JP4224695B2 - Method for producing nucleic acid having a plurality of target sequences - Google Patents

Description

  The present invention relates to a method for synthesizing a nucleic acid having a plurality of target sequences. The present invention also relates to a method for producing a highly sensitive sensor using a nucleic acid having an aptamer sequence multiple times. Furthermore, the present invention relates to an efficient protein production method.

  Polymerase chain reaction (PCR) is an important technology that is indispensable in the field of molecular biology. PCR makes it possible to easily amplify a target gene even from a small amount of gene sample, and to create a new artificial gene sequence.

  However, since PCR amplifies the sequence using a primer complementary to a part of the target sequence, if the target sequence contains many homologous sequences, the primer used tends to cause misannealing with the template DNA. For this reason, sequences of various lengths are amplified, and it is difficult to efficiently amplify the entire target sequence. In addition, there has been little reported overall amplification of sequences having the same sequence multiple times.

  The only reported method is to immobilize primers on beads and perform PCR on a solid phase (Non-patent Document 1). However, this method is rarely used because it is complicated and requires an operation different from a normal PCR protocol. Even when this method is used, it has not been reported to date that a nucleic acid having a target sequence repeated at high frequency or high density has been amplified.

It is already known that a nucleic acid in which a sequence complementary to a template is repeatedly arranged using a circular nucleic acid molecule as a template can be synthesized using φ29 DNA polymerase (Non-patent Document 2). However, in this method, a practically sufficient amount of nucleic acid having a repetitive sequence cannot be obtained in a short time.
Nucleic Acid Research, Vol. 20, 6743-6744, 1992 J. Biol. Chem., Vol. 264, 8935, 1989

  It is a first object of the present invention to provide a method capable of efficiently synthesizing nucleic acids having a plurality of identical or substantially identical sequences. Moreover, this invention makes it the 2nd subject to provide the method which can manufacture a highly sensitive sensor using this method. Moreover, this invention makes it the 3rd subject to provide the method which can manufacture protein efficiently using this method.

  In order to solve the above-mentioned problems, the present inventors have intensively studied and obtained the following knowledge.

  When rolling circle replication is performed using a circular DNA having a target base sequence as a template, a single-stranded nucleic acid having a complementary sequence of the target sequence can be obtained. By synthesizing the complementary strand of this single-stranded nucleic acid, double-stranded nucleic acid having a desired sequence and having various lengths can be obtained.

  In order to separate a double-stranded DNA having a desired length from the inside by means of electrophoresis or the like, a relatively large amount of nucleic acid of each length is required. In order to amplify each length of nucleic acid, PCR may be performed using a primer that can anneal only to the end of each length of double-stranded nucleic acid.

  However, since these double-stranded DNAs are obtained as a result of rolling circle replication, sense primers SP and antisense primers AP that have the same sequence repeatedly and can anneal only to the ends of double-stranded nucleic acids are designed. It is hard to do.

  Therefore, a sequence that can be hybridized with the sense primer SP is linked upstream of the primer for performing rolling circle type replication, and used when synthesizing complementary strands of single-stranded nucleic acids obtained as a result of rolling circle type replication. If a sequence capable of hybridizing with the antisense primer AP is linked to the upstream side of the primer, this primer set SP and AP was used using a double-stranded nucleic acid obtained by rolling circle replication and its complementary strand synthesis as a template. By performing PCR, the entire double-stranded nucleic acid of each length can be amplified.

  The present invention has been completed by further research based on the above findings, and provides the following nucleic acid production methods and the like.

Item 1. Preparing a circular DNA having a target sequence (1);
In order from the upstream side, a primer A having a first non-annealed sequence that does not anneal with the circular DNA and a first annealed sequence that can anneal with a part of the circular single-stranded DNA is used as a template. Performing the rolling circle type replication reaction (2),
In order from the upstream side, a second non-annealed sequence that does not anneal with the single-stranded nucleic acid obtained by the rolling circle type replication reaction, and a part of the single-stranded nucleic acid obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of the single-stranded nucleic acid using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded nucleic acid obtained in the step (3) is obtained. A method for producing a nucleic acid having a plurality of target sequences, comprising a step (4) of performing a polymerase chain reaction using a template.

  Item 2. Item 2. The method according to Item 1, further comprising the step (5) of separating a nucleic acid having a desired length.

  Item 3. Item 3. The method according to Item 2, wherein the nucleic acid having a desired length is separated in a single-stranded state in the step (5).

  Item 4. Item 4. The method according to Item 1, 2, or 3, further comprising a step (6) of amplifying the obtained nucleic acid by a polymerase chain reaction.

  Item 5. Item 5. The method according to Item 4, further comprising the step (6) of denaturing the obtained double-stranded DNA into a single strand.

  Item 6. Item 6. The method according to any one of Items 1 to 5, wherein a phage M2-derived DNA polymerase is used in the step (2).

  Item 7. Item 7. The method according to any one of Items 1 to 6, wherein a DNA polymerase having a calibration function is used in Step (3) and Step (4).

Item 8. Preparing a circular DNA having a sequence of a specific length (1);
From the upstream side, a rolling circle using this circular DNA as a template, using primer A having a first non-annealed sequence that does not anneal with this circular DNA and a first annealed sequence that can anneal with a portion of this circular DNA A step (2) of performing a mold replication reaction;
In order from the upstream side, a second non-annealed sequence that does not anneal with the single-stranded nucleic acid obtained by the rolling circle type replication reaction, and a part of the single-stranded nucleic acid obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of the single-stranded nucleic acid using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded nucleic acid obtained in the step (3) is obtained. A method for producing a nucleic acid ladder marker comprising a step (4) of performing a polymerase chain reaction using a template.

Item 9. Preparing a circular DNA having a base sequence encoding a target protein or a complementary sequence thereof (1);
From the upstream side, a rolling circle using this circular DNA as a template, using primer A having a first non-annealed sequence that does not anneal with this circular DNA and a first annealed sequence that can anneal with a portion of this circular DNA A step (2) of performing a mold replication reaction;
In order from the upstream side, a second non-annealed sequence that does not anneal with the single-stranded nucleic acid obtained by the rolling circle type replication reaction, and a part of the single-stranded nucleic acid obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of the single-stranded nucleic acid using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded nucleic acid obtained in the step (3) is obtained. A step (4) of performing a polymerase chain reaction using a template; and
A method for producing a nucleic acid encoding a target protein comprising

  Item 10. Item 10. The method for producing a nucleic acid encoding the target protein according to Item 9, wherein the target protein is metallothionein, isonitrile hydratase or nitrilase.

Item 11. (8) a step of obtaining an expression vector by inserting the nucleic acid obtained by the method of Item 9 into a vector;
Transforming the host with the expression vector (9),
A method for producing a target protein comprising culturing the transformant and recovering the target protein from the culture (10).

Item 12. (11) inserting the nucleic acid obtained by the method according to Item 9 into a host genome by homologous recombination;
A method for producing the target protein, comprising culturing the host and recovering the target protein from the culture (10).

  Item 13. Item 13. The method for producing a target protein according to Item 11 or 12, wherein the target protein is metallothionein, isonitrile hydratase or nitrilase.

Item 14. Preparing a circular DNA having a sequence complementary to the base sequence encoding the target protein and having the target sequence excluding the start codon and the stop codon; and
In order from the upstream side, this circular DNA is prepared by using primer A having a first non-annealed sequence that does not anneal with the target sequence of the circular DNA and a first annealed sequence that can anneal with a part of the target sequence of the circular DNA. (2) performing a rolling circle type replication reaction using as a template;
In order from the upstream side, a second non-annealed sequence that does not anneal with the single-stranded DNA obtained by the rolling circle replication reaction, and a part of the single-stranded nucleic acid that is different from the first annealed sequence (3) performing a complementary strand synthesis reaction of this single-stranded nucleic acid using primer B having a second annealable sequence that can be annealed;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded nucleic acid obtained in the step (3) is obtained. A method for producing a nucleic acid encoding a multimeric protein, comprising a step (4) of performing a polymerase chain reaction using a template.

  Item 15. Item 15. The method for producing a nucleic acid encoding a multimeric protein according to Item 14, wherein the target protein is metallothionein, isonitrile hydratase or nitrilase.

Item 16. (8) a step of obtaining an expression vector by inserting the nucleic acid obtained by the method of Item 14 into a vector;
Transforming the host with the expression vector (9),
And a step (10) of culturing the transformant and recovering the target protein from the culture.

Item 17. Inserting the nucleic acid obtained by the method of Item 14 into the host genome by homologous recombination (11);
And a step (10) of culturing the host and recovering the target protein from the culture.

  Item 18. Item 18. The method for producing a target protein according to Item 16 or 17, wherein the target protein is metallothionein, isonitrile hydratase or nitrilase.

Item 19. Preparing a circular DNA having an aptamer sequence (1);
From the upstream side, a rolling circle using this circular DNA as a template, using primer A having a first non-annealed sequence that does not anneal with this circular DNA and a first annealed sequence that can anneal with a portion of this circular DNA A step (2) of performing a mold replication reaction;
In order from the upstream side, a second non-annealed sequence that does not anneal with the single-stranded nucleic acid obtained by the rolling circle type replication reaction, and a part of the single-stranded nucleic acid obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of the single-stranded nucleic acid using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded nucleic acid obtained in the step (3) is obtained. A method for producing a nucleic acid having a plurality of aptamer sequences, comprising a step (4) of performing a polymerase chain reaction using a template.

  Item 20. Item 20. A method for producing a sensor chip, comprising immobilizing or adsorbing a nucleic acid repeatedly having an aptamer sequence obtained by the method according to Item 19 on a sensor chip surface.

  Item 21. Item 21. A method for producing a sensor capable of detecting an aptamer binding target by connecting a sensor chip obtained by the method according to Item 20 to a detection device.

  According to the method of the present invention, by using special primers, nucleic acids having the same or substantially the same sequence, which has been difficult in the past, can be easily obtained in large quantities using PCR and rolling circle replication, which are existing techniques. Can be obtained.

  For example, when the sequence is an aptamer sequence, a nucleic acid having an aptamer sequence is obtained repeatedly. Therefore, in a sensor in which this nucleic acid is fixed or attached to a sensor chip, the number of aptamers per unit area of the sensor chip is large. The detection sensitivity is improved accordingly. Moreover, when this sequence is a sequence encoding a protein, it can be efficiently produced by repeatedly transcribing the sequence at a time under one promoter.

  In addition, according to the method of the present invention, a mixture of nucleic acids containing various numbers of target sequences can be easily obtained in large quantities. Since this mixture is a mixture of nucleic acids whose lengths differ from each other by the length of the target sequence, it can be used as a ladder marker that is one type of molecular weight marker for nucleic acids.

Hereinafter, the present invention will be described in detail.
(I) Method for producing nucleic acid having a plurality of target sequences A method for producing a nucleic acid having a plurality of target sequences of the present invention includes:
Preparing a circular DNA having a target sequence (1);
In order from the upstream side, this circular single-stranded DNA is used as a template by using a primer A having a first non-annealed sequence that does not anneal with the circular DNA and a first annealed sequence that can anneal with a part of the circular DNA. Performing the rolling circle type replication reaction (2),
In order from the upstream side, a second non-annealed sequence that does not anneal with the single-stranded nucleic acid obtained by the rolling circle type replication reaction, and a part of the single-stranded nucleic acid obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of the single-stranded nucleic acid using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded nucleic acid obtained in the step (3) is obtained. And a step (4) of performing a polymerase chain reaction using a template.
Process (1)
In step (1), circular DNA having a target sequence is prepared.

  The length of the target sequence is not particularly limited. For example, in the case of a base sequence encoding a protein, it is usually about several thousand bases, particularly up to about 3000 bases, and in the case of a base sequence encoding an aptamer, it is usually about 200 bases, particularly up to about 100 bases. In the case of a unit sequence of a nucleic acid ladder marker, it is usually about 1000 bases, particularly about 200 bases. Whatever the target sequence, it is necessary to have two primers and a length that can be annealed, so a length of at least about 50 bases is necessary.

  The nucleic acid having the target sequence may be either linear or circular, and may be either DNA or RNA. When other than circular DNA is targeted, it may be converted into circular DNA in step (1). Further, the nucleic acid having the target sequence may be either double-stranded or single-stranded, but is preferably a single-stranded nucleic acid from the viewpoint that rolling circle type replication to be performed later easily occurs.

  When the nucleic acid having the target sequence is RNA, it may be converted to DNA using reverse transcriptase.

  When the nucleic acid having the target sequence is a double-stranded linear DNA, it may be converted into a circular double-stranded DNA using a DNA ligase.

  In addition, when the nucleic acid having the target sequence is a single-stranded linear DNA, the DNA is ligated using DNA ligase in a state of being hybridized with an auxiliary single-stranded linear nucleic acid having both end sequences of the linear DNA. A circular DNA partially hybridized with an auxiliary single-stranded nucleic acid may be obtained by linking both ends of the DNA. Hybridization and circularization by self-ligation of single-stranded DNA having the target sequence may be performed sequentially or simultaneously.

  As the auxiliary single-stranded linear nucleic acid, one having a sequence capable of hybridizing to the 5 ′ end region of DNA having the target sequence and a sequence capable of hybridizing to the 3 ′ end region can be used. The sequence capable of hybridizing to the 5 ′ end region and the sequence capable of hybridizing to the 3 ′ end region may be adjacent to each other, and an arbitrary sequence may be sandwiched therebetween.

  The sequence capable of hybridizing to the 5 ′ terminal region and the sequence capable of hybridizing to the 3 ′ terminal region are each preferably about 5 to 50 bp, particularly about 10 to 40 bp in length. If the length is too long, the annealing specificity is lowered, and if the length is too short, the annealing becomes unstable and ligation of DNA having the target sequence becomes difficult. In addition, the arbitrary sequence that may be sandwiched between them is preferably about 1000 bp or less, particularly about 10 bp or less in length. This is because if the length is too long, ligation of DNA having the target sequence is difficult to perform.

  The temperature at which the auxiliary single-stranded linear nucleic acid is annealed to both ends of the linear single-stranded DNA having the target sequence is selected so that the annealing selectivity for the linear single-stranded DNA having the target sequence is the highest. That's fine. The temperature at which the annealing selectivity is highest is a temperature at which the auxiliary single-stranded nucleic acid and the linear single-stranded DNA having the target sequence are specifically bound and non-specific binding due to mismatch is minimized, What is necessary is just to raise the temperature gradually for each combination of single-stranded nucleic acids. Usually, the temperature may be about 0 to 80 ° C.

  After circularizing the single-stranded linear DNA, it may be removed by denaturing the auxiliary single-stranded nucleic acid, but the rolling circle replication performed later will synthesize the primer extension while stripping the nucleic acid duplex. Thus, the auxiliary single-stranded nucleic acid does not necessarily have to be removed.

  There are no particular limitations on the DNA ligase used when ligating both ends of double-stranded DNA having the target sequence and linking both ends of single-stranded DNA having the target sequence. For example, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, or heat-resistant DNA ligase (Taq ligase, Pfu ligase, Amp ligase, Thermus thermophilus DNA ligase, etc.) can be used. When DNA is circularized using the auxiliary single-stranded nucleic acid described above, a DNA ligation reaction may be performed at a relatively high temperature to improve annealing selectivity, so use a heat-resistant DNA ligase. Is preferred. The DNA ligation reaction using DNA ligase may be performed according to a conventional method, and for example, the method described in JP-A-63-22197 or WO90 / 01069 can be used.

  It is preferable to add a phosphate group to the 5 ′ end of each strand of linear single-stranded DNA or linear double-stranded DNA having the target sequence. This facilitates self-ligation. The phosphate group can be added by, for example, allowing T4 polynucleotide kinase to act in the presence of ATP. Alternatively, it can be modified during nucleic acid synthesis.

The circular DNA prepared in step (1) only needs to have a target sequence, and usually may have an arbitrary sequence therebetween. The length of this arbitrary sequence is preferably about 1000 bases or less, particularly about 100 bases or less.
Process (2)
<Primer A>
Primer A has, in order from the upstream (5 ′ end side), a first non-annealed portion that does not anneal with the circular DNA and a first annealing sequence that can anneal with a portion of the circular DNA.

  When the circular DNA is double-stranded, usually any strand may be used as a template. However, for example, when producing a DNA encoding a multimer protein, it is only necessary to have a first non-annealed portion that does not anneal with a strand having a target sequence and a first annealed sequence that can anneal with a portion of this strand.

  Here, the first annealing portion only needs to be able to anneal with a part of the circular DNA under some condition. The first non-annealed portion may be any portion that does not anneal with the circular DNA under conditions where the first annealed portion is annealed to the circular DNA.

  The length of the first annealed portion only needs to be long enough to specifically bind to a part of the circular DNA, and is usually about 15 to 100 bases, particularly about 20 to 50 bases that can be used as a PCR primer. Good.

  Since the first non-annealed portion is used as a PCR primer in step (4), it may be about 15 to 100, particularly about 20 to 50 bases, which can be usually used as a PCR primer.

  In addition, if a long arbitrary sequence is added to the downstream side (3 ′ end side) of the first annealed sequence, the DNA replication reaction by DNA polymerase is inhibited, so it is preferable that such an arbitrary sequence does not exist.

Primers may be synthesized by a known method. Examples of such known methods include a phosphoramidite method, a phosphate triester method, an H-phosphonate method, and a thiophosphite method using a DNA synthesizer manufactured by ABI (Applied Biosystems Inc.). It may also be isolated from digests obtained by digesting biological sources such as bacterial DNA with restriction endonucleases.
<Rolling circle type replication reaction>
The DNA polymerase used in the rolling circle type replication reaction only needs to have a displacement activity. For example, a DNA polymerase having a displacement activity derived from a phage such as φ29 DNA polymerase or M2 DNA polymerase can be used. In particular, M2 DNA polymerase is preferable in terms of high reaction efficiency.

  The rolling circle replication reaction is performed by, for example, allowing DNA polymerase to act on the circular single-stranded DNA as a template in the presence of dNTP ((4 types of deoxyribonucleotides (dATP, dCTP, dGTP, dTTP)). It can be made to progress by performing. In detail, it can carry out by the method as described in patent 290723 or Journal of Molecular Biology (Vol.56, 341-361, 1971), for example.

In addition, if primer A contains a promoter sequence, the presence of four ribonucleotides, NTP (ATP, CTP, GTP, TTP), using the single-stranded DNA obtained by the rolling circle replication reaction as a template A single-stranded RNA having a sequence complementary to one strand of circular DNA is obtained by performing an RNA synthesis reaction using an RNA polymerase (eg, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, etc.) Can do. Furthermore, a large amount of RNA can also be synthesized in step (2) by repeating the synthesis of cDNA using reverse transcriptase using this RNA as a template and allowing RNA polymerase to act again on this cDNA. If the target sequence encodes a ribozyme, antisense, etc., a large amount of ribozyme, antisense, etc. can be obtained by this operation. This operation may be performed separately from the rolling circle replication by DNA polymerase, or may be performed simultaneously.
Process (3)
<Primer B>
Primer B is a second non-annealed part that does not anneal with the repetitive single-stranded nucleic acid obtained by rolling circle replication in order from the upstream (5 ′ end side), and a part of the repetitive single-stranded nucleic acid. A second annealing arrangement capable of annealing with a portion different from the first annealing arrangement portion is provided.
Here, the second annealing sequence only needs to be able to anneal with a part of the single-stranded nucleic acid that is repeatedly different from the first annealing sequence part under some conditions. The second non-annealed portion may be any one that does not repeatedly anneal with the single-stranded nucleic acid under conditions where the second annealed portion is repeatedly annealed with the single-stranded nucleic acid.

  The length of the second annealed portion only needs to be long enough to specifically bind to a part of the single-stranded nucleic acid, and is usually about 15 to 100 bases, particularly about 20 to 50 bases that can be used as a PCR primer. If it is.

  Since the second non-annealed portion is used as a PCR primer in the step (4), it may be about 15 to 100 bases, particularly about 20 to 50 bases, which can be usually used as a PCR primer.

If a long arbitrary sequence is added to the downstream side (3 ′ end side) of the second annealing sequence, the DNA synthesis reaction by DNA polymerase is inhibited, so it is preferable that such an arbitrary sequence does not exist.
<Complementary strand synthesis>
In complementary strand synthesis, primer B is annealed to a single-stranded nucleic acid having a repetitive sequence. The annealing conditions may be a temperature and a solution condition at which the maximum annealing selectivity can be obtained. Specifically, the single-stranded DNA and the primer B are specifically bound repeatedly, and non-specific binding due to mismatching is prevented. What is necessary is just to select by raising the temperature which becomes the minimum.
<Complementary strand synthesis reaction>
The type of DNA polymerase used in step (3) is not particularly limited. For example, known enzymes such as RNA polymerase such as E. coli polymerase I, Klenow Fragment, various heat-resistant DNA polymerases, reverse transcriptase, and Qβ replicase can be used.

  However, when DNA obtained by the method of the present invention is used for protein production or aptamer repetitive sequence production, it has a proofreading function because the base sequence itself is important, unlike when used for production of ladder markers, etc. It is preferable to use a DNA polymerase. A DNA polymerase having a proofreading function can correct an incorporation error of deoxyribonucleotides, and as a result, the mutation appearance rate of the synthesized DNA can be suppressed to an extremely low level. Examples of the DNA polymerase having a proofreading function include KOD DNA polymerase, Pfu polymerase, PfuTurbo polymerase, PfuUltra polymerase, Deep Vent polymerase, and Pfx polymerase. In particular, KOD DNA polymerase is preferable from the viewpoint of high calibration function.

  The annealing to the single-stranded nucleic acid having the repetitive sequence of primer B and the complementary strand synthesis may be performed sequentially or substantially simultaneously.

A single-stranded nucleic acid obtained by rolling circle replication is a mixture of a plurality of types of single-stranded nucleic acids containing various numbers of target sequences. In addition, since each single-stranded nucleic acid has a plurality of sequences that can be annealed with the second annealing sequence of primer B, the double-stranded nucleic acid produced in step (3) has two lengths including various numbers of target sequences. It is a mixture of double-stranded nucleic acids.
Process (4)
In order to separate a double-stranded nucleic acid having a desired length from double-stranded nucleic acids having various lengths, a relatively large amount of double-stranded nucleic acid having each length is required. However, if a required amount of double-stranded nucleic acid is obtained by rolling circle replication in step (2) and complementary strand synthesis in step (3), it takes a long time and is inefficient. Therefore, the method of the present invention can amplify each of the double-stranded nucleic acid mixture produced by the step (3) by PCR by providing the first non-annealed portion in primer A and the second non-annealed portion in primer B. Is the biggest feature.
<Primer C>
Primer C only needs to be capable of annealing to the first non-annealed sequence of primer A under some PCR conditions, and preferably may be a sequence complementary to the first non-annealed sequence of primer A.

Arbitrary sequence may be added to the 5 'end side of the annealed part of primer C, but if it has a long arbitrary sequence on the 3' end side, it has DNA strand elongation reaction, so it has Even if it is, it is preferable that it is about 2 bases or less. In primer C, a sequence that can anneal with the first annealed part of primer A may be added downstream of the part of primer A that anneals with the first non-annealed part.
<Primer D>
Primer D may be any primer that can anneal to the second non-annealed portion of primer B under some PCR conditions. An arbitrary sequence may be added to the 5 ′ end side of the annealed portion of primer D, but if it has a long arbitrary sequence on the 3 ′ end side, the DNA strand elongation reaction is inhibited, so it has Even if it is, it is preferable that it is about 2 bases or less. In primer D, a sequence capable of annealing with the second annealed portion of primer B may be added downstream of the second non-annealed portion of primer B and the annealed portion.

Primer C and Primer D are long enough to anneal with the first annealed part of Primer A and the second annealed part of Primer B, respectively, and are too long to reduce the annealing specificity or extend the DNA. The length may be such that the reaction is not hindered. Specifically, it is preferably about 15 bases or more, particularly about 20 bases or more, about 100 bases or less, particularly about 50 bases or less.
<PCR>
A polymerase chain reaction (PCR) is performed by using a double-stranded nucleic acid mixture as a template and primer C and primer D as a sense primer and an antisense primer, respectively, and allowing DNA polymerase to act in the presence of dNTP.

  The annealing conditions for Primer C and Primer D may be the temperature and solution conditions at which maximum annealing specificity is obtained.

  As the DNA polymerase, a heat-resistant DNA polymerase generally used for PCR can be used without limitation. Examples of such enzymes include Taq DNA polymerase and Tth DNA polymerase. However, when synthesizing DNA for protein production by the method of the present invention or when producing DNA having repeated aptamers, the sequence itself is important, so a heat-resistant DNA polymerase having a calibration function (for example, KOD DNA polymerase, etc.) ) Is preferably used.

Through the above steps, a mixture of double-stranded DNAs having the target sequence is obtained.
Process (5)
In the present invention, if necessary, the step (5) is performed.For example, the double-stranded DNA mixture is subjected to electrophoresis, chromatography such as HPLC, etc. to separate by length, and the DNA of the target length is separated. That's fine. For example, in the case of producing an aptamer, electrophoresis may be a mixture of nucleic acids having various numbers of aptamer sequences, but a collection of nucleic acids having the same number of aptamer sequences. Thus, when fixing or adhering to the sensor chip, the number of aptamers per unit area can be made uniform, and as a result, a sensor chip with no sensitivity unevenness can be obtained.

  In addition, when a ladder marker is produced using a specific length sequence as the target sequence, a nucleic acid having a certain number of repeats of the specific length sequence may be selected by electrophoresis or chromatography.

In this step, the nucleic acid having the target length may be separated in a double-stranded state under the conditions as described above, but the present inventors perform separation more efficiently when separated in a single-stranded state. Found out to get. For example, by subjecting a double-stranded nucleic acid mixture to alkaline agarose gel electrophoresis, a single-stranded nucleic acid having a desired length can be separated. Alkaline agarose gel electrophoresis can employ, for example, the method described in Molecular Cloning, A Laboratory Manual, Third Edition, Joseph Sambrook & David W. Russell, Cold Spring Harbor Laboratory Press.
Process (6)
In addition, when it is necessary to convert the obtained double-stranded DNA into a single-stranded DNA, such as in the case of producing aptamers or antisenses, the double-stranded DNA is converted into heat, alkali, acid, urea, formamide, A single-stranded DNA may be denatured by treatment with an enzyme such as DNA helicase. The alkali treatment can be performed by, for example, treating with 0.2 to 1 N NaOH for 1 to 30 minutes and then neutralizing with an equal amount of HCl. The acid treatment can be performed, for example, by treating with HCl of about 0.01 to 1 N for 1 to 30 minutes and then neutralizing with NaOH.

The DNA denaturation treatment may be performed simultaneously with the separation of the double-stranded DNA having a desired length, or may be performed after the separation of the double-stranded DNA having a desired length. For example, by subjecting the double-stranded DNA mixture to alkaline agarose gel electrophoresis, single-stranded DNA of the desired length can be separated. Alkaline agarose gel electrophoresis can employ the method described in, for example, Molecular Cloning, A Laboratory Manual, Third Edition, Joseph Sambrook & David W. Russell, Cold Spring Harbor Laboratory Press.
Process (7)
The DNA having a desired length obtained in step (5) or step (6) may be amplified by PCR as necessary. Primers may be appropriately designed based on the sequence of the terminal portion of the target DNA. For example, if a linear single-stranded DNA having the target sequence 5 times is separated and recovered, and PCR is carried out using it as a template, a large amount of linear double-stranded DNA having the target sequence 5 times can be obtained.

  When the target sequence is, for example, an aptamer sequence, a single-stranded nucleic acid having about 2 to 20 target sequences may be finally obtained. In addition, when the target sequence is a sequence encoding a protein, it may be a number that can be inserted into a vector as a whole, and depends on the type of protein, but is usually a double-stranded DNA having about 2 to 10 target sequences. Just get. In addition, when the target sequence is a unit of a nucleic acid ladder marker, it may be a mixture of nucleic acids usually having about 1 to 20 repeats.

  The final nucleic acid is double-stranded DNA when the target sequence is a protein-encoding sequence, and single-stranded DNA or RNA when the target sequence is an aptamer sequence, and the target sequence is a ladder marker. Is a single-stranded or double-stranded DNA or RNA.

Further, according to the present invention, a nucleic acid having a plurality of target sequences can be obtained. This nucleic acid has a plurality of identical sequences repeated, and a plurality of substantially identical sequences which are partially different depending on a base reading error by polymerase. Including both.
(II) Protein production method The protein production method of the present invention is a method for synthesizing a DNA having a plurality of the same sequences of the present invention as described above. After preparing a circular DNA having a specific sequence, and otherwise performing up to step (4) of the above method in the same manner, after obtaining a mixture of double-stranded DNA having a plurality of sequences encoding the target protein,
Inserting the double-stranded DNA into a vector to obtain an expression vector (8);
Transforming the host with the expression vector (9),
And a step (10) of culturing the transformant and recovering the target protein from the culture.

  In the present invention, the protein-encoding sequence includes a sequence encoding the full length of the protein and a sequence encoding a part of the protein.

The type of the target protein is not particularly limited, and may be any protein such as an enzyme, an antibody, a peptide antigen, or a receptor.
<Vector>
As the vector, a known vector can be selected from a wide range depending on the host. For example, pBR322, pUC18, pUC19, pUC119, pBluescript and the like are used when Escherichia coli is used as a host, and pUB110 and pC194 are used when a Bacillus bacterium is used as a host. When yeast is used as a host, YIp5, YEp24 and the like can be mentioned. AcNPV etc. are mentioned when using an insect cell as a host. When animal cells are used as hosts, examples include pERV3 and pEGSH.

An expression vector can be obtained by inserting double-stranded DNA into an expressible position downstream of the promoter of such a known vector.
<Transformation>
Any known host can be used without limitation. For example, E. coli (eg, JM109, HB101, C600, DH5, DH5α, W3110), bacteria such as Bacillus bacteria (eg, MI114), yeast (eg, AH22), insect cells (eg, Sf cells), animal cells (eg, COS-7) Cell, Vero cell, CHO cell, etc.).

The host transformation method may be selected from known methods according to the host. Known introduction methods include calcium phosphate method, electroporation method, lipofection method, DEAE dextran method and the like. It is also convenient to transform using a commercially available competent cell (for example, Competent high JM109 manufactured by Toyobo Co., Ltd.) according to the attached experimental procedure. There is also a method by Thompson et al. (Journal of Bacteriology, Vol.151, 668-677, 1982).
<Culture of transformant>
When the transformant is a microorganism, it may be cultured using a liquid medium or a plate medium usually used for culturing the microorganism. Usually, a medium containing a carbon source, a nitrogen source, inorganic ions, and, if necessary, nitrates, phosphates, etc. may be used. As the carbon source, starch or starch hydrolyzate, sugar tightness, peptones and the like are preferably used. As the nitrogen source, polypeptone, tryptone, meat extract, yeast extract and the like can be used. The culture temperature should just be in the temperature which can grow microorganisms, for example, about 15-40 degreeC is mentioned. The pH of the medium may be within the range in which microorganisms can grow, and examples thereof include about 6 to 8. The culture time may be a time when the amount of the target protein in the culture is maximized, and varies depending on other culture conditions, but is usually about 1 to 5 days, particularly about 1 to 2 days. When an induction type expression vector such as a temperature shift type or an IPTG induction type is used, the induction time may be within one day, particularly about several hours.

Even when the transformant is a mammalian cell, it may be cultured under conditions suitable for the cell. For example, DMEM medium (Nissui, etc.) supplemented with FBS can be used and cultured at a temperature of about 36 to 38 ° C. in the presence of 5% CO ₂ while being replaced with a new medium every few days. Once the cells have grown to confluence, for example, trypsin PBS solution may be added to disperse the cells, and the resulting cell suspension may be diluted several times and seeded in a new petri dish to continue passage. The culture days are usually about 2 to 5 days, particularly about 2 to 3 days.

  Even when the transformant is an insect cell, the culture conditions may be suitable depending on the cell type. For example, it can culture | cultivate at about 25-35 degreeC using culture media for insect cells, such as Grace's medium containing FBS and Yeastlate. The culture time is preferably about 1 to 5 days, particularly about 2 to 3 days. Further, in the case of a transformant containing a virus such as Baculovirus as a vector, the culture time should be until the cytoplasmic effect appears and the cells die (for example, about 3 to 7 days, particularly about 4 to 6 days). preferable.

  When the target protein is produced in the host after completion of the culture, the transformant cells are collected by centrifugation or the like, and if necessary, the cells are suspended in an appropriate buffer, and then polytron, What is necessary is just to crush by mechanical methods, such as a sonication, a homogenizer, a French press. When the host is a bacterium, it may be treated with a cell wall lytic enzyme such as lysozyme or β-glucanase.

The target protein is recovered in the supernatant by centrifuging the obtained disrupted liquid at about 2000 to 15000 g for about 5 to 10 minutes. When the target protein is secreted outside the host, the target protein is recovered in the culture supernatant.
<Purification of target protein>
If necessary, the obtained crude protein solution is subjected to known protein purification methods such as salting out, desalting by dialysis, ion exchange, hydrophobicity, gel filtration, affinity chromatography, etc. What is necessary is just to refine.
(III) Method for producing multimeric protein
Production of DNA encoding multimeric protein In producing multimeric protein, first, DNA encoding multimeric protein is produced according to the method for synthesizing DNA having a plurality of identical sequences of the present invention.

That is, a step (1) of preparing a circular DNA having a sequence complementary to a base sequence encoding a target protein and having a target sequence excluding a start codon and a stop codon;
In order from the upstream side, this circular DNA is prepared by using primer A having a first non-annealed sequence that does not anneal with the target sequence of the circular DNA and a first annealed sequence that can anneal with a part of the target sequence of the circular DNA. (2) performing a rolling circle type replication reaction using as a template;
In order from the upstream side, a second non-annealed sequence that does not anneal with the single-stranded DNA obtained by the rolling circle replication reaction, and a part of the single-stranded nucleic acid that is different from the first annealed sequence (3) performing a complementary strand synthesis reaction of this single-stranded nucleic acid using primer B having a second annealable sequence that can be annealed;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded nucleic acid obtained in the step (3) is obtained. A DNA encoding a multimeric protein is produced by a method comprising a step (4) of performing a polymerase chain reaction using a template.

In step (1), the target sequence is a base sequence that encodes a multimeric protein if the circular DNA is a circular single-stranded DNA and the single strand is the target sequence. A sequence and a sequence complementary to the base sequence are target sequences.
The sequence excluding the start codon and stop codon is obtained by arranging multiple sequences encoding the same protein between a set of start codons and stop codons to obtain a multimeric protein in which the same amino acid sequence is arranged in plural. Because it is.

In order to express a multimeric protein, it is necessary to add a new start codon and a stop codon. These can be included in the non-annealed portion of primer A and primer B, or can be included in advance in the vector described below. For example, a start codon may be included in the first non-annealed portion of primer A, and a stop codon complementary sequence may be included in the second non-annealed portion of primer B. Alternatively, a complementary sequence of a stop codon may be included in the first non-annealed portion of primer A, and a start codon may be included in the second non-annealed portion of primer B. Thereby, in the step (4), a double-stranded DNA in which a start codon and a stop codon are present in one strand and a base sequence encoding a protein is arranged in parallel (in tandem) is amplified.
<Manufacture of multimeric protein>
As described above, the obtained double-stranded DNA is inserted into a vector to obtain an expression vector, the host is transformed with this expression vector, the transformant is cultured, and the multimeric protein is recovered from the culture product. That's fine.
(IV) Sensor production method The sensor production method of the present invention comprises producing a single-stranded nucleic acid having a plurality of aptamer sequences, immobilizing or adsorbing this DNA on the surface of a sensor chip substrate, and producing a sensor chip. This is a method of connecting the sensor chip and the detection device.

DNA having a plurality of aptamer sequences is a step (1) of preparing a circular DNA having an aptamer sequence;
From the upstream side, a rolling circle using this circular DNA as a template, using primer A having a first non-annealed sequence that does not anneal with this circular DNA and a first annealed sequence that can anneal with a portion of this circular DNA A step (2) of performing a mold replication reaction;
In order from the upstream side, a second non-annealed sequence that does not anneal with the single-stranded nucleic acid obtained by the rolling circle type replication reaction, and a part of the single-stranded nucleic acid obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of the single-stranded nucleic acid using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded nucleic acid obtained in the step (3) is obtained. And a step (4) of performing a polymerase chain reaction using a template.

  The kind of aptamer is not particularly limited. For example, nucleic acids that can specifically bind to proteins such as enzymes and receptors, saccharides, lipids, complexes thereof, and the like are targeted. The nucleic acid may be either DNA or RNA. In the case of RNA, RNA may be synthesized after the PCR in step (4).

  The nucleic acid obtained by PCR is double-stranded DNA, but when used as an aptamer, it may be made single-stranded by denaturation. For example, a nucleic acid that can be used as an aptamer can be obtained by heat treatment at about 95 to 100 ° C. for about 2 to 5 minutes.

  In addition, since the obtained DNA is a mixture of double-stranded DNAs of various lengths with different aptamer sequence repeat numbers, the same length is obtained by denaturing agarose gel electrophoresis or the like when it is desired to make the lengths uniform. Single-stranded DNA may be separated. As a result, the number of aptamers per unit area can be uniform over the entire sensor chip obtained by applying and fixing or adsorbing the obtained aptamer to the surface of the sensor chip substrate, thereby reducing the sensitivity unevenness of the sensor chip. it can.

  The material of the sensor chip substrate surface is not particularly limited, and any known material can be used without limitation. An example is a sensor chip substrate having a metal layer on the surface. Methods for immobilizing or adsorbing nucleic acids on the substrate surface are already well known, such as J. Am. Chem. Soc., Vol. 121, 8044-8051 (1999); Anal. Chem., Vol. 71, 3928-3934 ( 1999); Annu. Rev. Phys. Chem., Vol. 51, 41-63 (2000); Anal. Chem., Vol. 73, 1-7 (2001); J. Am. Chem. Soc., Vol. 124, 6810-6811 (2002) can be employed.

  A sensor can be obtained by combining the sensor chip thus obtained with a detection device. The type of the detector is not particularly limited, and a known one can be used. Examples of the detection device to which the sensor chip is connected include a device using surface plasmon resonance and a device using a crystal microbalance method. Specific examples include MultiSPRinter (Toyobo), Biacore (Biacore), AffinixQ (Initium).

  When the sample is brought into contact with the sensor thus obtained, if the aptamer binding target substance is present in the sample, it is captured by the aptamer by binding and detected by the detection device.

  According to the method of the present invention, it is possible to reliably obtain DNA having a plurality of identical sequences, which has been difficult to produce conventionally. In addition, a nucleic acid having the same desired sequence multiple times can be synthesized using only a widely used apparatus, instrument and reagent without requiring a special apparatus, instrument and reagent.

  In particular, when this sequence is a sequence encoding a protein, a host is transformed with an expression vector obtained by cloning it into a vector, and the target protein is recovered from the culture by culturing the transformant. Thus, a desired protein can be produced efficiently.

  In addition, when this sequence is an aptamer sequence, a single-stranded DNA having an aptamer sequence is synthesized multiple times, and this is immobilized on a sensor chip to produce a sensor. Thus, the number of aptamers per unit area of the sensor chip is reduced. Therefore, the detection sensitivity is improved accordingly.

  Further, according to the method of the present invention, a mixture of nucleic acids having different lengths by the length of the unit sequence can be easily obtained, and can be suitably used as a ladder marker.

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.
Where to buy materials In the following examples, DNA (single-stranded oligonucleotide) was purchased from Hokkaido System Science Co., Ltd. or ICM Co., Ltd. Other reagents were purchased from Nacalai Tesque Co., Ltd., Wako Pure Chemical Industries, Ltd. or Nakayama Shoji Co., Ltd.
Preparation of DNA polymerase for rolling circle amplification reaction The M2 DNA polymerase for rolling circle amplification reaction used in the examples was expressed in J. Gen. Appl. Microbiol., Vol. 39, 467-478, 1993. Using the vector, it was prepared by the method described in J. Gen. Appl. Microbiol., Vol. 39, 467-478, 1993. At that time, the culture scale was 8 L, the cells were cultured at 37 ° C., and the cells were further cultured for 2 hours under the induction condition of 2 mM IPTG at 120 Klett units. For purification, a crude enzyme solution was prepared from the cell disruption solution by salting out ammonium sulfate, followed by column chromatography of Phosphocellulose, Heparin Sepharose CL-6B, Heparin Sepharose CL-6B, and DEAE-Toyopearl. M2 DNA polymerase was obtained.

Creation of double-stranded DNA having the same sequence multiple times
(1) Synthesis of circular single-stranded DNA
(1-1) The following reagent composition was prepared.
Linear single-stranded DNA (SEQ ID NO: RCA100-2) 250 pmol
Linear single-stranded DNA (SEQ ID NO: 2: lig-temp) 500pmol
Ampligase (epicentre) 3μl
(15U, enzyme concentration is 5U / μl)
10X Ampligase buffer 7.5 μl
75 μl total
(1-2) The end of linear single-stranded DNA (RCA100-2) was ligated to the reagent composition of (1-1) under the following reaction conditions to synthesize circular single-stranded DNA.

94 ° C 30 seconds 60 ° C 15 minutes Number of cycles 40 times
(2) Annealing to circular single-stranded DNA
(2-1) The following reagent composition was prepared.
About 15 pmol of circular single-stranded DNA synthesized in (1)
Primer A (SEQ ID NO: 3 RCA) 300 pmol
Tris-HCl (pH 8.2) 17 mM
MgCl ₂ 17mM
20 μl total
(2-2) After treating the reagent composition of (2-1) at 85 ° C. for 10 minutes and then lowering to 20 ° C. over 20 minutes, primer A was annealed to the circular single-stranded DNA.
(3) Rolling circle amplification reaction
(3-1) The following reagent composition was prepared.
20 μl of annealing product prepared in (2)
Tris-HCl (pH 8.5) 50 mM
dNTP 250μM
MgCl2 20 mM
M2DNA polymerase 0.8μg
30 μl total
(3-2) The reagent composition of (3-1) was reacted at 30 ° C. for about 30 minutes and then heated at 94 ° C. for 3 minutes to stop the reaction.
(4) Complementary strand synthesis
(4-1) The following reagent composition was prepared.
15 μl of rolling circle amplification product prepared in (3)
Primer B (SEQ ID NO: 4: R-pol) 300 pmol
dNTP 200μM
MgSO ₄ 1 mM
KOD-Plus DNA polymerase (Toyobo) 1 unit
10X KOD-Plus buffer 5μl
50 μl total
(4-2) The reagent composition of (4-1) was treated at 94 ° C. for 2 minutes, then lowered to 68 ° C. over 60 seconds, and reacted at 68 ° C. for 2 minutes.
(5) PCR reaction
(5-1) The following reagent composition was prepared.
5 μl of DNA product prepared in (4)
Primer C (SEQ ID NO: 5: PCR-S) 0.2 μM
Primer D (SEQ ID NO: 6: PCR-AS) 0.2 μM
dNTP 200μM
MgSO ₄ 1 mM
1 unit of KOD-Plus DNA polymerase
10X KOD-Plus buffer 5μl
50 μl total
(5-2) The reagent composition of (5-1) was first treated at 94 ° C. for 2 minutes and then subjected to PCR reaction under the following reaction conditions to obtain linear single-stranded DNA (SEQ ID NO: 1 RCA100). A double-stranded DNA having the same sequence derived from the sequence of -2) was synthesized several times.

94 ℃ 15 seconds 68 ℃ 15 seconds Number of cycles 35 times
(6) Detection by alkaline agarose gel electrophoresis
Using the double-stranded DNA having the same sequence multiple times synthesized in (5) as a sample, described in Molecular Cloning (Molecular Cloning, A Laboratory Manual, Third Edition, Joseph Sambrook & David W. Russell, Cold Spring Harbor Laboratory Press) Alkaline agarose gel electrophoresis was performed using a 2% agarose gel by the method. After electrophoresis, the gel was neutralized and further stained with SYBR Gold (Molecular Probes) to detect single-stranded DNA.

  The appearance of the gel is shown in FIG. A DNA corresponding to a single-stranded DNA having the same sequence multiple times derived from the sequence of a linear single-stranded DNA (SEQ ID NO: RCA100-2) has been detected, and a plurality of identical sequences are detected by the method of the present invention. It has been confirmed that single-stranded DNA can be produced.

Creation of multimeric DNA of metallothionein
(1) Preparation of linear single-stranded DNA
(1-1) The following reagent composition was prepared.
Linear single-stranded DNA (SEQ ID NO: 7: PCR189-part1) 1 nM
Linear single-stranded DNA (SEQ ID NO: 8: PCR189-part2) 1 nM
Linear single-stranded DNA (SEQ ID NO: 9: PCR189-part3) 1 nM
Primer (SEQ ID NO: 10: 189-sp) 0.2 μM
Primer (SEQ ID NO: 11: 189-as-b) 0.2 μM
dNTP 200μM
MgSO ₄ 1 mM
1 unit of KOD-Plus DNA polymerase
10X KOD-Plus buffer 5μl
50 μl total
(1-2) The reagent composition of (1-1) was first treated at 94 ° C. for 2 minutes and then subjected to PCR under the following reaction conditions to synthesize linear double-stranded DNA.

94 ° C 15 seconds 55 ° C 15 seconds 68 ° C 5 seconds Number of cycles 40
(1-3) The amplification product of (1-2) was subjected to agarose gel electrophoresis, and the amplified fragment was extracted as double-stranded DNA from the gel with MagExtractor-PCR & Gel Clean up- (Toyobo). Then, dissociate the double-stranded DNA in an alkaline buffer (0.1M NaOH, 0.1M NaCl), and capture the DNA strand with the biotinylated 5'-end on the beads using MAGNOTEX-SA (TAKARA). Only the target 5′-phosphorylated single-stranded DNA was recovered. The recovered DNA strand was purified by ethanol precipitation.
(2) Synthesis and purification of circular single-stranded DNA
(2-1) The following reagent composition was prepared.
About 500 pmol of linear single-stranded DNA prepared in (1)
Linear single-stranded DNA (SEQ ID NO: 2: lig-temp) 1000pmol
Ampligase 1μl (5U)
10X Ampligase buffer 2μl
20 μl total
(2-2) Circular single-stranded DNA was synthesized by ligating the ends of linear single-stranded DNA to the reagent composition of (2-1) under the following reaction conditions.

94 ° C 30 seconds 60 ° C 10 minutes Number of cycles 40 times
(1-3) Using the reaction product of (1-2) as a sample, 8% polyacrylamide gel electrophoresis is performed to cut out a circular single-stranded DNA band that appears at a position near 1100 base. Grind the excised gel, add elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA (pH 8.0)), incubate at 37 ° C for about 3 hours, precipitate the gel by centrifugation, and remove it. The supernatant was ethanol precipitated.
(3) Annealing to circular single-stranded DNA
(3-1) The following reagent composition was prepared.
Approximately 15 pmol of circular single-stranded DNA prepared in (2)
Primer A (SEQ ID NO: 12: RCA3) 300 pmol
Tris-HCl (pH 8.2) 17 mM
MgCl ₂ 17 mM
20 μl total
(3-2) After treating the reagent composition of (3-1) at 85 ° C. for 10 minutes and lowering to 20 ° C. over 20 minutes, primer A was annealed to the circular single-stranded DNA.
(4) Rolling circle amplification reaction
(4-1) The following reagent composition was prepared.
20 μl of annealing product prepared in (3)
Tris-HCl (pH 8.5) 50 mM
dNTP 250μM
MgCl ₂ 20 mM
M2DNA polymerase 0.8μg
30 μl total
After reacting the reagent composition (4-2) at 30 ° C. for about 30 minutes, the reaction was stopped by heating at 94 ° C. for 3 minutes.
(5) Complementary strand synthesis
(5-1) The following reagent composition was prepared.
15 μl of rolling circle amplification product prepared in (4)
Primer B (SEQ ID NO: 13: R-Pol3) 300 pmol
dNTP 200μM
MgSO ₄ 1 mM
1 unit of KOD-Plus DNA polymerase
10X KOD-Plus buffer 5μl
50 μl total
(5-2) The reagent composition of (5-1) was treated at 94 ° C. for 2 minutes, then lowered to 68 ° C. over 60 seconds, and reacted at 68 ° C. for 2 minutes.
(6) PCR reaction
(6-1) The following reagent composition was prepared.
5 μl of DNA product prepared in (5)
Primer C (SEQ ID NO: 14: PCR-S3) 0.2 μM
Primer D (SEQ ID NO: 15: PCR-AS3) 0.2 μM
dNTP 200μM
MgSO ₄ 1 mM
1 unit of KOD-Plus DNA polymerase
10X KOD-Plus buffer 5μl
50 μl total
(6-2) The reagent composition of (6-1) is first treated at 94 ° C. for 2 minutes, and then subjected to a PCR reaction under the following reaction conditions to have the same sequence derived from the metallothionein sequence multiple times. Double-stranded DNA was synthesized.

94 ° C 15 seconds 55 ° C 10 seconds 68 ° C 40 seconds Number of cycles 30
(7) Alkaline agarose gel electrophoresis and extraction of amplified DNA
Alkaline agarose gel electrophoresis was performed using a 2% agarose gel by the method described in Molecular Cloning using the double-stranded DNA having the same metallothionein gene sequence synthesized several times in (6) as a sample. After electrophoresis, the gel was neutralized and further stained with SYBR Gold to detect single-stranded DNA.

The appearance of the gel is shown in FIG. DNA corresponding to a single-stranded DNA having the same sequence derived from the metallothionein sequence multiple times was detected, and it was confirmed that a nucleic acid having the same enzyme gene sequence multiple times could be prepared by the method of the present invention.
(8) Gel extraction from alkaline agarose gel
The desired single-stranded DNA was excised from the electrophoresis gel of (7), finely crushed, added with an equal amount of saturated phenol, and frozen at -80 ° C. to recover the single-stranded DNA (Bio-Experiment Illustrated 2- Basics of gene analysis (Shujunsha)).
(9) PCR of target fragment
The following reagent compositions were prepared.
5 μl of DNA prepared in (8)
Primer C (described in SEQ ID NO: 14 in the sequence listing: PCR-S3) 0.6 μM
Primer D (SEQ ID NO: 15 in the sequence listing: PCR-AS3) 0.6 μM
dNTP 200 μM
MgSO4 1 mM
1 unit of KOD-Plus DNA polymerase
10X KOD-Plus buffer 5μl
50 μl total
This reagent composition was first treated at 94 ° C. for 2 minutes and then subjected to PCR under the following reaction conditions to synthesize double-stranded DNA having the same sequence derived from the metallothionein sequence multiple times.

94 ° C 15 seconds 55 ° C 10 seconds 68 ° C 10 seconds (Time adjusted according to the number of repetitions)
50 ° C. 5 seconds Number of cycles 15 times Since this PCR has a tendency to become smeared when the number of cycles is increased, it was set to the minimum number of cycles at which a sufficient amplification product was obtained.
(10) Gel extraction by agarose gel electrophoresis
The PCR amplification products of (9) were separated by agarose gel electrophoresis and extracted with MagExtaractor (manufactured by Toyobo). The PCR amplification product extracted with MagExtractor was subcloned into pUC18 cleaved with restriction enzyme HincII, and it was confirmed that the amplified sequence had no mutation.
(11) Construction of expression vector
The fragment subcloned into pUC18 was cleaved with restriction enzymes NdeI and SalI, and inserted into pET-21a (+) (Novagen) cleaved with the same enzymes to obtain an expression vector.

Creation of multimeric DNA of Taq aptamer
(1) Synthesis of circular single-stranded DNA
(1-1) The following reagent composition was prepared.
Linear single-stranded DNA (SEQ ID NO: 16: aptq25) 50 pmol
Linear single-stranded DNA (SEQ ID NO: 17: aptq-lig) 300 pmol
Ampligase 1μl (5U)
10X Ampligase buffer 2μl
20 μl total
(1-1) The ends of linear single-stranded DNA (aptq25) were ligated to the reagent composition of (1-2) under the following reaction conditions to synthesize circular single-stranded DNA.

94 ° C 10 minutes 60 ° C 10 minutes Number of cycles 20 times
(2) Annealing to circular single-stranded DNA
(2-1) The following reagent composition was prepared.
8 μl of circular single-stranded DNA synthesized in (1)
Primer A (SEQ ID NO: 17: aptq-lig) 100 pmol
Tris-HCl (pH 8.2) 17 mM
MgCl ₂ 17 mM
20 μl total
(2-2) After treating the reagent composition of (2-1) at 85 ° C. for 10 minutes and lowering to 20 ° C. over 15 minutes, primer A was annealed to the circular single-stranded DNA.
(3) Rolling circle amplification reaction
(3-1) The following reagent composition was prepared.
20 μl of annealing product prepared in (2)
Tris-HCl (pH 8.5) 50 mM
dNTP 400μM
MgCl ₂ 20 mM
M2DNA polymerase 0.8μg
30 μl total
(3-2) The reagent composition of (3-1) was reacted at 30 ° C. for about 15 minutes and then heated at 94 ° C. for 3 minutes to stop the reaction.
(4) Complementary strand synthesis
(4-1) The following reagent composition was prepared.
14 μl of rolling circle amplification product prepared in (3)
Primer B (SEQ ID NO: 18: aptq-comp) 300 pmol
dNTP 200μM
MgSO ₄ 1 mM
1 unit of KOD-Plus DNA polymerase
10X KOD-Plus buffer 5μl
50 μl total
(4-2) The reagent composition of (4-1) was treated at 94 ° C. for 2 minutes, then lowered to 68 ° C. over 60 seconds, and reacted at 68 ° C. for 2 minutes.
(5) PCR reaction
(5-1) The following reagent composition was prepared.
5 μl of DNA product prepared in (4)
Primer C (SEQ ID NO: 19: aptq-sb) 0.2 μM
Primer D (SEQ ID NO: 20: aptq-as) 0.2 μM
dNTP 200μM
MgSO ₄ 1 mM
1 unit of KOD-Plus DNA polymerase
10X KOD-Plus buffer 5μl
50 μl total
(5-2) The reagent composition of (5-1) was first treated at 94 ° C. for 2 minutes, and then subjected to PCR under the following reaction conditions to obtain linear single-stranded DNA (SEQ ID NO: in the sequence listing). Description 16: Double-stranded DNA having the same aptamer gene sequence derived from the Taq aptamer sequence of aptq25) was synthesized multiple times.

94 ° C 15 seconds 68 ° C 20 seconds Number of cycles 30
(6) Detection by alkaline agarose gel electrophoresis
Alkaline agarose gel electrophoresis was performed using a 2% agarose gel by the method described in Molecular Cloning using the double-stranded DNA having the same aptamer gene sequence synthesized several times in (5) as a sample. After electrophoresis, the gel was neutralized and further stained with SYBR Gold to detect single-stranded DNA.

The appearance of the gel is shown in FIG. DNA corresponding to single-stranded DNA having the same sequence derived from the Taq aptamer sequence multiple times was detected, and it was confirmed that a nucleic acid having the same aptamer gene sequence multiple times can be prepared by the method of the present invention. .
(7) Gel extraction from alkaline agarose gel electrophoresis
The desired single-stranded DNA was excised from the electrophoresis gel of (6), finely crushed, added with an equal amount of saturated phenol and frozen at −80 ° C. to recover the single-stranded DNA (Bio-Experiment Illustrated 2- Basics of gene analysis (Shujunsha)).
(8) PCR of target fragment
The following reagent compositions were prepared.
5 μl of DNA product prepared in (7)
Primer C (described in SEQ ID NO: 19 in the sequence listing: aptq-sb) 0.6 μM
Primer D (SEQ ID NO: 20 in the sequence listing: aptq-as) 0.6 μM
dNTP 200 μM
MgSO4 1 mM
1 unit of KOD-Plus DNA polymerase
10X KOD-Plus buffer 5μl
50 μl total
This reagent composition was first treated at 94 ° C. for 2 minutes and then subjected to a PCR reaction under the following reaction conditions to obtain a Taq aptamer sequence of linear single-stranded DNA (SEQ ID NO: 16 in the sequence listing: aptq25). A double-stranded DNA having the same aptamer gene sequence derived from it was synthesized multiple times.

94 ° C. 15 seconds 55 ° C. 10 seconds 68 ° C. 1-3 seconds (Time was adjusted according to the number of repetitions)
50 ° C. 5 seconds Cycle number 15 times In this PCR, since the tendency to become smear increases as the number of cycles is increased, the minimum cycle number at which a sufficient amplification product can be obtained was set.
(10) Alkaline agarose gel electrophoresis
Using the double-stranded DNA having the same aptamer gene sequence synthesized in (9) multiple times as a sample, alkaline agarose gel electrophoresis was performed using a 2% agarose gel. After electrophoresis, the gel was neutralized and further stained with SYBR Gold to detect single-stranded DNA.
(11) Gel extraction from alkaline agarose gel electrophoresis
The desired single-stranded DNA was excised from the electrophoresis gel of (10), crushed finely, added with an equal amount of saturated phenol, and frozen at -80 ° C. to recover the single-stranded DNA.

  In addition, since the Biacore that applies the obtained single-stranded DNA requires high purity in the sample, it was considered that the final purification should be performed by alkaline agarose gel electrophoresis. A part of the sample was subjected to agarose gel electrophoresis, and after confirming the purity sufficiently, it was fixed on the surface of a chip manufactured by Biacore by a streptavidin-biotin binding method, and used as a sensor chip for Biacore.

  The method of the present invention can be suitably used in fields such as aptamer production, sensor production using the aptamer, protein production, and nucleic acid ladder marker production.

The electrophoretic analysis result in Example 1 is shown. The electrophoretic analysis result in Example 2 is shown. The electrophoretic analysis result in Example 3 is shown.

Claims (21)

Preparing a circular DNA having a target sequence (1);
In order from the upstream side, a primer A having a first non-annealed sequence that does not anneal with the circular DNA and a first annealed sequence that can anneal with a part of the circular single-stranded DNA is used as a template. Performing the rolling circle type replication reaction (2),
A second non-annealed sequence that does not anneal with the single-stranded DNA obtained by the rolling circle type replication reaction in order from the upstream side, and a part of the single-stranded DNA obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of this single-stranded DNA using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded DNA obtained in the step (3) is obtained. A method for producing a double-stranded DNA having a plurality of target sequences, comprising a step (4) of performing a polymerase chain reaction using a template. The method according to claim 1, further comprising a step (5) of separating DNA having a desired length. The method according to claim 2, wherein in step (5), DNA having a desired length is separated in a single-stranded state. The method according to claim 1, 2 or 3, further comprising a step (6) of amplifying the obtained DNA by a polymerase chain reaction. Furthermore, the method of Claim 4 including the process (6) which denatures the obtained double stranded DNA and makes it single-stranded. The method according to any one of claims 1 to 5, wherein a phage M2-derived DNA polymerase is used in step (2). The method according to any one of claims 1 to 6, wherein a DNA polymerase having a calibration function is used in step (3) and step (4). Preparing a circular DNA having a sequence of a specific length (1);
From the upstream side, a rolling circle using this circular DNA as a template, using primer A having a first non-annealed sequence that does not anneal with this circular DNA and a first annealed sequence that can anneal with a portion of this circular DNA A step (2) of performing a mold replication reaction;
A second non-annealed sequence that does not anneal with the single-stranded DNA obtained by the rolling circle type replication reaction in order from the upstream side, and a part of the single-stranded DNA obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of this single-stranded DNA using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded DNA obtained in the step (3) is obtained. A method for producing a double-stranded DNA ladder marker comprising a step (4) of performing a polymerase chain reaction using a template. Preparing a circular DNA having a base sequence encoding a target protein or a complementary sequence thereof (1);
From the upstream side, a rolling circle using this circular DNA as a template, using primer A having a first non-annealed sequence that does not anneal with this circular DNA and a first annealed sequence that can anneal with a portion of this circular DNA A step (2) of performing a mold replication reaction;
A second non-annealed sequence that does not anneal with the single-stranded DNA obtained by the rolling circle type replication reaction in order from the upstream side, and a part of the single-stranded DNA obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of this single-stranded DNA using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded DNA obtained in the step (3) is obtained. A step (4) of performing a polymerase chain reaction using a template; and
A method for producing a double-stranded DNA encoding a protein of interest comprising The method for producing double-stranded DNA encoding the target protein according to claim 9, wherein the target protein is metallothionein, isonitrile hydratase or nitrilase. A step (8) of obtaining an expression vector by inserting the double-stranded DNA obtained by the method according to claim 9 into a vector;
Transforming the host with the expression vector (9),
A method for producing a target protein comprising culturing the transformant and recovering the target protein from the culture (10). Inserting the double-stranded DNA obtained by the method according to claim 9 into the genome of the host by homologous recombination (11);
A method for producing the target protein, comprising culturing the host and recovering the target protein from the culture (10). 13. The method for producing a target protein according to claim 11 or 12, wherein the target protein is metallothionein, isonitrile hydratase or nitrilase. Preparing a circular DNA having a sequence complementary to the base sequence encoding the target protein and having the target sequence excluding the start codon and the stop codon; and
In order from the upstream side, this circular DNA is prepared by using primer A having a first non-annealed sequence that does not anneal with the target sequence of the circular DNA and a first annealed sequence that can anneal with a part of the target sequence of the circular DNA. (2) performing a rolling circle type replication reaction using as a template;
In order from the upstream side, a second non-annealed sequence that does not anneal with the single-stranded DNA obtained by the rolling circle replication reaction, and a part of the single-stranded DNA that is different from the first annealed sequence (3) performing a complementary strand synthesis reaction of this single-stranded DNA using primer B having a second annealing sequence that can be annealed;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded DNA obtained in the step (3) is obtained. A method for producing a double-stranded DNA encoding a multimeric protein comprising a step (4) of performing a polymerase chain reaction using a template. 15. The method for producing a double-stranded DNA encoding a multimeric protein according to claim 14, wherein the target protein is metallothionein, isonitrile hydratase or nitrilase. Inserting the double-stranded DNA obtained by the method of claim 14 into a vector to obtain an expression vector (8);
Transforming the host with the expression vector (9),
And a step (10) of culturing the transformant and recovering the target protein from the culture. Inserting the double-stranded DNA obtained by the method according to claim 14 into the host genome by homologous recombination (11);
And a step (10) of culturing the host and recovering the target protein from the culture. 18. The method for producing a target protein according to claim 16 or 17, wherein the target protein is metallothionein, isonitrile hydratase or nitrilase. Preparing a circular DNA having an aptamer sequence (1);
From the upstream side, a rolling circle using this circular DNA as a template, using primer A having a first non-annealed sequence that does not anneal with this circular DNA and a first annealed sequence that can anneal with a portion of this circular DNA A step (2) of performing a mold replication reaction;
A second non-annealed sequence that does not anneal with the single-stranded DNA obtained by the rolling circle type replication reaction in order from the upstream side, and a part of the single-stranded DNA obtained by the rolling circle type replication, A step (3) of performing a complementary strand synthesis reaction of this single-stranded DNA using a primer B having a second annealing sequence that can anneal with a portion different from the annealing sequence portion;
Using the primer C having a sequence capable of annealing to the first non-annealed sequence in the primer A and the primer D having a sequence capable of annealing to the second non-annealed sequence in the primer B, the double-stranded DNA obtained in the step (3) is obtained. A method for producing double-stranded DNA having a plurality of aptamer sequences, comprising a step (4) of performing a polymerase chain reaction using a template. 20. A method for producing a sensor chip, comprising fixing or adsorbing an aptamer obtained from double-stranded DNA having the aptamer sequence repeatedly obtained by the method according to claim 19. 21. A method for producing a sensor capable of detecting an aptamer binding target by connecting a sensor chip obtained by the method according to claim 20 to a detection device.