
Unit / ml Thermococcus litoralis DNA polymerase was added to make the reaction mixture 2 ml, and the mixture was reacted at 74 ° C. for 3 hours. Then, the DNA was recovered by phenol treatment and ethanol precipitation, and dissolved in 20 μl of TE buffer. This crude DNA solution was purified by the cesium chloride-ethidium bromide equilibrium density gradient centrifugation method described above, and 20 μl of TE buffer solution was used.
Dissolved in. 20 ml of YPD medium (1% yeast extract, 2% peptone, 2% glucose) is inoculated with baker's yeast and cultured overnight at 30 ° C. Next, 2 ml of this culture solution is transferred to 200 ml of YPD medium and cultured again at 30 ° C. overnight. This culture solution is centrifuged at 400 × g for 5 minutes to collect yeast cells. 20 ml of SED solution (1 M sorbitol, 25 mM EDTA, 50 m
M disthreitol) is added, suspended, and incubated at 30 ° C. for 10 minutes. The yeast cells were collected again by the same centrifugation,
Suspend in 1 ml sorbitol. The yeast cells were collected by centrifugation again, and 20 ml of SCE solution [1 M sorbitol, 100 mM citric acid / NaOH (pH 5.
8) Suspend in 10 mM EDTA]. To this, 0.
Add 2 ml of gluculase and incubate at 30 ° C. for 1 hour. This solution is centrifuged at 300 × g for 5 minutes to collect yeast spheroplast cells. 20 ml of this precipitate
Wash twice with M sorbitol and finally with 20 ml of S
TC solution [1M sorbitol, 10 mM CaC
l ₂ , 10 mM Tris / HCl (pH 7.5)]. Suspend the precipitate in 1 ml of STC solution and
Divide by l. To this, add 5 μl of the above DNA solution,
Leave at room temperature for 10 minutes. Then, PEG solution [20% polyethylene glycol 3300, 10 mM CaC
1 ₂ , 10 mM Tris / HCl (pH 7.4)]
Add ml and let stand for 10 minutes at room temperature. After centrifugation at 300 × g for 5 minutes, the precipitate was added to 150 μl of SOS medium (10 ml of 2M sorbitol, 0.1 ml of 1M CaCl ₂ ,
YPD medium 6.7 ml, 1% leucine solution 27μ
l, distilled water is suspended in 3.17 ml). This is 30
6m that was pre-melted at 45 ℃ for 20 minutes at ℃
1 SYG agar medium [18.2% sorbitol, 2% agar, 0.67% yeast nitrogen base (no amino acids), 2% glucose, 0.001% adenine, 0.00
4% uracil] and layered on SYG agar plate medium,
Incubate at 30 ° C for 3 days. From the culture plate medium, 5 ml of YPD medium is inoculated with a colony and cultured at 30 ° C. overnight. Centrifuge at 400 × g for 5 minutes and precipitate to 250 μl of B
K buffer [100 mM Tris / HCl (pH 8.
0) 1 mM disthritol, 20% glycerin]. To this, 12.5 μl of PMSF solution (40 mM phenylmethylsulfonyl fluoride) is added and stirred vigorously. Add 250 μl of BK buffer again and stir well. Centrifuge at 17700 × g for 15 minutes and transfer the supernatant to another container. To 50 μl of this supernatant, 50 μl of BK buffer and Z
Buffer _{(1.61% Na 2 HPO 4 ·} 7H 2 O, 0.5
5% NaH ₂ PO ₄ , 0.075% KCl, 0.024
0.9 ml of 6% MgSO ₄ , 0.27% β-mercaptophenol) is added, and the mixture is incubated at 28 ° C. for 5 minutes. Then, 0.2 mL of ONPG solution (4 mg / ml o-nitrophenyl-β-D-galactopyranoside) is added to start the reaction, and the mixture is incubated at 28 ° C. After 30 minutes, 0.5 ml of 1M Na ₂ CO ₃ solution is added to stop the reaction. Then, the absorbance at 420 nm is measured.
Also, the amount of protein was determined by the Bradford dye-binding method [Bratford, Analytical Biochemistry, Vol. 72, Vol. 248].
~ 254 (1976)]. The β-galactosidase specific activity in the bacterial extract was calculated from the absorbance and the amount of protein. As a result, β-galactosidase activity could be confirmed in 4 out of 20 samples (Table 6). This proved that the β-galactosidase gene was introduced into the yeast chromosome via the ntp-DNA added to the end. Therefore, ntp-
It was confirmed that DNA has an activity of causing recombination at high frequency.

[0046]

INDUSTRIAL APPLICABILITY The present invention aims to creatively generate DNA, that is, genetic information by a DNA polymerase in a template / primer-independent manner.
A plays an important role in the field of genetic engineering. that is,
Genes can be created by creating DNA that does not exist in nature, and proteins can be created from this gene. Therefore, for example, non-naturally occurring physiologically active substances, enzymes, receptors, signal transduction substances, hormones, anticancer substances, antiviral substances, immunosuppressive substances, membrane transport proteins, development-related substances, cytoskeletal substances, cranial nerve-related substances It becomes possible to create substances and the like. Besides, this DN
The creation of A can catch a glimpse of the creation of genetic information in the process of biological evolution, even if it is fragmentary, and it can be used to reproduce it and of course to produce useful mutants. Also, for example, a repetitive sequence DN that is considered to exist in a part of the constituent elements of this created DNA.
Regarding A, it also includes the effect of elucidating the regulation of gene replication, transcription, expression, etc., and the effect of being a probe for screening this regulatory site from existing genes. or,
In addition, by adding this ntp-DNA to the end of an existing gene and inserting it into a host cell, homologous recombination with the host chromosome can be caused at high frequency to express a protein derived from the gene. It also includes possible effects. The present invention can be expected to play a major role in the field of genetic engineering in the future, even in that it is simple and inexpensive to implement, and does not require a special device.

[Brief description of drawings]

FIG. 1 is a photograph of ethidium bromide-stained agarose gel electrophoresis of template-primer-independent DNA (ntpDNA) synthesized in Example 1.

FIG. 2 is a photograph of agarose gel electrophoresis illustrating the effect of deoxyribonucleotides on ntpDNA synthesis in Example 4. Line 1 is dCTP, dGTP and d
TTP, line 2 dATP and dGTP and dTTP, line 3 dATP and dCTP and d T TP, line 4 d
ATP and dCTP and dGTP, line 5 is dATP and d
NtpDNA of using a C TP and dGTP and dTTP
Is a synthesis of.

FIG. 3 is a photograph of agarose gel electrophoresis illustrating the synthesis of ntpDNA by various DNA polymerases shown in Example 5. Line 1 is a natural DNA polymerase from Pyrococcus furiosus. Line 2 is a recombinant DNA polymerase from Pyrococcus furiosus. Line 3 is E. coli DNA polymerase I (DNA polymerase I). Line 4 is Thermococcus litoralis DNA polymerase ( Tli DNA polymerase).
Line 5 is Selmus thermophilus DNA polymerase ( Tth DNA polymerase). Line 6 is Selmus aquatics DNA polymerase ( Taq DNA polymerase).

FIG. 4 DNA and R mixed in the enzyme shown in Example 6
It is a photograph of agarose gel electrophoresis for confirming the presence or absence of NA. Line 1 is normal ntp DNA synthesis. Line 2 is Tli DNA polymerase pretreated with RNaseA.
Line 3 is tRNA treated with RNaseA. Line 4 is tRNA not treated with RNaseA. Line 5 is RN
λ / Hin dIII treated with aseA. Line 6 is RN
λ / Hin dIII without aseA treatment. Line 7
Is T1i DNA polymerase pretreated with DNaseI . Line 8 is tRNA treated with DNaseI. Line 9 DNaseI treated λ / Hin dIII.

FIG. 5 is a photograph of agarose gel electrophoresis showing the synthesis of ntp10 kbp DNA shown in Example 7. Line 1 is 10 kbp ntp DNA. Line 2 is normal ntpDNA.

FIG. 6 is a graph showing the time course of ntpDNA synthesis in Example 8.

FIG. 7 is a photograph of agarose gel electrophoresis showing cleavage by the single-strand-specific nuclease of ntpDNA shown in Example 10. Line 1 is ntpDNA treated with S1 nuclease. Line 2 is λ / Hin dIII treated with S1 nuclease. Line 3 is M13mp18 single strand treated with S1 nuclease. Line 4 is ntpDNA treated with mung bean nuclease. Line 5 is lambda treated with mung bean nuclease
/ Is a Hin dIII. Line 6 is M13mp18 single stranded DNA treated with mung bean nuclease.
Line 7, 8, 9 are not treated single-stranded-specific nuclease respectively ntpDNA, λ / Hin dIII, M
13mp18 single-stranded DNA.

8 is a circular dichroism spectrogram of ntpDNA of Example 11. FIG. The solid line is pPT1 / EcoRI and the wavy line is ntpDNA.

9 is an electron micrograph of ntpDNA of Example 12. FIG.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

[Table 5]

[Table 6]

[Table 7]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI C12Q 1/68 C12N 15/00 A (56) Reference JP 62-19096 (JP, A) JP 8-38198 ( JP, A) International publication 96/7669 (WO, A1) International publication 94/24314 (WO, A1) Nucleic Acids Research, 1988, Vol. 16, No. 20, p. 9677-9686 Nucleic Acids Research, 1998, Vol. 26, No. 20, p. 4657-4661 Nucleic Acids Research, 1998, Vol. 26, No. 20, p. 4652-4656 Biochem. J. , 1997, Vol. 324, No. 2, p. 667-671 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 19/34 C07H 21/04 C12N 15/00 C12P 21/00 C12Q 1/68 BIOSIS (DIALOG) JISST file (JOIS) MEDLINE ( STN) WPI (DIALOG)

Claims (10)

(57) [Claims] 1. A method for synthesizing a double-stranded polydeoxyribonucleotide, which comprises reacting a deoxyribonucleotide with a thermostable deoxynucleotide polymerase in the absence of a template and a primer. 2. The synthetic method according to claim 1, wherein the thermostable deoxyribonucleotide polymerase is a bacterial deoxyribonucleotide polymerase belonging to the genus Sermococcus or the genus Selmouth. 3. The method according to claim 1, wherein the heating is performed at a temperature of 60 ° to 90 ° C.
Alternatively, the synthetic method described in 2. 4. The synthetic method according to claim 1, 2 or 3, which is carried out under neutral or weakly alkaline conditions. 5. The method of synthesis according to claim 1 wherein the polydeoxyribonucleotide having a repeating unit of TATCTAGA in salt sequences. 6. CATCATCATATAT in the base sequence
The method of synthesis according to claim 1 wherein the polydeoxyribonucleotide having the repeating units. 7. A polydeoxyribonucleotide according to claim 1, wherein in the nucleotide sequence having the following sequence TAGATATCTAGATCTAGATATCTATCTAGATATC
Method of synthesis . 8. A plaque hybridization characterized by labeling a polydeoxyribonucleotide obtained by reacting a thermostable deoxyribonucleotide polymerase with a deoxyribonucleotide in the absence of a template and a primer, and using the labeled polydeoxyribonucleotide as a probe. A method for screening a polydeoxyribonucleotide homologous to or similar to the above polydeoxyribonucleotide from a cDNA library. 9. A polydeoxyribonucleotide obtained by allowing a thermostable deoxyribonucleotide polymerase to act on deoxyribonucleotide in the absence of a template and a primer is ligated to the end of a double-stranded DNA containing a gene of a biological cell, Alternatively, a DNA obtained by synthesizing this polydeoxyribonucleotide at the same end
To a biological cell and culturing the cell to insert into a chromosomal DNA and express a protein derived from the gene. 10. In the absence of template and primer,
A polydeoxyribonucleotide obtained by allowing a thermostable deoxyribonucleotide polymerase to act on deoxyribonucleotide is ligated to the end of double-stranded DNA containing a gene of a biological cell, or the above polydeoxyribonucleotide is synthesized at the same end, A method for transforming with the gene, comprising transfecting the obtained DNA into a biological cell and culturing.