HK1054967B - Method of amplifying nucleic acid by using double-stranded nucleic acid as template - Google Patents

Description

Method for amplifying nucleic acid using double-stranded nucleic acid as template

Technical Field

The present invention relates to a method for synthesizing a nucleic acid comprising a nucleotide sequence complementary to a template double-stranded nucleic acid.

Background

The template-dependent PCR (polymerase chain reaction) method for synthesizing nucleic acids has greatly advanced the research in the recent field of bioscience. The PCR method enables exponential amplification of a nucleic acid containing a nucleotide sequence complementary to a template nucleic acid using a small amount of the template. The PCR method is a tool widely used for cloning or detecting genes at present. In the PCR method, a pair of primers containing complementary nucleotide sequences are used for both ends of a target nucleotide sequence. The primer pair is designed such that one primer anneals to the extension product provided by the other primer. The synthesis reaction is performed by repeating annealing with each other's extension product and the complementary strand synthesis reaction, thereby obtaining exponential amplification.

In the PCR method, a single-stranded nucleic acid template is prepared by some method, and a primer is annealed to the template. Since the template-dependent DNA polymerase requires a primer as a replication origin, it is necessary to prepare a single-stranded template in order to anneal the primer to the template in the PCR method. The step of converting a double-stranded template nucleic acid to single strands is generally referred to as denaturation. Denaturation is generally carried out by heating. Since other reaction components required for synthesizing nucleic acid, including DNA polymerase, are heat resistant, annealing and subsequent complementary strand synthesis reactions can be performed by mixing all reaction components and heating the reaction mixture. However, the conventional method including the heat treatment step has the following problems.

First, in the PCR method, denaturation of double-stranded nucleic acid and annealing of primers must be performed at each cycle. For this reason, a special mechanism for controlling the temperature is required. For example, although a method of monitoring an increase in a reaction product during PCR has been developed, the method cannot be carried out using a conventional analytical instrument, and therefore, it is necessary to provide an instrument having a mechanism capable of controlling temperature to perform a PCR method and a mechanism capable of monitoring a reaction. Therefore, if all reactions of nucleic acid synthesis can be carried out at a constant temperature, the reactions can be easily monitored using a conventional analytical instrument. This convenient method not only simplifies the apparatus but also simplifies the experimental operation. However, the reaction principle of this method is not known at present.

The reaction specificity of PCR depends on the specificity of primer annealing. Primers are expected to anneal to single stranded nucleic acids with sufficient specificity at high temperatures near the melting temperature. When the template is annealed with not high enough specificity and non-specifically, the resulting non-specific complementary strand synthesis reaction often occurs. Since the PCR method is accompanied by complicated temperature changes, the reaction mixture may be exposed to a temperature at which non-specific reactions are liable to occur. This is one of the causes of non-specific reactions associated with the PCR method.

Several methods have been proposed to solve the problems of the temperature-dependent non-specific reaction. For example, one method that has been put into practice uses a DNA polymerase that does not function at or below a certain temperature. Specifically, it has been reported that a temperature-sensitive DNA polymerase inhibitor, an antibody against DNA polymerase, or a variant of DNA polymerase, etc. are used. Furthermore, a process is known in which the reaction components are placed in compartments separated by a barrier which is meltable at high temperatures, so that the components can only be mixed after heating to a sufficiently high temperature. In all cases, since the PCR method is accompanied by complicated temperature changes, it is necessary to use a special component to prevent non-specific reactions.

Methods for amplifying DNA having a sequence complementary to a target sequence using the target sequence as a template are also known, such as the Strand Displacement Amplification (SDA) method (Pro.N.A.S., 89, pp.392-396; 1992, Nucleic Acid, Res., 20, pp.1691-1696; 1992). In the SDA method, when a complementary strand is synthesized using a primer complementary to the 3 '-side of a certain nucleotide sequence as a synthesis origin, a unique DNA polymerase is capable of synthesizing a complementary strand that can displace the 5' -side double-stranded region. When the "5 '-direction" or "3' -direction" is referred to below, these two terms refer to the direction of the template strand. This method is called strand displacement amplification because the double-stranded portion in the 5' -direction is displaced by the newly synthesized complementary strand.

In the SDA method, a step of changing the temperature, which is necessary for the PCR method, can be omitted by inserting a restriction enzyme recognition sequence into a sequence to which a primer anneals. That is, the nick provided by the restriction enzyme gives a 3' -OH group, which becomes the origin of complementary strand synthesis. From this origin, strand displacement and complementary strand synthesis proceed, and the synthesized complementary strand is dissociated as a single strand and used as a template in the subsequent complementary strand synthesis. Therefore, the SDA method does not require complicated temperature control necessary for the PCR method.

Although temperature control is not necessary in the SDA method, heat treatment is still required for preparing a single strand required for primer annealing when a double-stranded nucleic acid is used as a template. In addition, this method requires both a restriction enzyme capable of providing a nick and a DNA polymerase having a strand displacement activity. The need for another enzyme results in increased costs. In addition, in order to introduce nicks and not cleave the double strand (i.e., cleave only one strand), a dNTP derivative, such as α -thiodntp, must be used as a synthetic substrate, making one of the strands resistant to enzymatic digestion. Thus, the amplification product obtained by SDA has a different configuration from the natural nucleic acid. Thus, the use of restriction enzyme cleavage and amplification products in gene cloning is limited. The use of dNTP derivatives also leads to increased costs.

A nucleic acid amplification method which does not require complicated temperature control, namely nucleic acid sequence-based amplification (NASBA), that is, a so-called TMA/transcription-mediated amplification method, is known. NASBA is a reaction system in which DNA synthesis is performed using a DNA polymerase, using a target RNA as a template, and using a probe to which a T7 promoter is added. The synthesized DNA was made double-stranded using the second probe, and transcription was performed using T7RNA polymerase. The resulting double-stranded DNA was used as a template to amplify a large amount of RNA (Nature, 350, pp.91-92, 1991). Transcription using T7RNA polymerase in NASBA is performed isothermally. However, NASBA requires RNA as a template and therefore cannot be applied to double-stranded nucleic acids. This reaction can be carried out if the double-stranded nucleic acid is made single-stranded; however, in this case, complicated temperature control similar to PCR is required. In addition, it is necessary to use a plurality of enzymes such as reverse transcriptase, RNaseH, DNA polymerase and T7RNA polymerase in combination, which is economically disadvantageous, as in the SDA method. Therefore, the known nucleic acid amplification reaction method has a problem of complicated temperature control or a problem of necessity of using a plurality of enzymes.

In order to solve the problem of temperature control in the known nucleic acid synthesis methods, a complementary strand is synthesized under specific conditions using a primer as a synthesis origin (published Japanese translation of International publication No. Hei 11-509406; WO 97/00330). This method reveals the fact that hybridization of nucleic acids having complementary nucleotide sequences occurs in a dynamic equilibrium (kinetic) state. In the prior art method, it is believed that a complementary strand synthesis reaction using a primer as a synthesis origin is likely to occur even at a temperature that causes complete denaturation or lower. The term "completely denatured" as used herein refers to conditions under which most of the double-stranded template nucleic acid becomes single-stranded.

In this report, when a primer and a DNA polymerase having a strand displacement activity are mixed with a double-stranded template nucleic acid and the temperature is increased, synthesis of a complementary strand is observed at a temperature that does not cause denaturation of the template nucleic acid. However, the reaction efficiency of complementary strand synthesis without thermal cycling is significantly lower than that obtained in the PCR method with thermal cycling. In fact, the present inventors have conducted supplementary experiments to further confirm that the reaction did occur, but the amount of the reaction product obtained by this method did not reach a level usable for the actual nucleic acid synthesis method.

As described above, a nucleic acid synthesis reaction which does not require temperature control and does not deteriorate the reaction specificity and efficiency has not been reported so far.

Disclosure of Invention

An object of the present invention is to provide a nucleic acid synthesis method using a double-stranded nucleic acid as a template, wherein the method does not require a change in temperature and does not cause deterioration in synthesis efficiency, operability, specificity and the like. More specifically, the object of the present invention is to provide a novel nucleic acid synthesis method in which a reaction is performed by incubating a double-stranded nucleic acid as a template with reaction components such as a primer and a DNA polymerase at a constant temperature. Another object of the present invention is to provide a method for efficiently amplifying a nucleic acid using the synthesis method.

In order to carry out complementary strand synthesis using a double-stranded nucleic acid as a template without thermal cycling, the present inventors investigated whether or not a complementary strand synthesis reaction can be carried out under isothermal conditions using a primer as a synthesis origin. Known complementary strand synthesis methods based on dynamic equilibrium between a double-stranded nucleic acid and a primer (published Japanese translation of International publication No. Hei 11-509406; WO97/00330) do not require a change in temperature. However, as described above, it is difficult to obtain synthesis efficiency that can be used in practice using this method. Therefore, the present inventors combined this method with an isothermal nucleic acid synthesis reaction to efficiently perform complementary strand synthesis based on dynamic equilibrium without deteriorating specificity. As a result, the inventors have found that a high level of amplification efficiency, which could not be achieved by the known methods, can be obtained, and have completed the present invention. Namely, the present invention relates to the following nucleic acid synthesis method and a nucleic acid amplification method based thereon.

[1] A method for synthesizing a nucleic acid using a double-stranded nucleic acid as a template, wherein the method comprises:

a) incubating a double-stranded nucleic acid template and an arbitrary primer in the presence of a DNA polymerase capable of catalyzing a complementary strand synthesis reaction accompanying strand displacement under conditions that ensure synthesis of a complementary strand using the arbitrary primer as an origin, so that a region of a target template nucleic acid to which the primer anneals is under conditions that allow the region to undergo base pairing, the primer being capable of amplifying the template nucleic acid at a constant temperature;

b) annealing a primer capable of amplifying a template nucleic acid at a constant temperature to the region obtained in step a) under conditions such that it undergoes base pairing; and

c) complementary strand synthesis is performed using the primer as a synthesis origin.

[2] The method according to [1], wherein the step a) is carried out in the presence of a melting temperature regulator.

[3] The method according to [2], wherein the melting temperature regulator is at least one compound selected from the group consisting of: betaine, proline, dimethyl sulfoxide and trimethylamine-N-oxide.

[4] A method for synthesizing a nucleic acid in which a plurality of nucleotides constituting a specific region of a double-stranded nucleic acid template composed of complementary nucleotide sequences are connected on a single strand, wherein the method comprises:

a) incubating a double-stranded nucleic acid template and an arbitrary primer in the presence of a DNA polymerase capable of catalyzing a complementary strand synthesis reaction accompanied by strand displacement under conditions ensuring synthesis of a complementary strand using the arbitrary primer as an origin, so that a region of the target template nucleic acid to which the second primer anneals is under conditions allowing the region to undergo base pairing;

b) annealing a second primer to the region obtained in step a) under conditions such that it can undergo base pairing, and carrying out complementary strand synthesis using the second primer as an origin, wherein the 3 ' -end of the second primer anneals to a region defining the 3 ' -side of one strand constituting the specific region, and the 5 ' -end of the second primer contains a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis reaction product obtained using the primer as an origin;

c) subjecting a region of the extension product of the second primer synthesized in step b), which region will anneal to the first primer, to conditions that enable the region to undergo base pairing, wherein the 3 '-end of the first primer anneals to a region that defines the 3' -side of the region in the extension product obtained using the second primer as an origin;

d) annealing the first primer to the region obtained in step c) under conditions such that it can undergo base pairing, and conducting complementary strand synthesis using the first primer as the origin; and

e) self-annealing the 3' -end of the extension product of the first primer synthesized in step d), and performing complementary strand synthesis using the extension product itself as a template to obtain a nucleic acid in which a plurality of nucleotides constituting the specific region are connected on a single strand.

[5] The method according to [4], wherein the arbitrary primer in the step a) is a first primer.

[6] The method according to [4], wherein the step c) is performed by displacement according to a complementary strand synthesis reaction using a fourth primer that anneals to the 3' -side of the region of the template to which the second primer anneals, as the origin.

[7] The method according to [4], wherein the step e) further comprises a step of converting the extension product of the first primer into a single strand by displacement according to a complementary strand synthesis reaction using a third primer that anneals to the 3' -side of the template region to which the first primer anneals as an origin.

[8] The method according to [4], wherein the 5' -end of the first primer contains a nucleotide sequence complementary to an arbitrary region of the complementary strand synthesis reaction product obtained using the first primer as the origin.

[9] A method for amplifying a nucleic acid in which a plurality of nucleotides constituting a specific region of a double-stranded nucleic acid template composed of complementary nucleotide sequences are connected on a single strand, wherein the method comprises:

1) self-annealing the 3' -end of the extension product of the first primer produced by the method according to [7], and carrying out a complementary strand synthesis reaction using the extension product as an origin;

2) annealing the second primer or the first primer to a loop region formed by self-annealing at the 3' -end, and performing complementary strand synthesis using the primer as an origin;

3) strand displacement of the 3 '-end extension product by the complementary strand synthesis reaction of step 2) so that the 3' -end can undergo base pairing;

4) performing a complementary strand synthesis reaction using the displaced strand itself obtained in step 3) that can undergo base pairing as a template and using the 3' -end thereof as an origin to displace the complementary strand synthesized in step 2) using the loop region as an origin, thereby producing a single-stranded nucleic acid; and

5) repeating steps 2) to 4) to amplify the desired nucleic acid.

[10] The method according to [9], wherein the method further comprises:

6) performing a complementary strand synthesis reaction by self-annealing of the 3' -end of the single-stranded nucleic acid produced in the step 4);

7) annealing the second primer or the first primer to a loop region formed by self-annealing at the 3' -end, and performing complementary strand synthesis using the primer as an origin;

8) strand displacement of the 3 '-end extension product by the complementary strand synthesis reaction of step 7) so that the 3' -end can be subjected to base pairing;

9) using the displaced strand itself obtained in step 8) that can undergo base pairing as a template and conducting a complementary strand synthesis reaction using the 3' -end thereof as an origin to displace the complementary strand synthesized in step 7) using the loop region as an origin, thereby producing a single-stranded nucleic acid; and

10) repeating steps 7) to 9) to amplify the desired nucleic acid.

[11] A method for detecting a target nucleotide sequence in a sample, the method comprising performing the amplification method according to [10] and observing whether an amplification reaction product has been produced.

[12] The method according to [11], wherein the method according to [10] is carried out in the presence of a nucleic acid detecting agent, the method further comprising determining whether an amplification reaction product has been produced based on a change in signal of the detecting agent.

[13] A method for detecting a mutation by the detection method according to [11], wherein the mutation in the nucleotide sequence to be amplified prevents complementary strand synthesis at least one 3 '-end, the 3' -end being the origin of complementary strand synthesis constituting the amplification method.

[14] A method for amplifying a nucleic acid in which a plurality of nucleotides constituting a specific region of a template double-stranded nucleic acid composed of complementary nucleotide sequences are connected on a single strand, wherein the method comprises the step of incubating the following components under conditions capable of synthesizing a complementary strand using a first primer as an origin:

a target comprising a double-stranded nucleic acid template, said template comprising a specific region to be amplified;

a DNA polymerase catalyzing a complementary strand synthesis reaction accompanied by strand displacement;

a first primer of which 3 ' -end anneals to a region defining the 3 ' -side of one strand constituting the specific region and of which 5 ' -end contains a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis reaction product obtained using the primer as a synthesis origin;

a second primer whose 3 ' -end anneals to a region defining the 3 ' -side of one strand constituting the specific region and whose 5 ' -end contains a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis reaction product obtained using the primer as a synthesis origin; and

a nucleotide substrate.

[15] The method according to [14], wherein the composition further comprises:

a third primer that becomes the origin of the complementary strand synthesis reaction using the 3' -side of the region of the template to which the first primer anneals as the origin; and

and a fourth primer that serves as an origin of the complementary strand synthesis reaction using the 3' -side of the region of the template to which the second primer anneals as an origin.

[16] The method according to [14], wherein the incubation is carried out in the presence of a melting temperature regulator.

[17] The method according to [16], wherein the melting temperature regulator is at least one compound selected from the group consisting of: betaine, proline, dimethyl sulfoxide and trimethylamine-N-oxide.

[18] A method for subjecting a region of a target template nucleic acid to which a primer capable of initiating a reaction for amplifying a template nucleic acid at a constant temperature to base pairing, wherein the method comprises the step of incubating a template double-stranded nucleic acid, an arbitrary primer, and a primer capable of amplifying the template nucleic acid at a constant temperature in the presence of a DNA polymerase capable of catalyzing complementary strand synthesis accompanied by strand displacement under conditions that ensure synthesis of a complementary strand using the arbitrary primer as an origin.

In the present invention, a primer capable of amplifying a nucleic acid template at a constant temperature is used. The primer capable of amplifying a nucleic acid template at a constant temperature refers to a primer used in a nucleic acid amplification method without thermal cycling, which uses, as a template, a nucleic acid having a region to which the primer anneals, the region being capable of undergoing base pairing. That is, the primer of the present invention is a primer for a nucleic acid amplification reaction which does not require thermal cycling. The primer of the present invention is not particularly limited as long as it can amplify isothermal nucleic acid. Therefore, any primer capable of isothermal nucleic acid amplification is included in the primer of the present invention regardless of whether the primer can be used for a reaction requiring thermal cycling. As described above, it has been reported that amplification has been carried out at a constant temperature using PCR primers. However, since this method does not allow amplification at a practical level, it cannot be said that PCR primers are primers capable of isothermal nucleic acid amplification. In particular, amplification of a template nucleic acid can be carried out by a method of continuously synthesizing complementary strands without thermal cycling. The primer used in this method can be used as the primer of the present invention.

In a preferred embodiment, the nucleic acid amplification method uses a reaction principle (LAMP method) in which a complementary strand synthesis reaction is repeated by self-annealing at the 3' -terminal region that is a template for itself. In addition, a known nucleic acid amplification reaction, namely, SDA method can be used. In the present invention, the term "region that can undergo base pairing" refers to a region that is not accompanied by a complementary strand. Therefore, it includes not only a single-stranded nucleic acid produced by denaturing a double-stranded nucleic acid but also a single-stranded nucleic acid contained in a double-stranded nucleic acid having a single-stranded portion in part.

In addition, in the present invention, the nucleic acid may be DNA, RNA or chimeric molecules thereof. The nucleic acid may be a natural nucleic acid or an artificially synthesized nucleic acid. In addition, the nucleic acid of the present invention may also include a nucleotide derivative having a partially or completely artificial configuration as long as it can undergo base pairing. Examples of such molecules are: polynucleotide derivatives whose backbone is formed by phosphorothioate linkages. The number of nucleotides constituting the nucleic acid used in the present invention is not limited. In this context, the term nucleic acid is synonymous with the term polynucleotide. On the other hand, the term oligonucleotide as used herein refers to a specific polynucleotide having a smaller number of constituent nucleotides. In general, an oligonucleotide refers to a polynucleotide having 2 to 100, more usually 2 to 50 nucleotides, but it is not limited by these numbers.

The target nucleotide sequence as used herein refers to the nucleotide sequence of the nucleic acid to be synthesized. That is, the nucleotide sequence constituting the nucleic acid to be synthesized in the present invention is the target nucleotide sequence. In addition, when nucleic acid amplification is performed based on the nucleic acid synthesis method of the present invention, the nucleotide sequence constituting the nucleic acid to be amplified is a target nucleotide sequence. In general, the nucleotide sequence of a nucleic acid is described from the 5 '-side to the 3' -side of the sense strand. The target nucleotide sequence of the present invention includes not only the sense strand but also the nucleotide sequence of the complementary strand thereof, i.e., the antisense strand. More specifically, the term "target nucleotide sequence" refers to at least the nucleotide sequence to be synthesized or the complementary strand thereof.

The nucleic acid synthesis method of the present invention uses a double-stranded nucleic acid as a template. In the context of the present invention, a double-stranded nucleic acid is a nucleic acid that exists as a complementary strand hybridized at least in a region containing a nucleotide sequence complementary to a primer that is used as a synthesis origin of complementary strand synthesis. Thus, the double-stranded nucleic acid used in the present invention also includes a target nucleotide sequence in which some portion is not double-stranded. Further, the double-stranded nucleic acid used in the present invention may be not only a dimer but also a hybridization product of two or more single-stranded nucleic acids as long as it satisfies the above conditions. Alternatively, it may be a hairpin loop consisting of a single-stranded polynucleotide containing a complementary nucleotide sequence within the molecule. The double-stranded nucleic acid used in the present invention includes, for example, cDNA, genomic DNA and DNA-RNA hybrids. In addition, various vectors into which these DNAs are inserted can also be used as the double-stranded nucleic acid used in the present invention. The double-stranded nucleic acid used in the present invention may be a purified or crude nucleic acid. Furthermore, the method of the invention is also applicable to nucleic acids in cells (in situ). In situ genomic analysis can be performed using double-stranded nucleic acid in the cell as a template.

When cDNA is used as a template in the present invention, cDNA synthesis can be performed under the same conditions as those used for the synthesis of the nucleic acid of the present invention. When the first strand of a cDNA is synthesized using RNA as a template, a double-stranded nucleic acid in the form of a DNA-RNA hybrid is formed. The nucleic acid synthesis method of the present invention can be carried out using the obtained double-stranded nucleic acid as a template. When the DNA polymerase used in the nucleic acid synthesis method of the present invention has reverse transcriptase activity, nucleic acid synthesis can be performed under the same conditions using only the enzyme. For example, Bca DNA polymerase is a DNA polymerase having strand displacement activity as well as reverse transcriptase activity. Of course, the nucleic acid synthesis method of the present invention may also be used after the formation of a complete double-stranded cDNA by synthesizing the second strand.

In the nucleic acid synthesis of the present invention, a polymerase that catalyzes a complementary strand synthesis reaction accompanied by strand displacement is used. As used herein, the reaction of complementary strand synthesis accompanied by strand displacement refers to the following reaction. That is, when a template for a complementary strand synthesis reaction using a primer as a synthesis origin is hybridized with another polynucleotide and is in a double-stranded form, complementary strand synthesis proceeds while the reaction of separating the polynucleotide from the template is called a strand displacement complementary strand synthesis reaction. At this time, phosphodiester bonds in the isolated polynucleotide are generally retained. Thus, the polynucleotide thus formed has a length corresponding to the length of the synthesized complementary strand, and can undergo base pairing.

The same type of DNA polymerase as that used in the method such as SDA can be used as the polymerase catalyzing strand displacement complementary strand synthesis. If a double-stranded region exists in the 5 '-side of the template, a known unique polymerase synthesizes a complementary strand by replacing the double-stranded region using a primer complementary to a region in the 3' -side of a certain nucleotide sequence as a synthesis origin. According to the present invention, a substrate required for complementary strand synthesis is also used.

In the present invention, an arbitrary primer is mixed with a double-stranded nucleic acid, and the mixture is incubated under conditions ensuring synthesis of a complementary strand using the primer as an origin. The arbitrary primer of the present invention makes a region to be annealed with a primer used for a nucleic acid amplification reaction at a constant temperature ready for base pairing. Therefore, the arbitrary primer must be capable of initiating complementary strand synthesis of the template double-stranded nucleic acid using the complementary strand of the nucleotide strand as a template, which anneals to the primer used for the amplification reaction. In addition, the strand extension in complementary strand synthesis using the arbitrary primer of the present invention as the synthesis origin should proceed toward the region to which the primer for amplification reaction anneals. In other words, the primer can provide a synthesis origin at an arbitrary portion of the region that serves as a template in complementary strand synthesis using a primer used in an amplification reaction as an origin. Any primer may contain a nucleotide sequence complementary to any region as long as it satisfies the above criteria. For example, one primer used for the amplification reaction may be used as an arbitrary primer. The use of primers is one of the preferred embodiments of the present invention due to the reduction in the number of necessary reaction components.

By replacing one of the two strands of the double-stranded nucleic acid in the complementary strand synthesis using an arbitrary primer as the origin, base pairing with the primer used for the amplification reaction can be ensured. By selecting the parameters, the synthesis reaction can be carried out without changing the temperature, which is a big feature of the present invention. The conditions which allow the complementary strand synthesis reaction to be carried out by an arbitrary primer using a double-stranded nucleic acid as a template are practically the same as those required for carrying out the following combination:

i) providing a synthesis origin to the template double-stranded nucleic acid by an arbitrary primer; and

ii) continuing the complementary strand synthesis reaction using the synthesis origin.

It is believed that a primer can provide a synthesis origin for a nucleic acid strand only when the region to which the primer anneals is single-stranded. Thus, previously when double-stranded nucleic acid was used as a template, the nucleic acid was subjected to denaturation, a step of converting the nucleic acid to single-stranded before primer annealing. However, the synthesis origin may be provided by incubating the template and primer under conditions that destabilize the double strand in some way but do not completely convert to single strand. Examples of such conditions for destabilizing the double strand include heating the double-stranded nucleic acid to almost a melting temperature (hereinafter abbreviated as Tm). Alternatively, the addition of a Tm regulator is also effective.

Carrying out a reaction comprising a sequence of steps in the presence of a buffer, said bufferThe wash solution can provide a pH suitable for the enzymatic reaction, as well as salts required for annealing the primers and maintaining the enzymatic activity, preservatives for the enzyme, and if necessary, melting temperature (Tm) regulators, and the like. The buffer solution with a buffering effect and a pH range from neutral to weakly alkaline is used in the present invention. The pH is adjusted according to the type of DNA polymerase used. Examples of salts added for maintaining the enzymatic activity and changing the melting temperature (Tm) of nucleic acid include KCl, NaCl, (NH)₄)₂SO₄And the like. The enzyme preservatives include bovine serum albumin and sugars.

In addition, typical melting temperature (Tm) regulators include betaine, proline, dimethyl sulfoxide (hereinafter abbreviated as DMSO), formamide, and trimethylamine-N-oxide (TMANO). When a melting temperature (Tm) regulator is used, annealing of the above oligonucleotide can be regulated within a limited temperature range. In addition, betaine (N, N, N-trimethylglycine) and tetraalkylammonium salts are effective in improving the efficiency of strand displacement due to their isostabilizing action. The addition of betaine to the reaction solution at a concentration of about 0.2 to about 3.0M, preferably about 0.5 to about 1.5M is expected to enhance the amplification of nucleic acids of the present invention. Since these melting temperature regulators can lower the melting temperature, conditions giving the desired stringency and reactivity can be empirically selected by considering reaction conditions such as salt concentration and reaction temperature.

Temperature conditions suitable for the enzymatic reaction can be easily selected by using a Tm regulator. Tm can be changed depending on the relationship between the primer and the target nucleotide sequence. Therefore, it is preferable to adjust the amount of the Tm regulator so that the conditions for maintaining the enzyme activity are consistent with the incubation conditions in accordance with the standard of the present invention. According to the present invention, those skilled in the art can easily select an appropriate amount of the Tm regulator to be added according to the nucleotide sequence of the primer. For example, Tm can be determined based on the length of the annealing nucleotide sequence, GC content, salt concentration and the concentration of the Tm regulator.

It is assumed that annealing of the primer to the double-stranded nucleic acid is unstable under such conditions. However, when DNA is incubated with a polymerase that catalyzes strand displacement complementary strand synthesis, complementary strand synthesis can be performed using an unstable primer as a synthesis origin. When complementary strand synthesis is performed, hybridization between the synthesized complementary strand and the template nucleic acid becomes more stable in this process. The following DNA polymerases can catalyze complementary strand synthesis using a primer as a synthesis origin of a template double-stranded nucleic acid.

A DNA polymerase catalyzing a strand displacement complementary strand synthesis reaction plays a central role in the nucleic acid synthesis method of the present invention. The DNA polymerase includes those listed below. In addition, various mutants of these enzymes can also be used in the present invention as long as they have sequence-dependent complementary strand synthesis activity and strand displacement activity. Such mutants include truncated forms of enzymes containing only the structure having catalytic activity, or mutant enzymes whose catalytic activity, stability or thermostability have been modified by amino acid mutation, and the like.

Bst DNA polymerase

Bca (exo-) DNA polymerase

Klenow fragment of DNA polymerase I

Vent DNA polymerase

Vent (Exo-) DNA polymerase (Vent DNA polymerase without exonuclease Activity)

DeepVent DNA polymerase

DeepVent (Exo-) DNA polymerase (DeepVent DNA polymerase without exonuclease Activity)

Phi 29 bacteriophage DNA polymerase

MS-2 phage DNA polymerase

Z-Taq DNA polymerase (Takara Shuzo)

KOD DNA polymerase (TOYOBO)

Among these enzymes, Bst DNA polymerase and Bca (exo-) DNA polymerase are particularly preferable because they have high levels of thermal stability and high catalytic activity. According to the present invention, the step of using a primer as a synthesis origin of a double-stranded nucleic acid and complementary strand synthesis reaction is performed under the same conditions. Since the reaction often requires some heating, the use of thermostable enzymes is preferred. The reaction can be carried out under a variety of conditions using thermostable enzymes.

For example, Vent (exo-) DNA polymerase is a thermostable enzyme with strand displacement activity. The addition of a single-stranded binding protein has been reported to accelerate the strand displacement complementary strand synthesis reaction catalyzed by DNA polymerase (Paul M. Lizardi et al, Nature Genetics 19, 225-232, July, 1998). By applying this method to the present invention, it is expected that complementary strand synthesis will be accelerated by adding a single-strand binding protein. T4 gene 32 is effective as a single strand-binding protein when Vent (exo-) DNA polymerase is used.

When a DNA polymerase lacking 3 ' -5 ' exonuclease activity is used, a phenomenon is known in the art in which complementary strand synthesis is not terminated even when the reaction reaches the 5 ' -end of the template and an additional nucleotide is added to the synthesized strand. This phenomenon is not preferred in the present invention because the subsequent complementary strand synthesis starts from the synthesized 3' -end complementary strand sequence. However, the nucleotide added to the 3' -end by the DNA polymerase is highly likely to be the nucleotide "A". Therefore, the sequence for complementary strand synthesis should be selected to start synthesis from the A at the 3' -end, thereby avoiding the problems caused by erroneous addition of a single-dATP nucleotide. Alternatively, even when the 3 ' -end protrudes during complementary strand synthesis, it can be digested into a blunt end by 3 ' → 5 ' exonuclease activity. For example, natural Vent DNA polymerase having such an activity can be used in combination with Vent (exo-) DNA polymerase to overcome the above-mentioned problems.

Unlike the above-mentioned DNA polymerases, DNA polymerases conventionally used for PCR and the like, such as Taq polymerase PCR, exhibit substantially no strand displacement activity under ordinary conditions. However, such DNA polymerases can be used in the present invention as long as they are used under conditions that ensure strand displacement.

A phenomenon has been reported in which a complementary strand is synthesized by incubating a primer under conditions under which a double-stranded nucleic acid becomes unstable (published Japanese translation of International publication No. Hei 11-509406; WO 97/00330). However, under the reported conditions, only trace amounts of the synthesized product are expected to be actually produced. Although complementary strand synthesis can be performed theoretically by using destabilization of a double-stranded nucleic acid using a primer as a synthesis origin, the reaction is not as efficient as a reaction using a single-stranded nucleic acid as a template. When combined with a complementary strand synthesis reaction requiring a change in temperature, such as the PCR method, the efficiency of the destabilized complementary strand synthesis reaction using the double strand affects all complementary strand synthesis reactions; therefore, it is difficult to achieve reaction efficiency usable in practice. This is believed to be the reason for the insufficient amplification efficiency of the known methods.

On the other hand, the present invention is based on the finding that the low efficiency of complementary strand synthesis based on destabilization of a double-stranded nucleic acid can be compensated for by applying a complementary strand synthesis reaction based on destabilization of a double-stranded nucleic acid to a nucleic acid amplification reaction that provides a region that can anneal to a primer used for an amplification reaction that has been originally performed at a constant temperature. Since the present invention utilizes a nucleic acid amplification reaction carried out at a constant temperature, if the region to which the primer anneals is placed under conditions that ensure base pairing, a complementary strand synthesis reaction that does not require destabilization of the double strand will be subsequently carried out. Therefore, the influence of the low efficiency of the complementary strand synthesis reaction based on the destabilization of the double strand can be minimized. By using these two methods in combination, a practically usable level of synthesis efficiency is obtained for the first time. In other words, a complementary strand synthesis reaction based on destabilization of a double strand can be used as a reaction providing a region to anneal with a primer for a nucleic acid amplification reaction performed isothermally. In contrast, it is difficult to apply the reaction to a nucleic acid amplification reaction that requires thermal cycling.

For example, in the isothermal amplification method of nucleic acid of the present invention, it is necessary to self-anneal the 3' -end and use it as a template for the complementary strand synthesis reaction. That is, the main feature of the present invention is that no temperature change is required, and particularly when the following primers are used, the optimum use thereof is embodied. The present inventors conceived a nucleic acid amplification method using primers having a specific configuration. Hereinafter, the method is described as a LAMP (Loop-mediated isothermal amplification) method. According to the LAMP method, the 3' -end of the template polynucleotide anneals to itself to serve as the origin of complementary strand synthesis, and a primer that anneals to the loop thus formed is used to perform an isothermal complementary strand synthesis reaction. The inventors have found that denaturation of the double stranded template is not required.

The present invention relates to a nucleic acid synthesis method using a primer that can amplify a nucleic acid isothermally, the primer being an oligonucleotide composed of at least two regions X2 and X1c, wherein X1c is linked to the 5' -side of X2.

Herein, X2 is defined as a region having a nucleotide sequence complementary to any region X2c of a nucleic acid having a particular nucleotide sequence.

In addition, X1c is defined as a region having a nucleotide sequence substantially identical to any region in the X2c 5' -side of the nucleic acid region having a specific nucleotide sequence.

In the explanation given below, primers are used as the first primer and the second primer. The first primer anneals to an extension product synthesized using the second primer as an origin to serve as an origin of a complementary strand synthesis reaction, and vice versa. The synthesis product prepared by the nucleic acid synthesis method using the primer as the synthesis origin enables the following nucleic acid amplification. The present invention relates to a nucleic acid synthesis method in which a plurality of nucleotides constituting a specific region of a double-stranded template nucleic acid composed of complementary nucleotide sequences are connected on a single strand, wherein the method comprises the following steps. Herein, the condition that a certain nucleotide sequence 1 and at least one nucleotide sequence 2 complementary thereto exist on the same strand is referred to as a condition that a plurality of complementary nucleotide sequences are contained on a single strand.

a) Incubating a template double-stranded nucleic acid and an arbitrary primer in the presence of a DNA polymerase capable of catalyzing a complementary strand synthesis reaction accompanying strand displacement under conditions that ensure synthesis of a complementary strand using the arbitrary primer as an origin, so that a region of a target template nucleic acid to which the primer anneals is under conditions that allow the region to undergo base pairing, the primer being capable of amplifying the template nucleic acid at a constant temperature;

b) annealing a second primer to the region that can undergo base pairing obtained in step a), and performing complementary strand synthesis using the second primer as an origin, wherein the 3 ' -end of the second primer anneals to a region that defines the 3 ' -side of one strand constituting the specific region, and the 5 ' -end of the second primer contains a nucleotide sequence that is complementary to an arbitrary region of a complementary strand synthesis reaction product obtained using the primer as an origin;

c) subjecting a region of the second primer extension product synthesized in step b), which region will anneal to the first primer, to conditions that allow the region to undergo base pairing;

d) annealing the first primer to the region that can undergo base pairing obtained in step c) and performing complementary strand synthesis using the first primer as the origin, wherein the 3 '-end of the first primer anneals to a region that defines the 3' -side of the region in an extension product obtained using the second primer as the origin; and

e) self-annealing the 3' -end of the extension product of the first primer synthesized in step d), and performing complementary strand synthesis using the extension product as a template to obtain a nucleic acid in which a plurality of nucleotides constituting the specific region are connected on a single strand.

In the above reaction, only the step a) is obtained by destabilization of the double-stranded nucleic acid. The extension product of the primer (i.e., the second primer) used in the amplification reaction, from which the annealing region has been obtained in this step, is used as a template for the subsequent reaction. It should be noted that: since the DNA polymerase catalyzing the strand displacement complementary strand synthesis reaction is used not only together with the primer used in the amplification reaction but also the complementary strand synthesis of the present invention is always using the enzyme, the double-stranded configuration occurring in front of the complementary strand synthesis reaction does not prevent the reaction if only the primer is annealed.

In addition, in the present invention, the reaction after this step is originally carried out at a constant temperature, and does not rely on complementary strand synthesis using destabilization of a double-stranded nucleic acid with low efficiency. That is, the nucleic acid amplification reaction is started at a constant temperature after the complementary strand synthesis is performed using the primer used in step a) as a synthesis origin.

Even after the nucleic acid amplification reaction is carried out starting at isothermal temperature, the primer for the target nucleotide sequence present in the reaction system may result in synthesis of a complementary strand according to destabilization of the double-stranded nucleic acid. It cannot be denied that such a reaction may occur, and it goes without saying that the occurrence of such a reaction contributes to improving the efficiency of the entire reaction. In the nucleic acid amplification reaction of the present invention, it is not necessary to carry out a complementary strand synthesis reaction based on destabilization of a double-stranded nucleic acid simultaneously with the amplification reaction.

It is advantageous to use an external primer in step c) of the above reaction. Herein, the outer primer provides the origin of a complementary strand synthesis reaction that proceeds toward a primer that anneals to the target nucleotide sequence (opposite to the outer primer, which is referred to as an inner primer). Therefore, the region to which the outer primer anneals is a region in the 5 '-side (3' -side of the template) of the inner primer. An oligonucleotide having a nucleotide sequence usable as a primer at least in the 3' -side thereof can be used as an external primer. The first primer and the second primer correspond to the inner primers in this example.

On the other hand, the inner primer contains at its 3' -end a nucleotide sequence of a double-stranded nucleic acid template, which is complementary to the nucleotide sequence of the region to be synthesized. The inner primer is usually used as a primer set consisting of two primers. However, if the region to be synthesized contains repeats of the same nucleotide sequence, the two primers may contain the same nucleotide sequence. The primer set can be designed such that one inner primer can anneal to the extension product of another inner primer. Methods for determining the nucleotide sequence to be used as a primer are also well known when at least the nucleotide sequence at both ends of the region to be amplified is known.

If the inner primer is a primer that anneals to the target nucleotide sequence in the 3 '-side, an arbitrary nucleotide sequence may be added in the 5' -side of the inner primer. The fact that an arbitrary sequence can be added to the 5' -side of the inner primer brings about many changes to the nucleic acid synthesis method of the present invention. Specific examples will be described below.

The inner primers of the invention can be nested. That is, the second inner primer set that can anneal to the second target nucleotide sequence may be further associated with the first inner primer set that can anneal to the first target nucleotide sequence. In this combination, the first target nucleotide sequence is contained within the second target nucleotide sequence. When the inner primers are nested, the outer primers can be designed to anneal to the 5 '-side of the second inner primer (the 3' -side of the template).

The inner primer generally contains a combination of two primers, and the number of outer primers may be any number. In general, the outer primer of the present invention contains two primers, thereby providing the origin of a complementary strand synthesis reaction that proceeds toward the region to which each inner primer anneals. However, the method of the present invention can be performed even if only the outer primer is applied to any one of the inner primers. Alternatively, multiple outer primers may be used with one inner primer. In all cases, when the complementary strand synthesis proceeds toward the region to which the inner primer anneals, the product of the complementary strand synthesis reaction using the inner primer as the synthesis origin can be efficiently produced.

Complementary strand synthesis driven by the outer primer of the present invention is designed to start after synthesis of a complementary strand for the synthesis start point using the inner primer. This is most easily achieved when the concentration of the inner primer is higher than the concentration of the outer primer. Specifically, when the difference in primer concentration is usually about 2 to 50 times, preferably about 4 to 10 times, complementary strand synthesis from the inner primer is mainly performed. In addition, by setting the melting temperature (Tm) of the outer primer lower than the Tm of the inner primer, the timing of the complementary strand synthesis reaction can be controlled. When all other conditions are unchanged, the melting temperature (Tm) can be theoretically calculated from the length of the annealed complementary strand and the combination of nucleotides constituting the base pair. Therefore, the skilled in the art can easily derive the required reaction conditions from the disclosure of the present specification.

In addition, a phenomenon called adjacent stacking can be used to adjust the timing of annealing of the outer primers. Adjacent stacking is a phenomenon in which oligonucleotides that cannot be independently annealed are able to anneal when they are placed adjacent to a double-stranded portion (Chiara Borghesi-Nicoletti et al, Bio Techniques 12, pp.474-477 (1992)). That is, the outer primer is placed adjacent to the inner primer, and the outer primer is designed so that it cannot anneal independently under the incubation condition. Thus, the outer primer can anneal only when the inner primer anneals. Therefore, annealing of the inner primer is inevitably an absolute advantage. Based on this principle, examples of setting the nucleotide sequences of primer oligonucleotides required for a series of reactions are described in examples.

In the explanation described below, X2 and X1c in one inner primer are tentatively referred to as F2 and F1c, and X2 and X1c in the other inner primer are referred to as R2 and R1 c. The inner primers used for the explanation are tentatively referred to as FA and RA. One of FA and RA is the first primer of the present invention, and the other functions as the second primer. The regions constituting FA and RA are as follows:

X2 X1c

FA F2F 1c

RA R2R 1c

in the nucleic acid amplification method of the present invention, by the above-mentioned steps a) to c), it is important to produce a nucleic acid having an F1 region at its 3' -end, the F1 being capable of partially annealing to F1c on the same strand, forming a loop containing an F2c region, and undergoing base pairing by annealing the F1 region to F1c on the same strand. Such a nucleic acid configuration can be provided by a nucleic acid synthesis reaction according to the present invention, which utilizes an inner primer having the following configuration. Details of the reaction are as described above.

That is, the inner primer used in the nucleic acid amplification reaction of the present invention is composed of at least the above-mentioned two regions X2 and X1c, and contains an oligonucleotide in which X1c is linked to the 5' -side of X2.

Herein, the nucleic acid having a specific nucleotide sequence, which determines the configuration of the inner primer of the present invention, refers to: when the inner primer of the present invention is used as a primer, it becomes a nucleic acid of a template. When nucleic acid amplification is performed according to the synthesis method of the present invention, the nucleic acid having a specific nucleotide sequence is a double-stranded nucleic acid to be amplified or a derivative thereof. The double-stranded nucleic acid having a specific nucleotide sequence refers to: a nucleic acid at least a part of which is clear or whose sequence can be deduced. The portions whose nucleotide sequence should be clearly clarified are the above-mentioned region X2c and the region X1c in the 5' -side thereof.

The two regions may be connected to each other or may exist independently. The state of the loop portion formed by self-annealing of the product nucleic acid depends on the relative positions of the two regions. Preferably, the two regions must be separated so that self-annealing of the product nucleic acid occurs more readily than annealing between two molecules. Thus, the preferred length of the spacer nucleotide sequence between the two regions is generally from 0 to 500 nucleotides. However, in some cases, regions that are too close to each other may be detrimental to forming the desired self-annealed ring. More specifically, the loop must have a structure capable of annealing to the new primer and a smooth strand displacement complementary strand synthesis reaction origin. Therefore, it is more preferable to design the primer such that the distance between the region of X2c and the region of X1c located in the 5' -side thereof is about 0 to 100 nucleotides, and more preferably about 10 to 70 nucleotides. The distance values recited herein do not contain the length of X1c and X2. The nucleotide length of the loop portion further includes a length corresponding to X2.

In addition, the terms "identical" and "complementary" as used herein to characterize the nucleotide sequences that make up the primers used in the present invention encompass examples of incomplete identity and incomplete complementarity. More specifically, a sequence identical to a certain sequence also includes a sequence complementary to a nucleotide sequence that can anneal to the certain sequence. On the other hand, the complementary sequence refers to a sequence that anneals under stringent conditions and provides an origin of complementary strand synthesis. In the present invention, the term "identical" means that the homology of the nucleotide sequences is, for example, 90% or more, usually 95% or more, and more preferably 98% or more. The term "complementary" refers to the same nucleotide sequence as a complementary sequence. That is, a nucleotide sequence may be said to be "complementary" when it has a homology of, for example, 90% or more, usually 95% or more, more preferably 98% or more with a complementary sequence. In addition, when a complementary nucleotide sequence is used as the origin of complementary strand synthesis, at least one nucleotide at the 3' -end must be completely identical to the complementary sequence.

The X2 and X1c regions constituting the inner primer used in the present invention are generally arranged consecutively without overlapping each other. Alternatively, if X2 and X1c share a common nucleotide sequence, they may be arranged in a partially overlapping manner. X2 should be placed without exception at the 3' -end to serve as a primer. On the other hand, X1c was placed at the 5 '-end as described below, thereby providing a primer function for the 3' -end of the complementary strand synthesized using X1c as a template. The complementary strand obtained using the oligonucleotide as the synthesis origin is used as a template for the next reverse complementary strand synthesis, and finally, the inner primer portion of the present invention is also copied as a template for the complementary strand. The copied 3' -end contains the nucleotide sequence X1 and anneals to X1c located on the same strand to form a loop.

The inner primers used in the present invention are oligonucleotides that meet two requirements: (1) capable of forming a base pair complementary to a target nucleotide sequence, and (2) capable of providing an-OH group at the 3' -end of the base pair to serve as the origin of complementary strand synthesis. The backbone of the primer is not limited to those composed of phosphodiester bonds. For example, the primer may contain a phosphorothioate. In addition, the nucleotide may be any nucleotide as long as it can form a complementary base pair. In general, there are five types of natural nucleotides, namely a, C, T, G and U; however, analogs such as bromodeoxyuridine, for example, are also included. The oligonucleotide used in the present invention can be used not only as a synthesis origin but also preferably as a template for complementary strand synthesis.

The inner primer used in the present invention is composed of nucleotides having an appropriate length so as to be able to base pair with a complementary strand by maintaining a desired specificity under given conditions in various types of nucleic acid synthesis reactions described below. Specifically, the primer contains 5 to 200 nucleotides, more preferably 10 to 50 nucleotides. The minimum length of a primer recognized by a polymerase known to catalyze sequence-dependent nucleic acid synthesis is about 5 nucleotides. Therefore, the length of the annealing portion must be at least 5 nucleotides or longer. In addition, in order to ensure a high possibility of nucleotide-sequence specificity, it is preferable to use a primer containing 10 or more nucleotides. On the other hand, it is difficult to chemically synthesize an excessively long nucleotide sequence. Therefore, the exemplified length of the primer is a preferable range. The exemplified primer lengths correspond only to the portions annealing to the complementary strands. The inner primer of the present invention contains at least two regions, namely, X2 and X1 c. Thus, the length of the primer exemplified above is to be understood as the length corresponding to each region constituting the inner primer.

In addition, the inner primer used in the present invention can be labeled with a known labeling substance. The label includes ligands having binding ability, such as digoxin and biotin; an enzyme; a fluorescent substance; luminescent substances and radioactive isotopes. In addition, techniques for converting nucleotides in the inner primers to fluorescent analogs are known (WO 95/05391; Proc. Natl. Acad. Sci. USA, 91, 6644-.

In addition, the inner primer used in the present invention may be immobilized on a solid phase. Alternatively, any portion of the inner primer is labeled with a ligand having binding capacity, such as biotin, and then immobilized indirectly via a binding partner, such as immobilized avidin. When the immobilized inner primer is used as a synthesis origin, the synthesized nucleic acid product is immobilized on a solid phase, so that the product can be easily separated. The isolated product may be detected by a nucleic acid-specific indicator or by further hybridization to a labeled probe. Alternatively, the desired nucleic acid fragment is recovered by digesting the nucleic acid with any restriction enzyme.

The term "template" as used herein refers to a nucleic acid that can serve as a template for complementary strand synthesis. Although the complementary strand having a nucleotide sequence complementary to the template is the strand corresponding to the template, the relationship between the two is merely relative. Specifically, a strand synthesized as a complementary strand can be used as a template. In other words, the complementary strand may also serve as a template.

The inner primer of the present invention may contain regions other than the above two regions. Specifically, X2 and X1c were placed at the 3 '-end and 5' -end, respectively, and an arbitrary sequence was placed between these two regions. The arbitrary sequence includes, for example, a restriction enzyme recognition sequence, a promoter recognized by RNA polymerase, DNA encoding ribozyme, and the like. The synthetic product of the present invention, that is, a single-stranded nucleic acid formed by connecting complementary nucleotide sequences, can be digested into two double-stranded nucleic acids having the same length by inserting a restriction enzyme recognition sequence. By inserting a promoter sequence recognized by RNA polymerase, the synthesized product of the present invention can be transcribed into RNA using the product as a template. Alternatively, by reinserting DNA encoding ribozymes, a self-cleaving system of transcripts can be established. All of the additional nucleotide sequences described above only function when the sequence is formed in a double-stranded nucleic acid. Therefore, these sequences do not play a role in the single-stranded nucleic acid of the present invention in the form of a loop. The additional sequence is only effective for the first time after the nucleic acid extension has continued and the sequence anneals to the complementary nucleotide sequence without forming any loops.

When the inner primer used in the present invention is linked to the promoter in the direction capable of transcribing the synthesized region, the reaction product of the present invention repeatedly having the same nucleotide sequence realizes a high-efficiency transcription system. Translation into protein is also possible by using a transcription system in combination with an appropriate expression system. I.e.it can be applied in bacterial or animal cells, or transcribed and translated into protein in vitro.

The plurality of primers used in the present invention can be chemically synthesized. Alternatively, the natural nucleic acid is digested with a restriction enzyme or the like to be modified or ligated, thereby containing the above-mentioned nucleotide sequence.

The basic principle of carrying out a double-stranded nucleic acid amplification reaction by using the above-mentioned inner primers FA and RA in combination with a DNA polymerase having a strand displacement activity will be described below with reference to FIGS. 1 to 4. According to the examples, the amplification primer set is composed of the inner primers FA and RA, and in addition, RA is used as an arbitrary primer of the present invention.

The above-mentioned arbitrary primer (RA in FIG. 1- (1)) first anneals to X2c (corresponding to R2c) on the template double-stranded nucleic acid to serve as the origin of complementary strand synthesis. Under such conditions, the double-stranded nucleic acid is unstable, and the primer directly serves as the origin of complementary strand synthesis on the double-stranded nucleic acid. In FIG. 1- (2), the complementary strand synthesized using RA as the origin displaces one strand of the template double-stranded nucleic acid, and the F2c region annealed by the primer FA used for the amplification reaction is ready for base pairing (FIG. 1- (2)).

Complementary strand synthesis is performed by annealing FA to the F2c region ready for base pairing. Herein, the outer primer F3, which initiates complementary strand synthesis from the 5 '-side of FA (3' -side of the template), also anneals to this region (FIG. 2- (4)). The outer primers are designed to initiate complementary strand synthesis in the 5 '-side of each inner primer (3' -side of the template). The outer primers also have a higher Tm and are used at a lower concentration than the inner primers. Therefore, the outer primer always initiates complementary strand synthesis with a lower probability than the inner primer. The result of complementary strand synthesis using the outer primer F3 as the origin is: the extension product synthesized using the inner primer FA as the origin is displaced and released in a single-stranded form (FIG. 2- (5)). Using the single strand as a template, RA and the outer primer R3 corresponding to RA anneal to each other and initiate complementary strand synthesis (FIG. 3- (6)). As a result, the extension product of RA has a structure enabling intramolecular annealing of the 3' -end F1 with itself (FIG. 3- (8)). In FIG. 3- (6), the 5' -end of the strand anneals intramolecularly with itself. However, since the 5' -end cannot serve as the origin of complementary strand synthesis, the amplification reaction cannot be initiated with this structure. The amplification reaction can be initiated only when the complementary strand of the strand shown in FIG. 3- (6) is synthesized and, subsequently, a structure that anneals to itself at the 3' -end thereof is provided (FIG. 3- (8)). These reaction steps may be referred to as initial steps of the amplification reaction of the present invention.

The nucleic acid amplification of the present invention will be specifically described below with reference to the illustration shown in the figure. The self-annealed 3' -end F1 (FIG. 3- (8)) was used as the origin of complementary strand synthesis. Annealing to the 3' -end occurs between F1 and F1c, and therefore, there is a possibility that it competes for annealing with FA also containing F1 c. However, in practical applications, the complementary nucleotide sequences F1/F1c present in adjacent regions on the same strand preferentially anneal to each other. Therefore, the complementary strand synthesis reaction using its own strand as a template is preferentially initiated. By this reaction, nucleic acid in which the target nucleotide sequence has been connected on a single strand is synthesized. In addition, F2c that could anneal to the inner primer FA was present in the loop forming region formed by self-annealing of the 3' -terminal F1. After FA anneals to this portion, complementary strand synthesis is initiated (FIG. 3- (8)). Complementary strand synthesis initiated from FA annealed to the loop displaces the complementary strand synthesis product initiated from the 3 '-end using itself as a template, and the 3' -end is again ready for self-annealing (FIG. 4- (9)). The subsequent reaction includes the alternative steps of complementary strand synthesis using its own 3' -end as the origin and its own strand as the template, and complementary strand synthesis using the loop portion as the origin and the inner primer FA or RA as the origin. As described above, the nucleic acid amplification reaction includes the alternative steps of repeated 3' -terminal extension using its own strand as a template, and newly initiated extension from a primer that anneals to a loop portion.

On the other hand, with respect to a nucleic acid strand complementary to a single-stranded nucleic acid synthesized by extension using its own strand as a template and an oligonucleotide annealed to a loop portion thereof as an origin, a nucleic acid having a plurality of complementary nucleotide sequences linked on a single strand is also synthesized on such a synthesized nucleic acid strand. Specifically, for example, complementary strand synthesis from the loop portion is completed when it reaches R1 shown in FIG. 4- (9). Then, another complementary strand synthesis is newly initiated using the 3' -end displaced by the nucleic acid synthesis as the origin (FIG. 4- (9)). The reaction gradually reached the loop portion which was the starting point of synthesis in the previous step to initiate the displacement again. Thus, the nucleic acid which initiates synthesis from the loop portion is also displaced, resulting in the 3' -end R1 which anneals on the self strand (FIG. 4- (11)). The 3' -end R1 initiates complementary strand synthesis after annealing to R1c present on the same strand. When F in this reaction is considered to be R and R is considered to be F, the reaction is the same as that shown in FIG. 3- (8). Therefore, the structure shown in FIG. 4- (11) serves as a novel nucleic acid that proceeds with the self-extension process and the synthesis of another nucleic acid.

As described above, according to the method of the present invention, the reaction for continuously providing a nucleic acid capable of initiating another extension is performed simultaneously with the elongation of the nucleic acid. As the nucleic acid is further extended, multiple loop forming sequences can be generated at both the end of the strand and within the same strand. When these loop forming sequences are ready for base pairing by a synthesis reaction involving strand displacement, the inner primer anneals to the loop forming sequences and serves as a synthesis origin for generating new nucleic acids. By combining synthesis from the inner region of the strand with strand synthesis from the strand end, more efficient amplification can be obtained. As described above, by using the LAMP method, strand extension accompanying synthesis of a novel nucleic acid can be performed. In addition, according to the LAMP method, a newly produced nucleic acid is itself extended, resulting in newly producing another nucleic acid. Theoretically, this series of reactions continues continuously, and thus extremely efficient nucleic acid amplification can be obtained. Alternatively, the process of the invention may be carried out under isothermal reaction conditions.

Herein, the accumulated reaction product has a structure in which the nucleotide sequence between F1 and R1 and its complementary sequence are linked to each other in plural numbers. Regions containing the nucleotide sequence of F2-F1(F2c-F1c) or R2-R1(R2c-R1c) continue at both ends of the sequence of repeating units. For example, FIG. 4- (10) shows a state in which nucleotide sequences are linked in the order of (R2-F2c) - (F1-R2c) - (R1-F1c) - (F2-R2c) from the 5' -side. This is because the amplification reaction of the present invention proceeds based on the principle that amplification is initiated from F2 (or R2) using the inner primer as the synthesis origin, and then the product is extended from F1 (or R1) by a complementary strand synthesis reaction using the 3' -end itself as the synthesis origin.

In the most preferred embodiment herein, the inner primers FA and RA are used as oligonucleotides annealing to the loop portion. However, the nucleic acid amplification of the present invention can be carried out only when an oligonucleotide having these restricted structures and an oligonucleotide capable of initiating complementary strand synthesis from the loop portion are used simultaneously. That is, if the 3' -end is replaced (which continues to extend) only by complementary strand synthesis from the loop start, another loop portion is provided again. Since the complementary strand synthesis starting from the loop portion uses a template nucleic acid in which a plurality of complementary nucleotide sequences are linked on a single strand, it is apparent that the nucleic acid required for the present invention can be synthesized. However, after the substitution, the synthesized nucleic acid forms a loop to perform complementary strand synthesis, but does not have a 3' -end that can be used subsequently to form a loop, and thus cannot be used as a new template. Thus, in contrast to the case of using a nucleic acid synthesized starting from FA or RA, exponential amplification cannot be obtained. For this reason, inner primers having structures similar to FA and RA can be used in the present invention for efficient nucleic acid synthesis.

A series of reactions can be easily performed by adding the following components to a double-stranded nucleic acid used as a template and incubating them under conditions that ensure annealing of the inner primers and the outer primers and complementary strand synthesis reaction using these primers as origins. The incubation conditions were as described above. In the present invention, an amplification reaction of a template nucleic acid can be obtained by incubating the following components at a temperature lower than that required for denaturing a double-stranded nucleic acid used as a template. At this time, a step of denaturing the template nucleic acid is not necessary. Herein, the temperature required for denaturing a double-stranded nucleic acid refers to a temperature at which a template nucleic acid is converted into a single strand after rapid cooling.

Four types of oligonucleotides:

FA，

RA，

outer primer F3, and

an outer primer R3;

a DNA polymerase catalyzing a strand displacement complementary strand synthesis reaction;

nucleotides used as substrates for DNA polymerases

As described in the explanation of the reaction principle, when the above-mentioned FA and RA are used as inner primers, the nucleic acid synthesis method of the present invention using a double-stranded nucleic acid as a template is necessary for a reaction using a nucleic acid derived from a sample as a template. When an inner primer having a specific structure similar to FA and RA is applied to the nucleic acid synthesis method of the present invention, the 3' -end of the resulting product anneals to itself and serves as the origin of complementary strand synthesis using itself as a template. Further, the new primer anneals to the loop portion formed by self annealing of the 3' -end of the above-mentioned product and serving as the synthesis origin, and the strand displacement complementary strand synthesis reaction proceeds. These reactions are performed independently of the nucleic acid synthesis method of the present invention using a double-stranded nucleic acid as a template.

That is, when FA and RA are used as the inner primers, the nucleic acid synthesis reaction of the present invention using a double-stranded nucleic acid as a template constitutes the initial reaction. Therefore, the initial reaction requires conditions for ensuring annealing of the inner primer and the outer primer and for carrying out the complementary strand synthesis reaction using these primers as the origin. The subsequent reaction may be carried out under more appropriate conditions. However, when a temperature change is required for this purpose, the advantages of the present invention, which do not require a denaturation step, cannot be fully utilized. Therefore, in the present invention, it is preferable to carry out the initial reaction as well as all subsequent reactions under preferable conditions.

When FA and RA are used as the inner primers of the present invention, it is important to note that a series of reaction steps can be performed only when the relative positions of the regions are maintained. Therefore, non-specific synthesis accompanying non-specific complementary strand synthesis can be effectively prevented. In particular, this helps to reduce the likelihood of the product being used as a starting material for subsequent amplification steps, even when some non-specific reactions occur. In addition, the fact that the reaction process is controlled by a number of different regions provides flexibility in constructing detection systems that ensure accurate discrimination of similar nucleotide sequences.

This aspect of the invention can be used to detect gene mutations. In the embodiment of the present invention using the outer primers, a total of four primers-two outer primers and two inner primers-are used. If the nucleotide sequences constituting the six regions contained in the four oligonucleotides and the target nucleotide sequence are not prepared as designed, any of the complementary strand synthesis reactions of the present invention will be inhibited. The nucleotide sequence near the 3 '-end of each oligonucleotide serving as the origin of complementary strand synthesis, and the nucleotide sequence near the 5' -end of the X1c region whose complementary nucleotide sequence serves as the origin of synthesis are particularly important for complementary strand synthesis. Therefore, if a nucleotide sequence essential for complementary strand synthesis is designed to correspond to a mutation to be detected, the presence or absence of a mutation, such as a deletion or insertion of a nucleotide or a genetic polymorphism, such as SNP, can be detected by observing the product of the synthesis reaction of the present invention.

More specifically, when a nucleotide having a mutation or polymorphism located near the 3 '-end of the oligonucleotide serving as the origin of complementary strand synthesis, or near the newly synthesized complementary strand is expected to become the synthesis origin, the nucleotide sequence should be designed to be identical to the sequence near the 5' -end of the strand serving as the template of complementary strand synthesis. When a mismatch is present at or near the 3' -end serving as the origin of complementary strand synthesis, the reaction of complementary strand synthesis of nucleic acid is significantly inhibited.

According to the LAMP method, when the reaction from the end structure of the product generated at the early stage of the reaction cannot be repeated many times, sufficient amplification cannot be performed. Therefore, since the complementary strand synthesis required for amplification is inhibited at some stage, sufficient amplification cannot be obtained with mismatches even if erroneous synthesis occurs. As a result, the mismatch effectively inhibits amplification, giving the correct result. That is, nucleic acid amplification by LAMP has a highly accurate nucleotide sequence check-mechanism. These features confer advantages over methods such as PCR, which can only amplify sequences between two regions.

In addition, when a complementary sequence is synthesized and the complementary sequence anneals to newly synthesized X1 within the same sequence to perform a synthesis reaction using the strand itself as a template, the characteristic region X1c of the oligonucleotide used in the present invention serves only as an origin. Therefore, the oligonucleotide of the present invention does not form a loop, which is often a serious problem in the prior art, and even if so-called primer-dimer is generated, the problem cannot be avoided. Therefore, non-specific amplification by primer-dimer does not occur in the present invention, which contributes to improvement of reaction specificity.

Nucleic acid amplification was efficiently performed by setting the Tm of the outer primer relative to the inner primer FA to (outer primer F3: F3 c). ltoreq.F 2 c/F2. It is necessary to design the respective regions constituting FA so that the annealing between F1c and F1 is more dominant than the annealing between F2c and F2. Tm, the constituent bases, etc. should be considered in designing. In addition, since annealing between F1c and F1 is an intermolecular reaction, it should also be noted that the annealing is likely to predominate. Needless to say, similar conditions should be considered when designing RA that anneals to the elongation product of FA. By ensuring this relationship, it is highly possible to obtain ideal reaction conditions.

In the present invention, the terms "synthesis" and "amplification" of nucleic acids are used. Nucleic acid synthesis herein refers to the extension of a nucleic acid from an oligonucleotide that serves as the point of initiation of synthesis. A series of reactions including consecutive reactions of forming other nucleic acids and extending the formed nucleic acids in addition to synthesis is collectively referred to as "amplification".

By using FA and RA as inner primers, an F1 region can be provided at the 3' -end, and F1 can partially anneal to F1c on the same strand. Annealing of the F1 region to the F1c region on the same strand produces a single-stranded nucleic acid that can form a loop containing the F2c region that can subsequently undergo base pairing. Single-stranded nucleic acids can be used as important starting materials in subsequent nucleic acid amplification reactions. Single-stranded nucleic acids can also be provided according to the following principles. Namely, complementary strand synthesis was carried out using a primer having the following structure as an inner primer:

5 '- [ X1 region annealing to X1c region located in the primer ] - [ Loop forming sequence under conditions ensuring base pairing ] - [ X1c region ] - [ region having a sequence complementary to the template ] -3'

Two nucleotide sequences were prepared for the region having a sequence complementary to the template, one complementary to F1 (primer FA) and one complementary to R1c (primer RA). In addition, the nucleotide sequence of the nucleic acid to be synthesized includes a nucleotide sequence from the region of F1 to the region of R1c, and a nucleotide sequence from the region of R1 to the region of F1c having a nucleotide sequence complementary to the nucleotide sequence. On the other hand, X1c and X1 that can anneal intermolecularly in the primer may be any sequences. However, the sequence of the X1c/X1 region needs to be different from that of the primers FA and RA.

First, the F2 region of the double-stranded nucleic acid to be a template is placed under conditions that ensure base pairing using an arbitrary primer. The primer FA then anneals to F2 which can undergo base pairing, and complementary strand synthesis proceeds. In this case, RA can be used as an arbitrary primer. Then, the R2c region of the synthesized complementary strand is placed under conditions that ensure base pairing, and another primer is annealed to the region to prepare a complementary strand synthesis origin. Since the 3 ' -end of the synthesized complementary strand has a nucleotide sequence complementary to the primer FA, which constitutes the 5 ' -end portion of the strand synthesized for the first time, the 3 ' -end thereof has the X1 region, and when subsequently annealed to the X1c region on the same strand, a loop can be formed. Thus, the characteristic 3' -terminal configuration of the invention as described above is provided, and the subsequent reaction is the reaction system that has been shown previously in the most desirable embodiment. It is noted that the oligonucleotide to which the loop portion is annealed has an X2 region complementary to the X2c region, an X2 region being located in the loop at the 3 '-terminus, and an X1 region being located at the 5' -terminus. In the previous reaction system, a loop configuration is provided to the 3' -end of the nucleic acid by synthesizing a strand complementary to the template nucleic acid using the primers FA and RA. This method effectively provides the terminal configuration characteristic of the present invention using a short primer. On the other hand, in the embodiment of the present invention, the entire nucleotide sequence of the loop is provided as a primer from the beginning, and it is necessary to synthesize a longer primer.

In addition, the principle of the present invention can also be applied to known nucleic acid amplification methods such as SDA and NASBA. In order to apply the principle of SDA to the present invention, a primer set for SDA, a DNA polymerase catalyzing a strand displacement complementary strand synthesis reaction, a restriction enzyme, and a substrate necessary for complementary strand synthesis (including a thionucleotide imparting ribozyme resistance) are incubated with a double-stranded nucleic acid as a template under the above-described conditions of the present invention. When either SDA primer initiates complementary strand synthesis by destabilizing the double strand, the region of the template nucleic acid that should be replaced to which the other primer anneals is in a condition that ensures base pairing. Then, primer annealing and synthesis of a complementary strand of the template nucleic acid are performed.

Next, annealing of the outer primer to the primer and synthesis of the complementary strand are performed, and the complementary strand previously synthesized from the SDA primer is substituted to generate a single-stranded nucleic acid. In addition, complementary strand synthesis using the above arbitrary primer is performed in the 5' -direction of the template nucleic acid. Therefore, the region to which the SDA primer should anneal and the region to which the outer primer should anneal are both under conditions that ensure base pairing. The complementary strand synthesized from the other primer using the single-stranded nucleic acid as a template is resistant to the ribozyme. Thus, the restriction enzyme acts only at the restriction enzyme recognition site in the primer, thereby creating a nick. Using this nick as a synthesis origin, complementary strand synthesis and substitution are repeated for amplification. At the same time, the SDA primers also anneal to the single-stranded nucleic acid generated by the substitution, and complementary strand synthesis proceeds.

The nucleic acid used as the template at this time is resistant to the ribozyme, but since the SDA primer is not resistant to the ribozyme, a nick by the restriction enzyme is generated in the primer. As a result, a nucleic acid amplification reaction was also obtained in the substituted single-stranded nucleic acid. Thus, by continuing the incubation under such conditions, a double-stranded DNA containing the nucleotide sequence of the region defined by the SDA primers can be successively synthesized, and as a result, nucleic acid amplification is obtained. Although the principle of SDA method is known, the present invention provides a novel finding that the denaturation step of double-stranded nucleic acids can be eliminated by initiating the reaction using a nucleic acid synthesis reaction based on the destabilization of double-strands.

In order to carry out the NASBA method according to the present invention, primers for the NASBA method are used in combination with a DNA polymerase and an RNA polymerase which catalyze the strand displacement complementary strand synthesis reaction. The primer for the NASBA method is composed of a first primer to which a promoter sequence is added, and a second primer annealing to a complementary strand synthesized using the first primer as an origin.

First, a complementary strand is synthesized for a double-stranded template nucleic acid using an arbitrary primer, and a region that should anneal to the first primer for NASBA is placed under conditions that ensure base pairing. Subsequently, the complementary strand synthesized using the NASBA first primer as the origin is substituted with an external primer so as to be single-stranded. When the NASBA second primer anneals to the resulting single strand to make it double-stranded, the promoter region added to the NASBA first primer becomes double-stranded. Transcription reaction using RNA polymerase is initiated by a promoter region that has become double-stranded, and RNA synthesis is performed using the target nucleotide sequence as a template.

The various reagents required for the nucleic acid synthesis method or amplification method of the present invention may be provided in the form of a prepackaged kit. Specifically, the kit of the present invention comprises oligonucleotides necessary for the inner and outer primers, dNTPs (which are substrates for complementary strand synthesis), a DNA polymerase for performing a strand displacement complementary strand synthesis reaction, a buffer solution for providing conditions preferable for the enzymatic reaction, and, if necessary, reagents necessary for detecting the synthesis reaction product, and the like. In particular, in a preferred embodiment of the present invention, since no reagent is required to be added during the reaction, it is possible to provide reagents required for one reaction in such a manner as to be put into a reaction tube, so that the reaction can be initiated by adding only a sample. If a system is established such that detection of the reaction product can be carried out in the reaction tube using a luminescent signal or a fluorescent signal, it is not necessary to open and close the tube after the reaction. This is necessary from the viewpoint of preventing contamination.

All publications describing the prior art mentioned herein are incorporated herein by reference.

Brief Description of Drawings

FIG. 1 illustrates the (1) th and (2) th parts of the reaction principle of a preferred embodiment of the present invention.

FIG. 2 illustrates the reaction principles of the preferred embodiment of the present invention in sections (3) to (5).

FIG. 3 illustrates the reaction principles of the preferred embodiment of the present invention in sections (6) to (8).

FIG. 4 illustrates the reaction principles of the preferred embodiment of the present invention in sections (9) to (11).

FIG. 5 is a photograph of an electrophoresis gel after an amplification reaction using HB V, HCV, and PSA gene sequences.

FIG. 6 is a photograph of an electrophoresis gel after an amplification reaction in the presence or absence of betaine.

FIG. 7 is a photograph of an electrophoresis gel after an amplification reaction in the presence of proline and DMSO.

FIG. 8 is a photograph showing the observation results of the influence of the external primer on the nucleic acid amplification method of the present invention. The vertical axis and the horizontal axis represent fluorescence intensity and reaction time, respectively.

Best Mode for Carrying Out The Invention

The present invention will be explained in further detail below with reference to examples.

Example 1 amplification of HBV, HCV and PSA Gene sequences

The nucleic acid synthesis method of the present invention is carried out using, as a template, DNA (double strand) prepared by incorporating each of HBV, HCV and PSA gene partial sequences into a plasmid. Four primers-inner F, inner R, outer F and outer R-were used in the experiments. The outer primers F and R are outer primers for replacing the first nucleic acid obtained using the inner primers F and R as synthesis origins, respectively.

The concentration of the inner primer F (or the inner primer R) is set high so that annealing of the primer can be dominant. The template sequences in this example, derived from HBV, HCV and PSA incorporated into the plasmids, are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. in addition, the sequences of the primers (inner primer F, inner primer R, outer primer F and outer primer R) used for amplifying the respective templates are shown below.

·HBV：

Inner primer F (SEQ ID NO: 4):

5’-GATAAAACGCCGCAGACACATCCTTCCAACCTCTTGTCCTCCAA-3’

inner primer R (SEQ ID NO: 5):

5’-CCTGCTGCTATGCCTCATCTTCTTTGACAAACGGGCAACATACCTT-3’

outer primer F (SEQ ID NO: 6):

5’-CAAAATTCGCAGTCCCCAAC-3’

outer primer R (SEQ ID NO: 7):

5’-GGTGGTTGATGTTCCTGGA-3’

·HCV：

inner primer F (SEQ ID NO: 8):

5’-GAGTGGGTTTATCCAAGAAAGGACTTTAGCCATAGTGGTCTGCGGA-3’

inner primer R (SEQ ID NO: 9):

5’-CTAGCCGAGTAGCGTTGGGTTGCTTTGCACTCGCAAGCACCCTATC-3’

outer primer F (SEQ ID NO: 10):

5’-GCAGAAAGCGTCTAGCCATGG-3’

outer primer R (SEQ ID NO: 11):

5’-CTAGCCGAGTAGCGTTGGGTTGC-3’

·PSA：

inner primer F (SEQ ID NO: 12):

5’-TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG-3’

inner primer R (SEQ ID NO: 13):

5’-TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG-3’

outer primer F (SEQ ID NO: 14):

5’-TGCTTGTGGCCTCTCGTG-3’

outer primer R (SEQ ID NO: 15):

5’-GGGTGTGTGAAGCTGTG-3’

in addition, the characteristics of the primer configuration are briefly described below.

An inner primer F:

5 '-oriented region/3' -oriented region of primer

The inner primer F is the same as the F1c region of the complementary strand synthesized using the inner primer F/complementary to the F2c region of the template DNA

Inner primer R is identical to the R1c region of the complementary strand synthesized using inner primer R/complementary to the R2c region of the complementary strand synthesized using inner primer F

The outer primer F is complementary to F3c adjacent to the 3' -side of the F2c region of the template DNA

The outer primer R is complementary to R3c adjacent to the 3' -side of the R2c region of the complementary strand synthesized using the inner primer F

These primers synthesized nucleic acids in which the region from F1c to R1c incorporating the partial sequence of each gene and the nucleotide sequence complementary thereto were repeatedly linked to a single strand flanked by the loop-forming sequence containing F2 c. The components of the reaction solution for the nucleic acid synthesis method using these primers of the present invention are shown below.

Composition of the reaction solution (25. mu.l)

20mM Tris-HCl，pH8.8

10mM KCl

10mM(NH₄)₂SO₄

4mM MgSO₄

1M betaine

0.1％Triton X-100

0.4mM dNTP

8U Bst DNA polymerase (NEW ENGLAND BioLabs)

Primer:

1600nM inner primer F

1600nM inner primer R

400nM outer primer F

400nM outer primer R

Template:

1×10^-20mol HBV DNA

1×10^-17mol HCV DNA

1×10^-22mol PSA DNA

a template which is not heat-denatured is prepared. The reaction solution was reacted at 65 ℃ for 1 hour.

The reaction was confirmed: mu.L of the loading buffer was added to 5. mu.L of the above reaction solution, and then, loaded on 2% agarose gel (0.5% TBE). Electrophoresis was performed at 100V for 0.5 hour. Phi X174Hae III was used as molecular size marker. After electrophoresis, the gel was stained with ethidium bromide (herein abbreviated as EtBr) to visualize the nucleic acids. The results are shown in FIG. 6. Each lane corresponds to the following sample.

Lane 1: phi X174Hae III

Lane 2: DNA (+), PSA

Lane 3: DNA (-), PSA

Lane 4: DNA (+), HBV

Lane 5: DNA (-), HBV

Lane 6: DNA (+), HCV

Lane 7: DNA (-), HCV

The experimental results are: even when any DNA such as HBV, HCV and PSA is used as a template, a ladder characteristic to the amplification product of the inner primers of the present invention can be observed. It was further confirmed that nucleic acid can be synthesized under isothermal conditions using double-stranded nucleic acid as a template, and an inner primer and an outer primer at the same time.

EXAMPLE 2 amplification in the Presence of betaine

DNA (1X 10) in which a partial sequence of HCV gene was incorporated into a plasmid was used^-17mol) as a template, the nucleic acid synthesis method of the present invention is performed. Four primers-inner F, inner R, outer F and outer R-were used in the experiments. At this time, a reaction solution containing no betaine was also prepared.

A template which is not heat-denatured is prepared. The reaction solution was allowed to react at 65 ℃ for 1 hour and 2 hours.

The reaction was confirmed: mu.L of the loading buffer was added to 5. mu.L of the above reaction solution, and then, loaded on 2% agarose gel (0.5% TBE). Electrophoresis was performed at 100V for 0.5 hour. Phi X174Hae III was used as molecular size marker. After electrophoresis, the gel was stained with EtBr to visualize the nucleic acids. The results are shown in FIG. 7. Each lane corresponds to the following sample.

Lane 1: phi X174Hae III

Lane 2: DNA (-), betaine (-), 1 hour

Lane 3: DNA (+), betaine (-), 1 h

Lane 4: DNA (-), betaine (+), 1 h

Lane 5: DNA (+), betaine (+), 1 h

Lane 6: phi X174Hae III

Lane 7: DNA (-), betaine (-), 2 hours

Lane 8: DNA (+), betaine (-), 2 h

The experimental results are: when the reaction time was 1 hour, amplification was only observed in the presence of betaine. On the other hand, when the reaction time was extended to 2 hours, amplification was observed even in the absence of betaine. It was confirmed that amplification was also observed in the usual reaction system.

EXAMPLE 3 amplification in the Presence of proline or DMSO

DNA (1X 10) in which a partial sequence of HCV gene was incorporated into a plasmid was used^-17mol) as a template, the nucleic acid synthesis method of the present invention is performed. Four primers-inner F, inner R, outer F and outer R-were used in the experiments. At this time, a reaction solution containing no betaine was also prepared.

Proline or DMSO was added to the reaction solution instead of betaine so that the final concentration of proline or DMSO was 1% or 5%. The other components in the reaction solution are the same as described above.

A template which is not heat-denatured is prepared. The reaction solution was allowed to react at 65 ℃ for 2 hours.

The reaction was confirmed: mu.L of the loading buffer was added to 5. mu.L of the above reaction solution, and then, loaded on 2% agarose gel (0.5% TBE). Electrophoresis was performed at 100V for 0.5 hour. Phi X174Hae III was used as molecular size marker. After electrophoresis, the gel was stained with EtBr to visualize the nucleic acids. The results are shown in FIG. 8. Each lane corresponds to the following sample.

Lane 1: phi X174Hae III

Lane 2: proline (+)

Lane 3: DMSO (+)

Lane 4: betaine (-)

The results of the amplification reaction using proline or DMSO which have a similar effect (melting temperature lowering effect) to betaine can be further confirmed: even when proline or DMSO is used, the amplification reaction can be carried out.

[ example 4] Effect of external primers

Lambda DNA (SEQ ID NO: 16, 1X 10) was used⁵Individual molecules) of the DNA, which is a linear strand and is used as a target nucleotide sequence, is subjected to the nucleic acid synthesis method of the present invention. Four primers-inner F, inner R, outer F and outer R-were used in the experiments. Reactions without the use of the outer primers F and R were performed simultaneously. Ethidium bromide (EtBr) was added to all reaction systems to a final concentration of 0.25. mu.g/ml.

A target that is not heat-denatured is prepared. The reaction solution was reacted at 65 ℃ for 1.5 hours, and a change in fluorescence intensity was observed over time using ABI 7700(Perkin Elmer). EtBr is a fluorescent stain specific for double-stranded nucleic acids. Therefore, the fluorescence intensity increases as the amount of double-stranded nucleic acid generated by nucleic acid amplification increases.

The measurement results are shown in FIG. 9. The amplification rate was found to be slower in reaction systems without external primers.

Nucleotide sequence of lambda DNA primer

Inner primer F (SEQ ID NO: 17):

CAGCCAGCCGCAGCACGTTCGCTCATAGGAGATATGGTAGAGCCGC

inner primer R (SEQ ID NO: 18):

GAGAGAATTTGTACCACCTCCCACCGGGCACATAGCAGTCCTAGGGACAGT

outer primer F (SEQ ID NO: 19):

GGCTTGGCTCTGCTAACACGTT

outer primer R (SEQ ID NO: 20):

GGACGTTTGTAATGTCCGCTCC

industrial applicability

The present invention provides a nucleic acid synthesis method which does not require a change in temperature and thus does not deteriorate the specificity and efficiency of the reaction. Although the present invention uses a double-stranded nucleic acid as a template, it is not necessary to change the temperature for denaturation. Thus, nucleic acid synthesis is performed without using an instrument having a special temperature control mechanism. In addition, according to the present invention which does not require thermal cycling, it is expected to prevent non-specific reactions due to temperature changes.

The nucleic acid synthesis method of the present invention can be applied to any nucleic acid synthesis method based on the principle of using a primer as a synthesis origin. In particular, when used in combination with a nucleic acid synthesis reaction based on the principle that initially no temperature change is required, higher synthesis efficiency can be obtained. A nucleic acid amplification method such as that described in examples, which can provide an amplification product having a configuration in which the 3' -terminal region can be partially annealed to itself, can achieve excellent operability and specificity when used in combination with the present invention. A high level of amplification efficiency can be obtained only by incubating a double-stranded nucleic acid as a template with a primer and a DNA polymerase at a constant temperature. The present invention enables a nucleic acid amplification method that does not require a change in temperature, but at the same time maintains high specificity and high amplification efficiency.

Since the nucleic acid synthesis method based on the present invention does not require a change in temperature, the reaction can be easily monitored. I.e., the reaction can be monitored using an incubation mechanism that provides a constant temperature and an instrument with an optical reading mechanism. This mechanism is a general mechanism that conventional optical analysis instruments can provide. Thus, the nucleic acid amplification method based on the present invention can be monitored by a conventional analytical instrument.

As described above, the nucleic acid synthesis method of the present invention does not require complicated temperature control, which is a problem associated with known methods such as PCR method, and also significantly simplifies the experimental operation. In addition, the present invention can also perform a nucleic acid amplification method which can be used in general because a special temperature control instrument is not required. In addition, in the present invention, non-specific reactions due to a change in temperature can be prevented.

Sequence listing

<110> Rongyan Chemical Co., Ltd (Eiken Chemical Co., Ltd.)

<120> method for amplifying nucleic acid Using double-stranded nucleic acid as template

<130>E2-A0001P

<140>

<141>

<150>JP 2000-111939

<151>2000-04-07

<160>20

<170>PatentIn Ver.2.0

<210>1

<211>198

<212>DNA

<213> hepatitis B Virus

<400>1

caaaattcgc agtccccaac ctccaatcac tcaccaacct cttgtcctcc aatttgtcct 60

ggctatcgct ggatgtgtct gcggcgtttt atcatattcc tcttcatcct gctgctatgc 120

ctcatcttct tgttggttct tctggactac caaggtatgt tgcccgtttg tcctctactt 180

ccaggaacat caaccacc 198

<210>2

<211>279

<212>DNA

<213> hepatitis C Virus

<400>2

gcagaaagcg tctagccatg gcgttagtat gagtgtcgta cagcctccag gcccccccct 60

cccgggagag ccatagtggt ctgcggaacc ggtgagtaca ccggaattac cggaaagact 120

gggtcctttc ttggataaac ccactctatg tccggtcatt tgggcgtgcc cccgcaagac 180

tgctagccga gtagcgttgg gttgcgaaag gccttgtggt actgcctgat agggtgcttg 240

cgagtgcccc gggaggtctc gtagaccgtg catcatgag 279

<210>3

<211>178

<212>DNA

<213> human (Homo sapiens)

<400>3

tgcttgtggc ctctcgtggc agggcagtct gcggcggtgt tctggtgcac ccccagtggg 60

tcctcacagc tgcccactgc atcaggaaca aaagcgtgat cttgctgggt cggcacagcc 120

tgtttcatcc tgaagacaca ggccaggtat ttcaggtcag ccacagcttc acacaccc 178

<210>4

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>4

gataaaacgc cgcagacaca tccttccaac ctcttgtcct ccaa 44

<210>5

<211>46

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>5

cctgctgcta tgcctcatct tctttgacaa acgggcaaca tacctt 46

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>6

caaaattcgc agtccccaac 20

<210>7

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>7

ggtggttgat gttcctgga 19

<210>8

<211>46

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>8

gagtgggttt atccaagaaa ggactttagc catagtggtc tgcgga 46

<210>9

<211>46

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>9

ctagccgagt agcgttgggt tgctttgcac tcgcaagcac cctatc 46

<210>10

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>10

gcagaaagcg tctagccatg g 21

<210>11

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>11

ctagccgagt agcgttgggt tgc 23

<210>12

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>12

tgttcctgat gcagtgggca gctttagtct gcggcggtgt tctg 44

<210>13

<211>45

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>13

tgctgggtcg gcacagcctg aagctgacct gaaatacctg gcctg 45

<210>14

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>14

tgcttgtggc ctctcgtg 18

<210>15

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>15

gggtgtgtga agctgtg 17

<210>16

<211>245

<212>DNA

<213> lambda phage (Bacteriophage lambda)

<400>16

ggcttggctc tgctaacacg ttgctcatag gagatatggt agagccgcag acacgtcgta 60

tgcaggaacg tgctgcggct ggctggtgaa cttccgatag tgcgggtgtt gaatgatttc 120

cagttgctac cgattttaca tattttttgc atgagagaat ttgtaccacc tcccaccgac 180

catctatgac tgtacgccac tgtccctagg actgctatgt gccggagcgg acattacaaa 240

cgtcc 245

<210>17

<211>46

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>17

cagccagccg cagcacgttc gctcatagga gatatggtag agccgc 46

<210>18

<211>51

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>18

gagagaattt gtaccacctc ccaccgggca catagcagtc ctagggacag t 51

<210>19

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>19

ggcttggctc tgctaacacg tt 22

<210>20

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: artificially synthesized primer sequence

<400>20

ggacgtttgt aatgtccgct cc 22

Claims (9)

1. A method for synthesizing a nucleic acid in which a plurality of nucleotides constituting a specific region of a double-stranded nucleic acid template having a complementary nucleotide sequence are linked on a single strand, wherein the method comprises:

a) incubating a double-stranded nucleic acid template containing a specific region to be synthesized and a first primer and Bst DNA polymerase so that a region of a target template nucleic acid to be annealed to the second primer is single-stranded, as shown in FIG. 1 (2);

wherein the first primer anneals to a region defining the 3 '-side of one strand constituting the specific region, and the 5' -end of the first primer includes a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis reaction product obtained using the primer as a synthesis origin;

b) annealing a second primer to the single-stranded region obtained in step a) and performing complementary strand synthesis using the second primer as a synthesis origin, wherein the 3 ' -end of the second primer anneals to a region defining the 3 ' -side of one strand constituting the specific region and the 5 ' -end of the second primer contains a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis reaction product obtained using the primer as a synthesis origin, as shown in FIG. 2 (3);

c) converting a region of the extension product of the second primer synthesized in step b), which will anneal to the first primer, into a single strand, to be displaced by a complementary strand synthesis reaction using a fourth primer as an origin, which anneals to the 3' -side of the region of the template annealed by the second primer, as shown in FIG. 2 (5);

d) annealing the first primer to the single-stranded region obtained in step c) and performing complementary strand synthesis using the first primer as a synthesis origin, as shown in FIG. 3 (6); and

e) converting the extension product of the first primer into a single strand, displacing by a complementary strand synthesis reaction using a third primer as an origin, annealing the third primer to the 3 '-side of the region of the template annealed by the first primer and self-annealing the 3' -end of the extension product of the first primer synthesized in step d), and performing complementary strand synthesis using the extension product itself as the template to obtain a nucleic acid in which a plurality of nucleotides constituting the specific region are ligated on the single strand, as shown in fig. 3 (8).

2. A method for amplifying a nucleic acid in which a plurality of nucleotides constituting a specific region of a double-stranded nucleic acid template having a complementary nucleotide sequence are linked on a single strand, wherein synthesis of the complementary strand is performed by Bst DNA polymerase, and wherein the method comprises:

1) self-annealing the 3' -end of the first primer extension product produced by the method of claim 1, and performing complementary strand synthesis using the extension product as an origin, as shown in FIG. 3 (8);

2) annealing the second primer or the first primer to the loop region formed by self-annealing at the 3' -end, and performing complementary strand synthesis using the primer as the origin, as shown in FIG. 3 (8);

3) strand displacement is performed from the 3 '-end extension product by the complementary strand synthesis of step 2) so that the 3' -end is single-stranded, as shown in FIG. 4 (9);

4) performing complementary strand synthesis using the displaced strand itself, which is single-stranded obtained in step 3), as a template and the 3' -end thereof as an origin, as shown in FIG. 4(9), and displacing the complementary strand synthesized in step 2) using the loop region as an origin, thereby generating a single-stranded nucleic acid, as shown in FIG. 4 (11); and

5) repeating steps 2) to 4) to amplify the desired nucleic acid.

3. The method of claim 2, wherein the method further comprises:

6) complementary strand synthesis by self-annealing of the 3' -end of the single-stranded nucleic acid generated in step 4), as shown in FIG. 4 (9);

7) annealing the second primer or the first primer to the loop region formed by self-annealing at the 3' -end, and performing complementary strand synthesis using the primer as the origin, as shown in FIG. 4 (10);

8) performing strand displacement from the 3 '-end extension product by the complementary strand synthesis reaction of step 7) so that the 3' -end is single-stranded;

9) using the displaced strand itself, which is single-stranded, obtained in step 8) as a template and performing complementary strand synthesis using the 3' -end thereof as an origin, displacing the complementary strand synthesized using the loop region as an origin in step 7), thereby producing a single-stranded nucleic acid; and

10) repeating steps 7) to 9) to amplify the desired nucleic acid.

4. A method for detecting a target nucleotide sequence in a sample, the method comprising performing the amplification method of claim 3, and observing whether an amplification reaction product has been produced.

5. The method of claim 4, wherein the method of claim 3 is performed in the presence of a nucleic acid detection reagent, the method further comprising determining whether an amplification reaction product has been produced based on a change in signal from the detection reagent.

6. A method for detecting a mutation by the detection method as set forth in claim 4, wherein the mutation in the nucleotide sequence to be amplified prevents complementary strand synthesis at least one 3 '-end, which is the origin of complementary strand synthesis constituting the amplification method, wherein the 3' -end is selected from the group consisting of:

i) the 3' -end of the first primer is,

ii) the 3' -end of the second primer,

iii) the 3 '-end of the newly synthesized complementary strand at the 5' -end of the first primer, which serves as a template in the complementary strand synthesis, or

iv) the 3 '-end of the newly synthesized complementary strand at the 5' -end of the second primer, which serves as a template in the complementary strand synthesis.

7. A method for amplifying a nucleic acid in which a plurality of nucleotides constituting a specific region of a double-stranded nucleic acid template having a complementary nucleotide sequence are ligated on a single strand, wherein the method comprises the step of incubating the following components under conditions capable of synthesizing a complementary strand using a first primer as an origin:

a target comprising a double-stranded nucleic acid template, said template comprising a specific region to be amplified;

bst DNA polymerase;

a first primer of which 3 ' -end anneals to a region defining the 3 ' -side of one strand constituting the specific region and of which 5 ' -end contains a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis product obtained using the primer as a synthesis origin;

a second primer whose 3 ' -end anneals to a region defining the 3 ' -side of one strand constituting the specific region and whose 5 ' -end contains a nucleotide sequence complementary to an arbitrary region of a complementary strand synthesis product obtained using the primer as a synthesis origin;

nucleotide substrates

A third primer that becomes the origin of complementary strand synthesis using the 3' -side of the region of the template to be annealed by the first primer as the origin; and

a fourth primer that serves as an origin of complementary strand synthesis using the 3' -side of the region of the template to be annealed by the second primer as an origin.

8. The method according to claim 7, wherein the incubation is carried out in the presence of a melting temperature regulator.

9. The method according to claim 8, wherein the melting temperature regulator is at least one compound selected from the group consisting of: betaine, proline, dimethyl sulfoxide or trimethylamine-N-oxide.