JPH05292967A - Method for enzymatic synthesis of oligonucleotides and use of oligonucleotides as primers - Google Patents

Abstract

(57) [Abstract] [PROBLEMS] To provide a method for relatively easily synthesizing an oligonucleotide that can be used as a primer for DNA synthesis and a probe for identifying a DNA sequence, and a primer for DNA synthesis and a probe for identifying a DNA sequence comprising the oligonucleotide. .. [Structure] Ligation of a pentamer (5 mer) having a ribonucleotide group at the 3 ′ end, a 10 mer or 15 mer and a tetramer (4 mer) having a phosphate group at the 5 ′ end (4 mer) or a 9 mer by RNA ligase, AT in buffer
It is carried out in the presence of P, magnesium ion, polyethylene glycol and hexaamminecobalt chloride. Oligonucleotide synthesis. 9 from 5 and 4
Mar dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ dN ₆ dN ₇ dN
₈ dN ₉ (dN represents deoxyribonucleotide, r
N represents a ribonucleotide). A DNA synthesis primer and a DNA sequence identification probe comprising these oligonucleotides.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for enzymatically synthesizing an oligonucleotide, and more particularly to a method for enzymatically synthesizing an oligonucleotide using a tetramer block. Furthermore, the present invention relates to a primer for DNA synthesis of an oligonucleotide containing a ribonucleotide in the chain and a probe for identifying a DNA sequence.

[0002]

2. Description of the Related Art Nowadays, with the development of genetic engineering, there are many opportunities to require many kinds of oligomers little by little. For example, it is used as a primer for enzymatic DNA synthesis or as a probe for searching a specific sequence. Therefore, a DNA synthesizer has been developed for in-situ synthesis as needed. But,
Even if a synthesizer is used, a huge project such as the determination of the total DNA base sequence of a human gene (about 3 billion bases long) requires a methodological improvement from the viewpoint of efficiency and economical efficiency. In this case, it is possible to directly handle a long (about 10,000 bases long) DNA fragment, and a DN like a simple primer relay method is used.
It is necessary to develop an efficient experimental method for A sequencing. Further, in preparing various kinds of DNA oligomers required there, an efficient synthesis method and operation method are required even when using an automatic DNA synthesizer. By nature, expensive machine-independent methods are important in and of themselves.

Specific DN for DNA having 10,000 bases
The length of A synthetic primer and the probe for identification is
In many cases, it has been shown that a length of octamer (hereinafter, abbreviated as "8-mer") is required.
As a method for efficiently preparing such an 8-mer functional DNA oligomer, a synthetic method using a 4-mer as a basic block has been reported (Yamamoto et al., Nikka Journal 1988 (2)).
p189-193). This method involves the condensation of two tetramers, previously protected with protecting groups, into organic chemistry.

However, although this method can efficiently synthesize an 8-mer, it requires an operation such as deprotection, and an improvement in simplicity is desired.

[0005]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method for synthesizing an oligonucleotide which can be used as a primer for DNA synthesis and a probe for identifying a DNA sequence relatively easily.

Further, another object of the present invention is to provide a primer for DNA synthesis and a probe for identifying a DNA sequence using a novel oligonucleotide.

[0007]

SUMMARY OF THE INVENTION The present invention is the number of nucleotides 2-20, and the 5 ^'end an oligonucleotide of one or two or more having a phosphoric acid group, the number of nucleotides 2-20
One or more of the oligonucleotides are
A method for synthesizing an oligonucleotide by ligation in the presence of NA ligase, the method comprising ligating in the presence of adenosine triphosphate and a divalent metal ion.

[0008] The present invention further 3 ^'5-mer has a ribonucleotide group at the end _{dN 1 dN 2 dN 3 dN 4} rN 5
(In the formula, dN ₁ , dN ₂ , dN ₃ and dN ₄ represent the same or different deoxyribonucleotides, rN ₅ represents a ribonucleotide) and 4 having a phosphate group at the 5 ^′ end.
Mar pdN ₆ dN ₇ dN ₈ dN ₉ (in the formula, dN ₆ , dN
₇ , dN ₈ and dN ₉ represent the same or different deoxyribonucleotides, and pdN ₆ represents a deoxyribonucleotide whose 5 ^′ end is phosphorylated) in the presence of RNA ligase to form a 9-mer dN ₁ dN ₂ dN ₃ dN ₄ rN
A method for synthesizing ₅ dN ₆ dN ₇ dN ₈ dN ₉
The present invention relates to a method for synthesizing an oligonucleotide, which comprises performing the above ligation in the presence of adenosine triphosphate and a divalent metal ion.

The present invention further relates to a 10-mer dN ₁ dN ₂ dN ₃ dN ₄ rN having a ribonucleotide group at the 3'end.
₅ dN ₆ dN ₇ dN ₈ dN ₉ rN ₁₀ (in the formula, dN ₁ , d
N ₂ , dN ₃ dN ₆ rN ₇ dN ₈ and dN ₉ represent the same or different deoxyribonucleotides, rN ₅ and rN ₅
N ₁₀ represents the same or different ribonucleotide) and 5 ′
4-mer pdN ₁₁ dN ₁₂ dN ₁₃ d having a phosphate group at the end
N ₁₄ or 9-mer pdN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄ rN ₁₅ dN
₁₆ dN ₁₇ dN ₁₈ dN ₁₉ (in the formula, dN ₁₁ , dN ₁₂ , d
N ₁₃ , dN ₁₄ , dN ₁₆ , dN ₁₇ , dN ₁₈ and dN ₁₉ represent the same or different deoxyribonucleotides, and rN ₁₅
Represents a ribocleotide, and pdN ₁₁ represents a deoxyribonucleotide having a phosphorylated 5 ′ end) in the presence of RNA ligase to form a 14-mer dN ₁ dN ₂ dN ₃ d
N ₄ rN ₅ dN ₆ dN ₇ dN ₈ dN ₉ rN ₁₀ dN ₁₁ dN
₁₂ dN ₁₃ dN ₁₄ or 19-mer dN ₁ dN ₂ dN ₃ dN ₄
rN ₅ dN ₆ dN ₇ dN ₈ dN ₉ rN ₁₀ dN ₁₁ dN ₁₂
Method for synthesizing dN ₁₃ dN ₁₄ rN ₁₅ dN ₁₆ dN ₁₇ dN ₁₈ dN ₁₉ or 15-mer dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ dN ₆ dN ₇ having a ribonucleotide group at the 3 ^′ end
dN ₈ dN ₉ rN ₁₀ dN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄ rN
₁₅ (wherein dN ₁ , dN ₂ , dN ₃ , dN ₆ , dN ₇ ,
dN ₈ , dN ₉ , dN ₁₁ , dN ₁₂ , dN ₁₃ and dN ₁₄ represent the same or different deoxyribonucleotides, and rN
_5, rN ₁₀ and rN ₁₅ is 4-mer pdN having the same or different showing a ribonucleotide) and 5 ^'end phosphate group
₁₆ dN ₁₇ dN ₁₈ dN ₁₉ (in the formula, dN ₁₆ , dN ₁₇ , dN ₁₈
And dN ₁₉ represent the same or different deoxyribonucleotides, and pdN ₁₆ represents a deoxyribonucleotide phosphorylated at the 5 ^′ end) in the presence of RNA ligase to form a 19-mer dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ dN ₆
dN ₇ dN ₈ dN ₉ rN ₁₀ dN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄ r
The present invention relates to a method for synthesizing N ₁₅ dN ₁₆ dN ₁₇ dN ₁₈ dN ₁₉ , which comprises ligating in the presence of adenosine triphosphate and a divalent metal ion.

A preferred embodiment of the synthesis of oligonucleotides, 5 having a ribonucleotide base in the 3 ^'end of the
Mer, 10 mer or 4-mer or 9-mer and 9-mer linked presence of RNA ligase has a 15-mer and a phosphate group at the 5 ^'end of the above, a method of synthesizing a 14-mer or 19-mer, A method for synthesizing an oligonucleotide, which comprises performing the above ligation in the presence of adenosine triphosphate (ATP), magnesium ion, polyethylene glycol and hexaamminecobalt chloride in a buffer solution.

Furthermore, the present invention has a nucleotide number of 9 to 1
9 oligonucleotides, at least one nucleotide is a ribonucleotide, and the remaining nucleotides are deoxyribonucleotides, to a primer for DNA synthesis and a probe for identifying a DNA sequence.

The present invention will be described below. Synthesis of the raw material of the present invention, the nucleotide number 2-20, preferably 4-10, and 5 ^'end an oligonucleotide having a phosphate group (hereinafter, sometimes referred donor) and ^3' ends An oligonucleotide having a ribonucleotide group of 2 to 20 and preferably 4 to 15 nucleotides (hereinafter may be referred to as acceptor).
In particular, an oligonucleotide having a phosphate group at the 5 ^'end, preferably a DNA oligomer containing a single DNA oligomers and ribonucleotides group. The other oligonucleotide 3 having a ribonucleotide base in the 'end DNA oligomers and 3 having a ^{ribonucleotide'} end and one ribonucleotide group or 2
It is preferable that it is a DNA oligomer containing one.

More preferably, the starting material for the synthesis method of the present invention is a 5-mer (pentamer), a 10-mer or 15-mer (hereinafter sometimes referred to as an acceptor) having a ribonucleotide at the 3'end, and a phosphate at the 5'end. It is a 4-mer (tetramer) or a 9-mer (hereinafter sometimes referred to as a donor) having a group.

The bases constituting the deoxyribonucleotides and the bases constituting the ribonucleotides in the raw material oligonucleotides serving as acceptors and donors are any of adenine (A), cytosine (C), guanine (G) and thymine (T). It is.

In the present invention, an acceptor oligonucleotide, for example, dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ and a donor oligonucleotide, for example, pdN ₆ dN.
Ligation with ₇ dN ₈ dN ₉ by RNA ligase is performed at least in the presence of ATP and divalent metal ions. By performing the ligation in this system, the ligation reaction between the acceptor and the donor, which are relatively short oligomers, can easily proceed.

[0016] Preferably, the ligation is performed in an AT buffer.
P, magnesium ion, polyethylene glycol (P
EG) and hexaamminecobalt chloride (HC
In the presence of C). By performing the ligation in this system, the ligation reaction proceeds almost quantitatively even when the amounts of the acceptor and the donor used are close to the stoichiometric ratio. This is
It is very advantageous when the synthesized 9-mer oligonucleotide is used as a primer for DNA synthesis without purification. This is because the content of unreacted acceptor or donor that may function as a pseudo-primer can be drastically reduced by ligating at a usage amount close to the stoichiometric ratio.

The acceptor and donor that are the raw materials can be synthesized by a known method. For example, as an acceptor, 3 ′ such as dN ₁ dN ₂ dN ₃ dN ₄ rN ₅
A DNA oligomer having a ribonucleotide at the end is
A ribonucleotide is added to a DNA oligomer such as 4-mer dN ₁ dN ₂ dN ₃ dN ₄ and a terminal deoxynucleotidyl transferase (hereinafter abbreviated as TdT).
Can be synthesized by ligating in the presence of The donor can also be synthesized by phosphorylating an oligonucleotide such as 4-mer dN ₆ dN ₇ dN ₈ dN _{9 in} the presence of polynucleotide kinase.

In the present invention, the acceptor and the donor are synthesized under the same conditions as in the ligation reaction, that is, the same buffer containing magnesium ion, polyethylene glycol and hexaamminecobalt chloride is used. It is preferable from the viewpoint of facilitating the ligation reaction.

That is, for example, the acceptor dN ₁
Synthesis of dN ₂ dN ₃ dN ₄ rN ₅ was performed using 4-mer dN ₁ dN
₂ dN ₃ dN ₄ and ribonucleotide triphosphate rN ₅ TP
It is preferable to carry out by linking and in the presence of TdT, magnesium ion, polyethylene glycol and hexaamminecobalt chloride in a buffer solution.

Further, for example, 10-mer dN ₁ dN ₂ d
N ₃ dN ₄ rN ₅ dN ₆ dN ₇ dN ₈ dN ₉ rN ₁₀ and 15-mer dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ dN ₆ dN ₇
dN ₈ dN ₉ rN ₁₀ dN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄ rN
₁₅ are each 9-mer dN ₁ dN ₂ dN ₃ dN ₄ rN
₅ dN ₆ dN ₇ dN ₈ dN ₉ and ribonucleotide triphosphate rN ₁₀ TP, and 14-mer dN ₁ dN ₂ dN ₃ d
N ₄ rN ₅ dN ₆ dN ₇ dN ₈ dN ₉ rN ₁₀ dN ₁₁ dN
₁₂ dN ₁₃ dN ₁₄ and ribonucleotide triphosphate rN ₁₅ TP
Is preferably obtained by ligating and in the presence of terminal deoxynucleotidyl transferase, magnesium ion, polyethylene glycol and hexaamminecobalt chloride in a buffer solution.

Further, for example, the donor pdN ₆ dN
The synthesis of ₇ dN ₈ dN ₉ was carried out in a buffer solution using polynucleotide kinase, adenosine triphosphate, magnesium ion,
It is preferably carried out by phosphorylating dN ₆ dN ₇ dN ₈ dN ₉ in the presence of polyethylene glycol and hexaamminecobalt chloride.

Further, for example, 4-mer pdN ₁₁ dN ₁₂ d
N ₁₃ dN ₁₄ , 9-mer pdN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄ rN ₁₅
dN ₁₆ dN ₁₇ dN ₁₈ dN ₁₉ and 4-mer pdN ₁₆ dN ₁₇ d
N ₁₈ dN ₁₉ is a 4-mer dN ₁₁ dN ₁₂ dN ₁₃ dN
₁₄ , 9-mer dN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄ rN ₁₅ dN ₁₆ dN
₁₇ dN ₁₈ dN ₁₉ and 4-mer dN ₁₆ dN ₁₇ dN ₁₈ dN
₁₉ is preferably obtained by phosphorylating ₁₉ in the presence of polynucleotide kinase, adenosine triphosphate, magnesium ion, polyethylene glycol and hexaamminecobalt chloride in a buffer solution.

Acceptor dN ₁ dN ₂ dN ₃ d
The synthesis reaction conditions such as N ₄ rN ₅ may vary depending on the type of TdT, but may be, for example, 35 to 40 ° C. for 10 minutes to 2 hours. Donor pdN ₆ dN ₇ dN ₈
The synthesis reaction conditions such as dN ₉ vary depending on the type of polynucleotide ligase, but are 10 to 35 ° C., for example.
It can be from 1 minute to 2 hours.

Acceptor dN ₁ dN ₂ dN ₃ d
Reaction mixture containing N ₄ rN _{5 and the} like, and pd as a donor
The reaction mixture containing N ₆ dN ₇ dN ₈ dN ₉ or the like can be mixed as it is, and RNA ligase or the like can be further added to be used for the ligation reaction. However, before the addition of RNA ligase, in order to inactivate the enzyme TdT and the kinase contained in each reaction mixture, for example, heat treatment is performed at 100 ° C. for 2 minutes. This heat treatment is performed for each reaction mixture.

DAN oligomers such as 4-mer dN ₁ dN ₂ dN ₃ dN ₄ and dN ₆ dN ₇ dN ₈ dN ₉ and R
Each NA oligomer can be easily obtained by synthesizing it using a commercial product or a DNA synthesizer (for example, 381A manufactured by Applied Biosystems). In addition, the donor 9-mer can be synthesized by the method of the present invention. Further, the acceptor 10-mer can be synthesized by linking a ribonucleotide to the 9-mer using TdT. Further, the acceptor 15-mer can also be synthesized by linking the 14-mer synthesized by the method of the present invention with ribonucleotide using TdT.

In the ligation, it is preferable to use the acceptor and the donor in a substantially stoichiometric ratio. However, either one can be used in excess. However, it is not preferable to use one of them in a large excess because the content of unreacted acceptor or donor increases as described above. Usually, it is preferable to set the molar ratio of the acceptor to the acceptor 1 to 5 in the range of 1.

It is preferable to use T4 RNA ligase as the RNA ligase because it is easily available. T4R
The amount of NA ligase used is appropriately 1 to 50 units.

In the present invention, the buffer solution is not particularly limited as long as it can maintain the pH in the vicinity of neutrality (pH: about 7 to 8). For example, Tris buffer, Hepes buffer, phosphate buffer and the like can be mentioned. The Tris buffer solution is a Tris-hydrochloric acid buffer solution, and the concentration is, for example, in the range of 10 to 100 mM, and the pH is appropriately adjusted to 7 to 8.

The concentration of ATP (adenosine triphosphate) is 1
It is suitable to set it to 0 to 1000 μM, preferably 50 to 500 μM.

Polyethylene glycol (PEG) has a molecular weight of, for example, 2,000 to 10,000, preferably 6,000.
~ 8000, 1-40 wt% of the total buffer
%, Preferably 15 to 30 wt%.

Examples of the divalent metal ion include magnesium ion and manganese ion. Especially, it is preferable to use magnesium ion. As the magnesium ion source, for example, magnesium chloride can be used. The concentration is 1 to 50 mM, preferably 5 to
20 mM is appropriate.

Hexaamminecobalt chloride (HC
C) is suitably 0.5 to 20 mM, preferably 1 to 10 mM.

Dithiothreitol (DTT), bovine serum albumin (BSA) and the like can be further added to the buffer solution. Dithiothreitol is 1-50 mM
It is suitable to set the concentration to. In addition, BSA is 1 to
It is suitable to set it to 50 μg / ml.

The ligation reaction temperature varies depending on the type of RNA ligase, but when T4 RNA ligase is used, it is suitable to set it at around room temperature, for example, 20 to 30 ° C. The reaction time may vary depending on the reaction temperature, but can be, for example, 1 to 10 hours.

The 9-mer to 19-mer oligonucleotides synthesized by the method of the present invention can be used as a primer for DNA synthesis and a probe for identifying a DNA sequence.

In the synthesis primer of the present invention, for example, an oligonucleotide is a 9-mer dN ₁ dN ₂ dN ₃ dN ₄
rN ₅ dN ₆ dN ₇ dN ₈ dN ₉ , 14-mer dN ₁ dN
₂ dN ₃ dN ₄ rN ₅ dN ₆ dN ₇ dN ₈ dN ₉ rN ₁₀
dN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄ or 19-mer dN ₁ dN ₂ d
N ₃ dN ₄ rN ₅ dN ₆ dN ₇ dN ₈ dN ₉ rN ₁₀ dN
₁₁ dN ₁₂ dN ₁₃ dN ₁₄ rN ₁₅ dN ₁₆ dN ₁₇ dN ₁₈ dN ₁₉
(In the formula, dN ₁ , dN ₂ , dN ₃ , dN ₄ , dN ₆ , d
N ₇ , dN ₈ , dN ₉ , dN ₁₁ , dN ₁₂ , dN ₁₃ , dN
₁₄ , dN ₁₆ , dN ₁₇ , dN ₁₈ , and dN ₁₉ represent the same or different deoxyribonucleotides, and rN ₅ , rN
₁₀ and rN ₁₅ represent the same or different ribonucleotides).

The DNA sequence discrimination probe of the present invention is, for example,
For example, the oligonucleotide is 9mer dN₁dN₂dN
₃dN_FourrN_FivedN₆dN₇dN₈dN₉, 14 Mar d
N₁dN₂dN₃dN_FourrN_FivedN₆dN₇dN₈dN
₉rN_TendN₁₁dN₁₂dN₁₃dN₁₄Or 19-mer dN₁
dN₂dN₃dN_FourrN_FivedN₆dN₇dN₈dN₉r
N_TendN₁₁dN₁₂dN₁₃dN₁₄rN₁₅dN₁₆dN₁₇dN
₁₈dN₁₉(Where dN₁, DN₂, DN₃, DN_Four, D
N₆, DN₇, DN₈, DN₉, DN₁₁, DN ₁₂, DN
₁₃, DN₁₄, DN₁₆, DN₁₇, DN₁₈, And dN₁₉Is
The same or different deoxyribonucleotides, rN
_Five, RN_TenAnd rN₁₅Are the same or different ribonucleoti
Is shown).

The synthesis of DNA using the primer for synthesizing DNA of the present invention can be carried out, for example, by using Current Protocols in Molecular Biology.
in Molecular Biology) (John Wiley & Sons, Inc.) 7.4.1-9. Specifically, the template DNA and the DNA synthesis primer of the present invention having a base sequence complementary to the base sequence at a predetermined position of the template DNA are mixed and heat-denatured (for example, at 60 to 80 ° C.). 5-30 minutes)
And then annealed (eg, 10 at 40-50 ° C).
-60 minutes) to bind the primer to the predetermined position of the template DNA. Next, 4 types of dNT
P, ie dATP, dCTP, dGTP and dTTP
In the presence of Klenow fragment, eg 40
By reacting at -60 ° C for 10 to 60 minutes, a DNA having a base sequence complementary to the template DNA at the starting position of the primer can be synthesized.

Further, the use of the probe for identifying a DNA sequence of the present invention can be carried out, for example, in Current Protocols in Molecular Biology.
ularBiology) (John Wiley & Sons, Inc.) 2.9.
It can be performed based on the method described in 10 to 13.
The use of the probe for identifying a DNA sequence of the present invention can be carried out, for example, by
Although a part of the base sequence is known, the whole base sequence is useful for identification of unknown DNA. For example, it is useful in screening a library of cDNA. For example, by contacting the DNA sequence-identifying probe of the present invention to which a fluorescent chromophore is added, with a colony of a host transformed with a vector containing a cDNA library,
A cDNA having a base sequence complementary to the DNA sequence identification probe can be distinguished from other cDNA. Alternatively, a DNA having a nucleotide sequence complementary to the DNA before and after the restriction enzyme cleavage site of the plasmid.
By using a sequence identification probe, it is possible to distinguish between a plasmid in which a foreign gene is incorporated at this cleavage site and a plasmid other than that.

[0040]

EXAMPLES The present invention will be described below with reference to examples.

The enzymes used in the following examples are as follows. T4 RNA ligase (Takara): T4amN82-infected E. coli B strain-derived T4 polynucleotide (Takara): E. coli 594 (λCM21) NM575-derived E. coli A19 strain-derived NEB: T4 polynucleotide kinase having T4pseT gene. Recombinant E. coli W3110 strain (λRC4) derived from overproduction TdT (Takara): calf thymus origin

Example 1 (Preparation of GTTGrAGTTG) (Preparation of GTTGrA) 500 pmol of commercially available GTTG,
10 μl of a standard solution having the following composition containing 1 nmole of rATP and TdT10 unit was prepared. This solution was allowed to stand at 37 ° C. for 1 hour, and then production of GTTGrA was confirmed by electrophoresis.

(GTTG phosphorylation (preparation of pGTTG)) 500 pmol of commercially available GTTG, 1 nmol of rATP and 10 units of polynucleotide kinase were dissolved in the following standard solution to make a total volume of 10 µl. Add this solution to 3
The mixture was allowed to stand at 7 ° C for 1 hour, and then generation of pGTTG was confirmed by electrophoresis.

(Ligation of GTTGrA and pGTTG) 500 pmol of GTTGrA obtained above and pGTTG10
10 μl of the standard solution shown below containing 0 pmol, 1 nmol rATP and 50 units of T4 RNA ligase (Takara) was reacted at 25 ° C. for 4 hours.

As a result, it was confirmed by electrophoresis that GTTGrA and rGTTG were linked to each other to produce GTTGrAGTTTG.

Standard solution 50 mM Tris-HCl buffer (pH 8.0) 10 mM MgCl ₂ 5 mM DTT (dithiothreitol) 25% PEG6000 1 mM HCC (hexaamminecobalt chloride) 10 μg / ml BSA (bovine serum albumin)

Example 2 (Preparation of TGAGrCCATT) (TGAG + rC → TGAGrC) 500 pmol of TGAG, 1 nmol of rCTP and TdT13 unit synthesized by a commercially available DNA synthesizer were dissolved in the standard solution shown in Example 1 to make the total amount 10 μl. .. Five samples were prepared, one immediately after preparation, and the other four were allowed to react at 37 ° C. for 30 minutes, 1, 2 or 4 hours, respectively, and then, generation of TGAGrC was confirmed by electrophoresis. .. As a result, the total amount of TGAG reacted and T
GAGrC had been produced.

(Phosphorylation of CATT) 500 pmol of CATT, rATP1n synthesized by a commercially available DNA synthesizer
Molar and 10 units of polynucleotide kinase (New England Biolabs (New England
d Biolabs) was dissolved in the standard solution shown in Example 1 to make the total volume 10 μl. Five of these samples were prepared, one immediately after preparation, and the other four were allowed to react at 37 ° C. for 30 minutes, 1, 2 or 4 hours, respectively, and then generation of pCATT was confirmed by electrophoresis. As a result, the total amount of CATT was phosphorylated and p
It was converted to CATT.

(TGAGrC + pCATT → TGAGr
CCATT) 5 μl of a reaction solution corresponding to 250 pmol of TGAGrC previously synthesized, 5 μl of a reaction solution corresponding to 250 pmol of pCATT synthesized above, 1 μl of a solution containing 500 pmol of rATP, and 1 μl of a solution containing 50 units of T4 RNA ligase were used as the standard shown in Example 1. The total volume was adjusted to 12 μl by mixing with 5 μl of the solution.

Five of these samples were prepared, one immediately after preparation and the other four at 25 ° C. for 30 minutes, 1, 2 or 4 hours, respectively, and then subjected to TGAGrC by electrophoresis.
Generation of CATT was confirmed. As a result, 30
The ligation reaction was completed within minutes.

Example 3 (Preparation of TGAGrCCATT) TGAGrC was obtained under the same conditions as in Example 2 using 1.5 nmole of TGAG, 2 nmole of rCTP and 13 units of TdT at 37 ° C. for 1 hour. Apart from this, CATT1
pCATT was obtained under the same conditions as in Example 2 at 37 ° C. for 1 hour using nmole, 2 nmole of rATP and 10 units of polynucleotide kinase.

The ligation reaction was carried out with the molar ratio of TGAGrC and pCATT being 1: 1, 3: 1 or 5: 1. That is, 1 μ of the reaction solution of pCATT corresponding to 100 pmol
1, 1 mM of 2 mM rATP solution and 50 units of T4 RNA ligase at 100 pmol, 300 pmol or 5
Mix with a reaction solution of TGAGrC corresponding to 00 pmol,
The reaction was performed under the same conditions as in Example 1 for 1 hour to give TGAGrC.
I got CATT. It was confirmed by electrophoresis that the ligation reaction proceeded almost quantitatively under all conditions.

Example 4 (Confirmation of primer activity) The primer activities of three kinds of reaction products containing TGAGrCCATT obtained in Example 3 and having different reaction conditions were confirmed. Single-stranded DNA of Escherichia coli bacteriophage M13
(M13ssDNA) was used as a template.

The above TGAGrCCATT product is the D
Perfectly complementary to # 2607- # 2615 of NA, and the nearest restriction enzyme HaeII downstream from this primer is
The cleavage fragment up to the recognition cleavage site (# 2556) of I (Takara Shuzo) is the priming indicator (6
0 base length).

As a template, M13ssDNA1
pmol and pCATT and TGAGrC as primers
Reaction solutions having different molar ratios of ((A) 1: 1, (B) 3;
1 (C) 5: 1) is equivalent to 1 pmol of pCATT or 2p
Used in molar equivalent. Or as a reference a mixture of 5 pmol TGAGrC and 1 pmol pCATT or TGAGrC1
A mixture of 0 pmol and 2 pmol pCATT was also used as a primer. 7 mM Tris-H as extension reaction buffer
Cl (pH 7.5), 0.1 mM EDTA, 20 mM
Using NaCl and 7 mM MgCl ₂ , the total volume was 18 μl including the above template and primer, and the temperature was 70 ° C.
It was denatured for 10 minutes and then annealed at 45 ° C. for 30 minutes.

1 dNTP (4 mM each) was added to the reaction mixture.
4 units of Klenow fragment (1 μl of Takara Shuzo Co., Ltd.) was added to make the total volume 20 μl, and the temperature was 45 ° C. 30.
It was made to react for minutes. Then, the enzyme was inactivated by treatment at 80 ° C. for 5 minutes. Next, 12 units of restriction enzyme HaeIII (1 μl of Takara Shuzo Co., Ltd.) was added to the reaction solution and reacted at 37 ° C. for 1 hour. Half of the reaction mixture was electrophoresed (8% polyacrylamide / 8M urea gel, 300 V, 1 hour) and detected by silver staining.

The results are shown in Reference Photo 1. Lane 1
Table 1 below summarizes the conditions from 1 to 9 and whether or not a priming indicator (PI) appeared at a length of 60 bases.

Table 1 ─────────────────────────────────── Lane condition PI appearance ───── ─────────────────────────────── 1 M13ssDNA without HaeIII treatment 2 Primer (A) 1 pmol used 3 Primer (B) 1 pmol used 4 Primer (C) 1 pmol used 5 TGAGrC 5 pmol, pCATT 1 pmol used 6 Primer (A) 2 pmol used 7 Primer (B) 2 pmol used 8 Primer (C) 2 pmol used 9 TGAGrC5p Mol, pCATT1p mol not used ────────────────────────────────────

Example 5 (Preparation of GTTGrCGTTG) (Preparation of GTTGrC) 250 pmol of commercially available GTTG,
rCTP 500 pmol, TdT13 unit in Example 1
The total volume was adjusted to 10 μl with the standard solution shown in. This sample is 4
Prepare one, immediately after preparation, and the remaining three are each 3
Generation of GTTGrC was confirmed by electrophoresis after 10 minutes, 20 minutes, or 30 minutes at 7 ° C. As a result, GT in 30 minutes
It was confirmed that the production of TGrC was almost completed.

(Phosphorylation of GTTG) Commercially available GTTG2
The total amount of 50 pmol, 500 pmol of rATP, and 10 units of kinase was adjusted to 10 µl with the standard solution shown in Example 1. Four of these samples were prepared, one of which was immediately after preparation, and the other of which was 10 minutes, 20 minutes, or 30 minutes after preparation, respectively, and the generation of pGTTG was confirmed by electrophoresis. As a result, the phosphorylation of GTTG was almost completed 10 minutes after preparation.

(Connection of GTTGrC and pGTTG) G
Reaction solutions corresponding to 75 pmol of TTGrC and 25 pmol of pGTTG or 25 pmol of GTTGrC and pGTTG2
Each reaction solution corresponding to 5 pmol and rATP 500 pmol T
50 units of 4 RNA ligase in standard solution
μl. Prepare 2 or 4 of these samples and prepare GTGTr immediately after preparation or after 10 minutes, 20 minutes or 30 minutes at 25 ° C.
Generation of CGTTG was confirmed. As a result, GTTGrC
It was confirmed that the product band appeared after 30 minutes at / pGTTG = 3.

Example 6 (Preparation of GTTGrAGTTG) GTTGrA50 obtained by the same method as in Example 1
0 pmol, pGT obtained by the same method as in Example 1
TG 10 pmol, rATP 1 nmol and T4 RNA
50 mM Hepes-NaOH buffer (pH 7.9) containing 50 units of ligase (Takara) (10 mM Mn
Cl ₂ , 10 mM DTT (dithiothreitol), 5
0 μg / ml BSA (bovine serum albumin)) 10 μl
Was reacted at 17 ° C. for 5 hours.

As a result, it was confirmed by electrophoresis that GTGTrA and rGTTG were linked to each other to produce GTTGrAGTTTG.

Example 7 (Preparation of TGAGrCTGAG) TGAGrC50 obtained by the same method as in Example 2
0 pmol, pTG obtained by the same method as in Example 1
10 pmol AG, 1 nmol rATP and T4 RNA
50 mM Hepes-NaOH buffer (pH 7.9) containing 50 units of ligase (Takara) (10 mM Mn
Cl ₂ , 10 mM DTT (dithiothreitol), 5
0 μg / ml BSA (bovine serum albumin)) 10 μl
Was reacted at 17 ° C. for 5 hours.

As a result, it was confirmed by electrophoresis that TGAGrC and rTGAG were linked to produce TGAGrCTGAG.

Example 8 (Preparation of CGGArGTGAGrAATAG) (CGGA + rG → CGGArG) 250 pmol of CGGA synthesized with a commercially available DNA synthesizer, 1 nmol of rGTP and TdT10 unit were dissolved in the standard solution shown in Example 1 to bring the total amount to 10 μl. .. 37 this sample
The reaction was carried out at ℃ for 1 hour. Generation of CGGArG was confirmed by electrophoresis.

(Phosphorylation of TGAG) 250 pmol of TGAG, rATP1n synthesized by a commercially available DNA synthesizer
Molar and 10 units of polynucleotide kinase (New England Biolabs (New England
d Biolabs) was dissolved in the standard solution shown in Example 1 to make the total volume 10 μl. 37 this sample
The reaction was carried out at ℃ for 4 hours. Generation of pTGAG was confirmed by electrophoresis.

(CGGArG + pTGAG → CGGAr
GTGAG) 8 μl of the reaction solution corresponding to 250 pmol of CGGArG synthesized above, 4 μl of the reaction solution corresponding to 250 pmol of pTGAG synthesized above, 1 μl of a solution containing 1 nmole of rATP, and 1 μl of a solution containing 50 units of T4 RNA ligase were used as the standard shown in Example 1. The total volume was 17 μl by mixing with 5 μl of the solution.

This sample was reacted at 25 ° C. for 1 hour. C
GGArGTGAG was confirmed by electrophoresis.

(CGGArGTGAG + rA → CGGA
rGTGAGrA) CGGArGTGAG previously synthesized
The reaction solution (14 μl) and rATP (1 mol) and 10 units of TdT dissolved in 1 μl of the standard solution of Example 1 were mixed to obtain a total volume of 16 μl. 37 this sample
The reaction was carried out at 0 ° C for 1 hour. Generation of CGGArGTGAGrA was confirmed by electrophoresis.

(ATAG phosphorylation) 250 pmol ATAG, rATP1n synthesized by a commercially available DNA synthesizer
Molar and 10 units of polynucleotide kinase (New England Biolabs (New England
d Biolabs) was dissolved in the standard solution shown in Example 1 to make the total volume 10 μl. 37 this sample
The reaction was carried out at ℃ for 1 hour. Generation of pATAG was confirmed by electrophoresis.

(CGGArGTGAGrA + pATAG
→ CGGArGTGAGrAATAG) C previously synthesized
Reaction solution 10 equivalent to 250 pmol of GGArGTGAGrA
2 μl of the reaction solution corresponding to 250 pmol of pATAG synthesized above, 1 μl of a solution containing 1 nmole of rATP, and 1 μl of a solution containing 50 units of T4 RNA ligase were mixed with 5 μl of the standard solution shown in Example 1 to bring the total amount to 16 μ
It was set to l.

This sample was reacted at 25 ° C. for 1 hour. C
Generation of GGArGTGAGrAATAG was confirmed by electrophoresis.

Example 9 (Confirmation of primer activity) CGGArGTGAGrAATAG obtained in Example 8
The primer activity of the reaction product containing was confirmed. Single-stranded DNA of Escherichia coli bacteriophage M13 (M13ssD
NA) was used as a template.

The above CGGArGTGAGrAATAG product is completely complementary to # 1619- # 1633 of this DNA, and the recognition site (# 139) of the nearest restriction enzyme HaeIII (Takara Shuzo) downstream of the primer from this product.
The cleaved fragment up to 8) becomes a priming indicator (236 bases long).

As a template, M13ssDNA1
pmol and CGGArGTGAGrA as primer
In addition to ATAG, those shown in the table below were used as references. 7 mM Tris-HCl (p
H7.5), 0.1 mM EDTA, 20 mM NaC
1, 7 mM MgCl ₂ was used to make a total volume of 18 μl including the template and the primer, and the mixture was denatured at 70 ° C. for 10 minutes and then annealed at 45 ° C. for 30 minutes.

DNTPs (4 mM each) 1 were added to the reaction mixture.
μl and 4 units of Klenow fragment (1 μl (Takara Shuzo)) were added and reacted at 45 ° C. for 30 minutes. Then, the enzyme was inactivated by treatment at 80 ° C. for 5 minutes. Next, 12 units of restriction enzyme HaeIII (Takara Shuzo, 1
μl) was added and the mixture was reacted at 37 ° C. for 1 hour. Half of the reaction mixture was electrophoresed (4% polyacrylamide / 8M urea gel, 300 V, 1 hour) and detected by silver staining.

The result is shown in Reference Photograph 2. Lane 1
Table 2 below summarizes the conditions from 1 to 10 and whether or not a priming indicator (PI) appeared at a length of 236 bases.

Table 2 ──────────────────────────────────── Lane condition PI appearance ──── ──────────────────────────────────────── 1 CGGArG (6 pmol) + pTGAG (3 pmol) None 2 CGGArGTGAG reactant (3p Mole equivalent) Yes (weak) 3 CGGArGTGA reaction product (9 pmol equivalent) Yes (weak) 4 CGGArG (6 pmol) + pTGAG (3 pmol) + pATAG (3 mol) None 5 CGGArGTGAGrAATAG reaction product (3 pmol equivalent) Yes (strong) 6 CGGArGTGAGrAATAG reaction product (corresponding to 9 pmol) Yes (strong) 7 GGAGTGAG (known DNA oligomer) (1 pmol) Yes (strong) 8 No (template) No 9 CG ArG (18 pmol) + pTGAG (9 pmol) None 10 CGGArG (18 pmol) + pTGAG (9 pmol) + pATAG (9 pmol) None ─────────────────────── ──────────────

Example 10 (Application to PCR) Oligonucleotide CGGArGT obtained in Example 8
The application of GAGrAATAG as a PCR primer was examined. The reaction and analysis were performed under the following conditions.

Application of CGGArGTGAGrAATAG synthesis reaction to PCR Reaction buffer: 10 mM Tris-HCl (pH 7.5),
50 mM KCl, 3 mM MgCl ₂ , 5 μg / ml
Gelatin, each 50 μM dNTP template: M13ssDNA (1 fmol) Synthase: AmpliTaq (Takara, 0.5 unit) Primer 1: CGGArGTGAGrAATAG synthesis reaction product (9 pmol equivalent) Primer 2: GCTGAGGGTGACGATCCCCC
GC (P1, 2 pmol) Reaction condition: 40 cycles (denaturation: 92 ° C., 70 seconds, annealing: 45 ° C., 300 seconds, synthesis: 60 ° C., 150 seconds / cycle) Analysis: 10 μl of reaction solution was 8 M urea / 4% It was analyzed by polyacrylamide gel electrophoresis.

Reference photograph 3 shows the result of electrophoresis. still,
The primers used for each lane are shown in Table 3 below. P
1 corresponds to the sequence of # 1369-1389 of M13ssDNA, and the tetramer triple adduct (14mer) is # 1.
Since it was complementary to 619-1633, generation of a band having a length of 264 bases was expected, and the fact that it was detected showed that the oligonucleotide of the present invention can be applied to PCR (lane 2).

Table 3 ──────────────────────────────────── Lanes Used primers ───── ────────────────────────────────────── 1 CGAGTGAG (2 pmol) + Pl (2 pmol) 2 CGGArGTGAGrAAATA Reaction (equivalent to 9 pmol ) + Pl (2 pmol) 3 CGGArGTGAG (corresponding to 9 pmol) + Pl (2 pmol) 4 CGGArG (18 pmol) + pTGAG (9 pmol) + pATAG (9 pmol) 5 Pl (2 pmol) 6 GGAGTGAG (2 pmol AT 7 CGAr product TCGAR) (Equivalent to 9 pmol) 8 None ─────────────────────────────────────

Claims (5)

[Claims] 1. A number of nucleotides are 2-20, and 5 ^'end phosphate group oligonucleotide one or two or more and a number of nucleotides oligonucleotide one or more 2 to 20 And a method of synthesizing an oligonucleotide by ligating in the presence of RNA ligase, the method comprising synthesizing the oligonucleotide in the presence of adenosine triphosphate and a divalent metal ion. 2. A 5-mer dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ having a ribonucleotide group at the 3 ^′ end (wherein d
N ₁ , dN ₂ , dN ₃ and dN ₄ represent the same or different deoxyribonucleotides, rN ₅ represents a ribonucleotide) and a 4-mer pdN having a phosphate group at the 5 ^′ end.
₆ dN ₇ dN ₈ dN ₉ (in the formula, dN ₆ , dN ₇ , dN ₈
And dN ₉ represent the same or different deoxyribonucleotides, and pdN ₆ represents a deoxyribonucleotide phosphorylated at the 5 ^′ end) in the presence of RNA ligase to form a 9-mer dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ dN ₆ d
A method for synthesizing N ₇ dN ₈ dN ₉ , which comprises ligating in the presence of adenosine triphosphate and a divalent metal ion. 3. A 10-mer dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ dN ₆ dN ₇ having a ribonucleotide group at the 3 ^′ end.
dN ₈ dN ₉ rN ₁₀ (in the formula, dN ₁ , dN ₂ , dN ₃ ,
dN ₄ , dN ₆ , dN ₇ , dN ₈ and dN ₉ represent the same or different deoxyribonucleotides, and rN ₅ and rN ₅
N ₁₀ represents the same or different ribonucleotide) and 5 ^'
4-mer pdN ₁₁ dN ₁₂ dN ₁₃ d having a phosphate group at the end
N ₁₄ or 9-mer pdN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄ rN ₁₅ dN
₁₆ dN ₁₇ dN ₁₈ dN ₁₉ (in the formula, dN ₁₁ , dN ₁₂ , d
N ₁₃ , dN ₁₄ , dN ₁₆ , dN ₁₇ , dN ₁₈ and dN ₁₉ represent the same or different deoxyribonucleotides, and rN ₁₅
Represents a ribonucleotide, and pdN ₁₁ represents a deoxyribonucleotide having a phosphorylated 5 ^′ end).
14-mer dN ₁ dN ₂ dN ₃ ligated in the presence of ligase
dN ₄ rN ₅ dN ₆ dN ₇ dN ₈ dN ₉ rN ₁₀ dN ₁₁ d
N ₁₂ dN ₁₃ dN ₁₄ or 19-mer dN ₁ dN ₂ dN ₃ dN
₄ rN ₅ dN ₆ dN ₇ dN ₈ dN ₉ rN ₁₀ dN ₁₁ dN ₁₂
Method for synthesizing dN ₁₃ dN ₁₄ rN ₁₅ dN ₁₆ dN ₁₇ dN ₁₈ dN ₁₉ or 1 having a ribonucleotide group at the 3 ^′ end
5-mer dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ dN ₆ dN ₇ d
N ₈ dN ₉ rN ₁₀ dN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄ rN ₁₅ (wherein dN ₁ , dN ₂ , dN ₃ , dN ₆ , dN ₇ , dN
N ₈ , dN ₉ , dN ₁₁ , dN ₁₂ , dN ₁₃ and dN ₁₄ represent the same or different deoxyribonucleotides, and r
N ₅ , rN ₁₀ and rN ₁₅ represent the same or different ribonucleotides) and a 4-mer pd having a phosphate group at the 5 ^′ end
N ₁₆ dN ₁₇ dN ₁₈ dN ₁₉ (in the formula, dN ₁₆ , dN ₁₇ , dN
₁₈ and dN ₁₉ represent the same or different deoxyribonucleotides, and pdN ₁₆ represents ^5′- terminally phosphorylated deoxyribonucleotides) in the presence of RNA ligase to form a 19-mer dN ₁ dN ₂ dN ₃ dN ₄ rN ₅ dN
₆ dN ₇ dN ₈ dN ₉ rN ₁₀ dN ₁₁ dN ₁₂ dN ₁₃ dN ₁₄
A method for synthesizing rN ₁₅ dN ₁₆ dN ₁₇ dN ₁₈ dN ₁₉ , which comprises ligating in the presence of adenosine triphosphate and a divalent metal ion. 4. A primer for DNA synthesis, which is an oligonucleotide having 9 to 19 nucleotides, at least one nucleotide is a ribonucleotide, and the remaining nucleotides are deoxynucleotides. 5. A probe for identifying a DNA sequence, which is an oligonucleotide having 9 to 19 nucleotides, at least one nucleotide is a ribonucleotide, and the remaining nucleotides are deoxynucleotides.