JP2002345499A - Method for detecting nucleic acid sequence and kit usable for the method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a target nucleic acid sequence, and further to provide a kit therefor. SOLUTION: The method comprises mixing and incubating a template polynucleotide capable of forming a loop capable of forming a base pairing when a complemental base sequence is hybridized therewith, and capable of allowing the 3' terminus to form a loop by annealing with itself and to allow the 3' terminus annealed to itself to become the starting point of a complemental chain synthesis by using itself as the template, mixing and incubating one or more kinds of fixed primers capable of providing the starting point of the complemental chain synthesis at the position different from the primer in two or more kinds of primer-extended products capable of providing the starting points of the complemental chain synthesis at different positions in the loop of the template polynucleotide, a DNA polymerase and a substrate, and observing the agglutination reaction accompanied by the amplification reaction of the polynucleotide. The kit is used for the method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a nucleic acid sequence and a kit for detecting a target base sequence used in the method.

[0002]

2. Description of the Related Art A genome draft has been identified, and the era is shifting to post-sequencing. There is an increasing need for functional analysis of genes such as SNPs and genetic diagnosis based on the analysis results. The development of technology that can analyze the nucleotide sequence of a gene more accurately and more quickly is necessary to advance functional analysis quickly and to put the results of gene functional analysis to practical use in actual medical practice. Has become an important issue.

[0003] A method of synthesizing a template-dependent nucleic acid by the PCR (Polymerase Chain Reaction) method has been a great driving force for research in the life science field in recent years. The PCR method is
Using a small amount of double-stranded nucleic acid as a template, exponential amplification of a nucleic acid composed of a base sequence complementary to the template was enabled. Now, the PCR method is widely used as a tool for cloning and detecting genes. In the PCR method, a set of primers consisting of a base sequence complementary to both ends of a target base sequence is used. The extension product provided by one primer is designed to anneal to the other primer. Thus,
A synthesis reaction in which annealing of the extension products with each other and synthesis of a complementary strand are repeatedly performed proceeds, and exponential amplification is achieved.

[0004] In the PCR method, complicated temperature control is indispensable. In order to cope with complicated temperature control, a special reactor must be used. Therefore, it is difficult to perform PCR at the bedside or outdoors. Also,
Improvement of the reaction specificity was also an important issue of the known complementary strand synthesis reaction. For example, in the PCR method, when a complementary strand synthesis product is used as a new template, the region to which the primer anneals is not exactly a base sequence derived from the sample but a transcript of the base sequence of the primer.
Therefore, it is generally difficult to recognize slight nucleotide sequence differences using PCR primers.

[0005] As one method for solving these problems, the present inventors have proposed the LAMP method (Loop-mediated isotherm).
mal amplification) (Nucleic Acid Res.
2000 Vol.28 No.12 e63, WO 00/28082). In the LAMP method, the 3 'end of the template polynucleotide is annealed to serve as a starting point for complementary strand synthesis, and a complementary strand synthesis reaction can be performed at isothermal temperature by combining a primer that anneals to the loop formed at this time. did. In the LAMP method, the base sequence check mechanism repeatedly functions because the 3 ′ end always anneals to a region derived from the sample. As a result, a slight discrimination of the nucleotide sequence became possible.

When a target base sequence is detected based on a complementary strand synthesis reaction such as the LAMP method or the PCR method, there is a close relationship between the time required for the reaction and the detection sensitivity.
That is, it is a condition for achieving a high detection sensitivity that the reaction is performed for as long as possible until the reaction reaches a plateau. Known complementary strand synthesis reactions such as the LAMP method and the PCR method reach a plateau in about 1 hour. In other words, it can be said that a reaction time of about one hour is required to obtain the maximum sensitivity. One hour is not so long as a reaction time, but it is useful if a shorter reaction time can be realized without sacrificing detection sensitivity or operability.

[0007] To detect an amplification product obtained by the nucleic acid amplification method, a nucleic acid reaction mixture is developed by gel electrophoresis, and the amplified product is separated from a non-detection target component such as a template nucleic acid or a primer. It has been performed by determining the band of the amplification product by staining based on its molecular weight and the like, and then measuring the fluorescence intensity of the band.

However, in the detection of nucleic acid using the nucleic acid amplification method, the reaction solution contains a template nucleic acid and a large excess of primers, and depending on the type of nucleic acid sequence to be amplified, non-detection target components may be used. In many cases, it is difficult to perform a separation operation between gel and amplification product by gel electrophoresis. In addition, when the number of specimens increases, performing complicated gel electrophoresis involves a great deal of labor and waste of time, which is an obstacle to performing an efficient detection operation. In particular, in gene analysis in clinical practice and the like, it is required that many samples can be processed efficiently in a shorter time, and there has been a problem that conventional methods cannot sufficiently meet such a demand.

In order to solve the above problems, development of a simpler method for detecting a nucleic acid amplification product has been attempted. JP 9
-168400 describes a method in which primers used for a nucleic acid amplification reaction are immobilized on an insoluble carrier or can be immobilized, and a nucleic acid amplification product is detected by an agglutination reaction.

In Japanese Patent Application Laid-Open No. Hei 9-168400, nucleic acid is amplified by PCR. To detect an amplified product by PCR, it is necessary to immobilize two types of primers or to immobilize them. . In addition, when PCR is performed using primers immobilized on an insoluble carrier, the nucleic acid amplification product repeatedly aggregates and disperses with the temperature cycle peculiar to PCR, so that the nucleic acid amplification reaction is monitored in real time by the aggregation reaction. Not suitable for. As described above, a method of aggregating and detecting a nucleic acid amplification product with one type of immobilized primer and a method of monitoring a nucleic acid amplification reaction in real time by an agglutination reaction are not yet known.

[0011]

An object of the present invention is to observe the presence or absence of an agglutination reaction accompanying a polynucleotide amplification reaction using a polynucleotide as a template.
A method for rapidly detecting a target nucleic acid sequence in a sample is provided.

[0012]

Means for Solving the Problems The present inventors have repeated studies on the conditions for complementary strand synthesis in order to solve the above problems. Then, they found that the combination of the primer used for complementary strand synthesis and the 3 ′ end serving as the starting point of complementary strand synthesis was closely related to the reaction rate of complementary strand synthesis. Furthermore, by combining a template polynucleotide having a specific structure with a primer immobilized on an insoluble carrier capable of providing a starting point for complementary strand synthesis at a specific position on the polynucleotide, the reaction efficiency of complementary strand synthesis is improved. As a result, the present inventors have discovered that an agglutination reaction occurs with the amplification reaction of the polynucleotide, and thus completed the present invention. That is, the present invention relates to a method for detecting the following nucleic acid sequence, and a detection kit used for the method.

1. A method for detecting a target nucleic acid sequence in a sample, comprising mixing and incubating the following elements [1] to [5], and observing the presence or absence of an agglutination reaction accompanying the amplification reaction of the polynucleotide. [1]: a template polynucleotide having the following conditions (a) to (d): (a) a complementary polynucleotide of (b) and (a) having a target base sequence consisting of at least one set of complementary base sequences; When the base sequence hybridizes, it can form a loop capable of base pairing, (c) the 3 'end can anneal to itself to form a loop, (d) anneal to itself The 3 'end can serve as a starting point for complementary strand synthesis using itself as a template [2]: at least two types of primers capable of providing starting points for complementary strand synthesis at different positions in a loop of a template polynucleotide; 3]: In a template polynucleotide and / or an extension product generated by annealing a primer of [2] to a template polynucleotide, a starting point of complementary strand synthesis can be provided at a position different from that of the primer of [2]. At least one kind of primer having a 5 'end immobilized on an insoluble carrier (immobilized primer), [4]: a DNA polymerase capable of catalyzing complementary strand synthesis involving strand displacement, [5]: 1. a substrate for complementary strand synthesis; The above-mentioned immobilized primer [3] is complementary to a position different from the primer of [2] in the loop formed by the template polynucleotide and / or an extension product generated by annealing of the primer of [2] to the template polynucleotide. 2. The method according to 1, wherein the primer is a primer (fixed loop primer) that can provide a starting point for strand synthesis. 3. 3. The method according to 1 or 2, wherein the insoluble carrier is a natural polymer carrier, a synthetic polymer carrier, a metal colloid, or a magnetic particle. 4. 4. The method according to 3, wherein the insoluble carrier is a latex of a synthetic polymer carrier. 5. 5. The method according to any one of 1 to 4, wherein the template polynucleotide has a base sequence complementary to an arbitrary region of its own base sequence at its 5 ′ end. 6. 6. The method according to any one of 1 to 5, wherein the template polynucleotide is produced by the following steps. a) annealing a first primer to a target base sequence;
A step of conducting a complementary strand synthesis reaction using this as a starting point; wherein the first primer gives a starting point of complementary strand synthesis to a region defining the 3 ′ side of one of the strands constituting the target base sequence at its 3 ′ end And a base sequence complementary to any region of the complementary strand synthesis reaction product starting from this primer is provided on the 5 ′ side of the first primer. B) Step a) B) making the region to be annealed by the second primer in the extension product starting from the first primer synthesized in the step (1) ready for base pairing; 3 ′ of the target base sequence in the extension product starting from the first primer
A base sequence capable of providing a starting point for complementary strand synthesis in a region defining the side, and a 5 ′ side of the second primer is provided with an optional region of a complementary strand synthesis reaction product starting from this primer. C) a step of annealing a second primer to the region where the base pairing has become possible in step b) and synthesizing a complementary strand starting from the second primer, d) ) Annealing the 3 'end of the extension product starting from the second primer synthesized in step c) to itself,
6. Step of performing complementary strand synthesis using itself as a template 7. The method according to 6, wherein the two kinds of primers [2] are a first primer and a second primer. 8. The immobilized primer [3] provides a starting point for complementary strand synthesis between a region derived from the first primer in the extension product starting from the first primer and the arbitrary region relative to the first primer. And / or a starting point for complementary strand synthesis between the region derived from the second primer in the extension product originating from the second primer and the optional region for the second primer. 8. The method according to 7, which comprises a second immobilized primer capable of providing the following. 9. In step b) and / or step c), the first primer and / or the first primer may be synthesized by complementary strand synthesis from an outer primer which provides a starting point for complementary strand synthesis upstream of the first primer or the second primer. The extension product originating from primer 2 is replaced to
5. The method according to 4, wherein each product starting from the primer or the second primer is single-stranded. 10. In step a), the target base sequence is present as a double-stranded polynucleotide, and the region where the first primer anneals is in a state where base pairing can be performed by a complementary strand synthesis reaction starting from any primer. Be done 4
The method described in. 11. 11. The method according to 10, wherein step a) is performed in the presence of a melting temperature modifier. 12. 12. The method according to 11, wherein the melting temperature regulator is at least one compound selected from betaine, proline, dimethyl sulfoxide, and trimethylamine N-oxide. 13. 13. The method according to any one of 1 to 12, wherein the amplification reaction of the polynucleotide is carried out in the presence of a pyrophosphate formation inhibitor. 14． 14. The method according to 13, wherein the pyrophosphate formation inhibitor is Pyrophosphatase. 15. A method for detecting a mutation in a target base sequence by the method according to any one of 15.1 to 14, wherein when the target base sequence is not the predicted base sequence, complementary strand synthesis constituting the amplification method At least one complementary strand synthesis reaction selected from the reaction is obstructed, and it is observed whether or not an agglutination reaction accompanying the amplification reaction has occurred. Determining that they are not. 16. A method for detecting a mutation in a target nucleotide sequence by the method according to any one of 16.1 to 14, wherein it is observed whether or not an agglutination reaction accompanying the amplification reaction has occurred. A method comprising determining that the sequence is a predicted nucleotide sequence. 17． A kit for detecting a target nucleotide sequence, comprising: a) a first primer; wherein the first primer is
A starting point for complementary strand synthesis can be given to a region defining the 3 ′ side of one of the strands constituting the target base sequence at the 3 ′ end, and this primer is used as the starting point on the 5 ′ side of the first primer. B) a second primer; wherein the second primer is the second primer;
At the 3 ′ end, a base sequence capable of providing a starting point for complementary strand synthesis to a region defining the 3 ′ side of the target base sequence in the extension product starting from the first primer,
And the 5 ′ side of the second primer is provided with a base sequence complementary to an arbitrary region of a complementary strand synthesis reaction product starting from the primer. C) At least one type of 5 ′ An immobilized primer having an end immobilized on an insoluble carrier; wherein the immobilized primer provides a starting point for complementary strand synthesis at a position different from each primer in an extension product starting from the first primer or the second primer D) a DN capable of catalyzing complementary strand synthesis with strand displacement
17. A polymerase, e) a substrate for complementary strand synthesis, f) a pyrophosphate formation inhibitor, In the loop formed by the immobilized primer and a template polynucleotide and / or an extension product generated by annealing of the first or second primer to the template polynucleotide, complementary strand synthesis is performed at a position different from each primer. A primer (fixed loop primer) which can provide a starting point.
The kit according to 1. 19. The immobilized primer c) may provide a starting point for complementary strand synthesis between a region derived from the first primer in the extension product starting from the first primer and the arbitrary region for the first primer. A first immobilized primer that can be provided; and providing an origin of complementary strand synthesis between a region derived from the second primer in the extension product originating from the second primer and the arbitrary region relative to the second primer. 19. The kit according to 17 or 18, which comprises a second immobilized primer. 20. The kit according to any one of 17 to 19, further comprising the following element g): g) outer primer; the outer primer can provide a starting point for complementary strand synthesis upstream of the first primer and / or the second primer. 21. The kit according to any one of 17 to 20, wherein the pyrophosphate formation inhibitor is Pyrophosphatase.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention combines a template polynucleotide having a specific structure with a primer having a 5 ′ end immobilized on an insoluble carrier, which can provide a starting point for complementary strand synthesis to a specific region of the polynucleotide. As a result, rapid complementary strand synthesis can be performed, and the target nucleic acid sequence in the sample can be rapidly detected by observing the presence or absence of an agglutination reaction accompanying the amplification reaction of the polynucleotide. First, the template polynucleotide used in the present invention has the following structure. (a) having a target base sequence consisting of at least one set of complementary base sequences, (b) forming a loop capable of base pairing when the complementary base sequences of (a) are hybridized. (C) 3 ′ end can anneal to itself to form a loop, (d) 3 ′ annealed to itself
The end can be a starting point for complementary strand synthesis using itself as a template.

In the present invention, a polynucleotide is
It can be DNA, or RNA, or a chimeric molecule thereof. Polynucleotides can be natural or artificially synthesized. In addition, even if the nucleotide derivative is partially or wholly entirely composed of an artificial structure, it can form a base pair bond, and can be used as a template for complementary strand synthesis as necessary. Where possible, it is included in the polynucleotide of the present invention. Such molecules include
For example, a PNA having a backbone formed by peptide bonds can be shown. The number of constituent bases of the polynucleotide in the present invention is not limited. Polynucleotide is synonymous with the term "nucleic acid". On the other hand, the term "oligonucleotide" in the present invention is used as a term indicating a polynucleotide having a particularly small number of constituent bases among polynucleotides. Generally, an oligonucleotide refers to a polynucleotide having a base number of 2 to 100, more generally about 2 to 50, and is referred to as an oligonucleotide, but is not limited to these numerical values.

In the present invention, the term "target nucleotide sequence" means the nucleotide sequence of a polynucleotide to be synthesized. Generally, the base sequence of a polynucleotide describes the base sequence of a sense strand from the 5 'side to the 3' side. The target nucleotide sequence in the present invention is, in addition to the nucleotide sequence of the sense strand, its complementary strand,
That is, it also includes the base sequence of the antisense strand. That is,
The term “target base sequence” is used as a term that means at least one of a base sequence to be synthesized and a complementary strand thereof. The present invention provides a method that enables not only synthesis and amplification of a polynucleotide but also detection of a target base sequence. When a polynucleotide is amplified according to the present invention, the base sequence to be amplified is referred to as a target base sequence. The target base sequence can be any continuous base sequence selected from long polynucleotides, or the entire length of a single-stranded or circular polynucleotide can be the target base sequence. The synthesis (synthesi
s) means an act of increasing one target base sequence at least twice or more. On the other hand, when a new target base sequence is continuously synthesized based on the synthesized target base sequence, it is particularly referred to as amplification. Amplification can also be referred to as continuous synthesis. Further, in the present invention, providing a starting point for complementary strand synthesis refers to hybridizing a 3 ′ end of a polynucleotide that functions as a primer necessary for complementary strand synthesis to a polynucleotide as a template. When a starting point for complementary strand synthesis is given in a specific region, it means that the polynucleotide is hybridized such that the 3 ′ end serving as the starting point for complementary strand synthesis is located at an arbitrary position in the region. I do. At this time, as long as the 3 'end is located in the target region, a part of the base sequence required for hybridization can be arranged outside the target region. Further, in the present invention, the 3 ′ end or 5 ′ end: 3 ′ end or 5 ′
The terminus means not only one terminal at any terminal but also a region including one terminal at the terminal and located at the terminal. More specifically, 500 bases from either end,
Desirably, 100 bases, or at least 20 bases, are included at the 3 'end or 5' end. On the other hand, in order to indicate one base at the end or a base at a specific position near the end, the position is indicated by specifying a numerical value.

The target base sequence of the present invention comprises at least one set of complementary base sequences. In the present invention, the terms identical or complementary include cases where neither is completely identical or which is not completely complementary.
That is, the same as a certain sequence may include a sequence complementary to a base sequence capable of annealing to a certain sequence. On the other hand, complementary refers to sequences that can be annealed under stringent conditions and provide a 3 ′ end from which to synthesize complementary strands. Specifically, for a certain base sequence, generally 50-
100%, usually 70-100%, preferably 80-1
Base sequences having 00% identity can be said to be substantially identical. Identity can be determined based on known algorithms such as BLASTN.

The template polynucleotide of the present invention can form a loop when the complementary base sequence hybridizes. After the complementary base sequence has hybridized, it is difficult to generate a new base pair bond under the condition that the base pair bond is stably maintained. On the other hand, the loop can form a new base pair bond with another polynucleotide having a nucleotide sequence complementary to the nucleotide sequence constituting the loop. The base sequence constituting the loop is arbitrary.

The term “hybridization” used in the present invention means that polynucleotides consisting of complementary base sequences bind by base pairing. The polynucleotides that base-pair can be different molecules or the same molecule. When the hybridization that occurs between different molecules is eliminated, the polynucleotide is dissociated into a plurality of polynucleotide molecules. On the other hand, a polynucleotide hybridized on the same molecule remains a single-molecule polynucleotide even if base pairing is eliminated. In the present invention, the term annealing is also used. When a polynucleotide hybridizes to a polynucleotide having a complementary base sequence to give a 3 ′ end which is a starting point of complementary strand synthesis, the term “anneal” may be particularly used.

The template polynucleotide of the present invention has its 3 ′
The end can anneal to itself to form a loop, and the 3 'end annealed to itself can serve as a starting point for complementary strand synthesis using itself as a template. The base sequence for annealing is not limited as long as it allows complementary strand synthesis from its 3 'end. Specifically, for example, 3 'to 100-200 bases, usually 50-
80 bases, preferably 20-30 bases, have a base sequence complementary to any region in the target base sequence.
At this time, it is desirable that the annealed 3′-terminal base is completely complementary to the target base sequence. Complete complementarity of the bases at the 3 'end is not essential, but is an important condition for efficient complementary strand synthesis. The template polynucleotide of the present invention can form a loop by annealing the 3 ′ end to itself. The loop consists of an arbitrary base sequence like the above-mentioned loop, and exists in a state capable of base pairing with another polynucleotide.

[0021] The template polynucleotide of the present invention may have a base sequence complementary to an arbitrary region of the template polynucleotide at the 5 'end. When the complementary strand of such a template polynucleotide is synthesized, the 3 ′ end of the synthesized new polynucleotide anneals to an arbitrary region of the new polynucleotide to form a complementary strand synthesis reaction using itself as a template. Can be a starting point. The 3 ′ end anneals to itself to form a loop. The primer thus formed can be annealed to the loop thus formed. In this manner, by setting the 5 ′ end of the template polynucleotide to a base sequence complementary to an arbitrary region of the template polynucleotide, a product of complementary strand synthesis can be reused as the template polynucleotide. Accordingly, such a template polynucleotide is a desirable structure for achieving a highly efficient complementary strand synthesis reaction in the present invention.

The template polynucleotide of the present invention comprises:
It can be synthesized enzymatically or chemically. For example, a template polynucleotide can be synthesized through the following steps a) to d). The following steps a) -d)
Is a method for synthesizing a polynucleotide using the LAMP method. a) annealing a first primer to a target base sequence;
A step of conducting a complementary strand synthesis reaction using this as a starting point; wherein the first primer gives a starting point of complementary strand synthesis to a region defining the 3 ′ side of one of the strands constituting the target base sequence at its 3 ′ end And a base sequence complementary to any region of the complementary strand synthesis reaction product starting from this primer is provided on the 5 ′ side of the first primer. B) Step a) B) making the region to be annealed by the second primer in the extension product starting from the first primer synthesized in the step (1) ready for base pairing; 3 ′ of the target base sequence in the extension product starting from the first primer
A base sequence capable of providing a starting point for complementary strand synthesis in a region defining the side, and a 5 ′ side of the second primer is provided with an optional region of a complementary strand synthesis reaction product starting from this primer. C) a step of annealing a second primer to the region where the base pairing has become possible in step b) and synthesizing a complementary strand starting from the second primer, d) ) Annealing the 3 'end of the extension product starting from the second primer synthesized in step c) to itself,
A step of performing complementary strand synthesis using itself as a template.

The above steps will be described more specifically with reference to FIG. In the following description, it is assumed that a first primer (RA) consisting of R2 and R1c, and F2 and F1
The process of generating a template polynucleotide in the present invention using the second primer (FA) consisting of c is illustrated. In the following description, the first primer and the second primer are tentatively named RA and FA, respectively. The first primer RA can provide a starting point for complementary strand synthesis to a region defining the 3 ′ side of one of the strands constituting the target base sequence at the 3 ′ end, and the 5 ′ of the first primer RA On the side, a base sequence complementary to any region of the complementary strand synthesis reaction product starting from this primer is provided. The region on the 3 ′ side of RA is referred to as R2, and the region on the 5 ′ side is referred to as R1c. On the other hand, the second primer FA can provide a starting point for complementary strand synthesis to a region defining the 3 ′ side of the target base sequence in the extension product starting from the first primer RA at the 3 ′ end. On the 5 ′ side of the second primer FA, a base sequence complementary to an arbitrary region of a complementary strand synthesis reaction product starting from this primer is provided. 3 'side of FA is F
The 2, 5 'side is called F1c. In addition, the 3 'side of RA and FA
Each of the regions constituting the 5 'side has a base sequence complementary to the following regions. 3 'side of RA (R2): R2c 5' side of RA (R1c): R1 3 'side of FA (F2): F2c 5' side of FA (F1c): F1

Eventually, RA is the target base sequence R2c and R1
Depending on the F2c and F2c in the target sequence.
1 will determine its structure. Therefore, in the present invention, the target base sequence is required to be a base sequence in which at least a part of the base sequence is clear or can be estimated. The portion where the nucleotide sequence is to be clarified is a region which determines the structure of RA and FA, or a region comprising a complementary nucleotide sequence thereof. R2c and R1c (or F2c and F1
c) can be assumed to be continuous and distant. The relative positional relationship between the two determines the state of the loop portion formed when the product polynucleotide self-anneals. In the present invention, self-annealing means that a region including the 3 ′ end of a single-stranded polynucleotide hybridizes to a complementary base sequence of the polynucleotide itself, and the origin of complementary strand synthesis using itself as a template. It means becoming. In order for the product polynucleotide to preferentially perform self-annealing rather than intermolecular annealing, it is desirable that the distance between the two is not unnecessarily large. Therefore, it is desirable that the positional relationship between the two is usually continuous over a distance of 0 to 500 bases. However, in the formation of a loop by self-annealing, which will be described later, if both are too close together, it may be disadvantageous to form a loop in a desirable state.

The loop is required to have a structure capable of smoothly initiating annealing of a new oligonucleotide and a complementary strand synthesis reaction involving strand displacement starting from the annealing. Therefore, more preferably, the region R2c and its region
The distance from the region R1c located on the 5 ′ side is designed to be 0 to 100 bases, more preferably 10 to 70 bases. Note that this numerical value indicates a length that does not include R1c and R2c. The number of bases constituting the loop portion is further R2
Is obtained by adding a region corresponding to. Note that the same condition is applied to F2c and F1c in FA.

RA and FA for the target base sequence
R2 and R1c (or F2 and F1c)
Are usually arranged consecutively without overlapping. Alternatively, if there is a common portion in both base sequences, they can be partially overlapped. R2
Since (or F2) needs to function as a primer, it must always be at the 3 'end. On the other hand, R1c (or F1c) is placed at the 5 ′ end because it is necessary to give the 3 ′ end of the complementary strand synthesized using the R1c as a template as a primer, as described later. In the next step, the complementary strand obtained using this oligonucleotide as a starting point of synthesis serves as a template for complementary strand synthesis from the opposite direction, and RA and FA are finally copied to the complementary strand as templates. The 3 'end generated by copying has the base sequence R1 (or F1), anneals to R1c (or F1c) on the same strand, and forms a loop.

First, R2c of the first primer anneals to R2c in the single-stranded target base sequence, and the complementary strand is synthesized [FIG. 1- (1)]. The extension product originating from the first primer is, for example, an outer primer R
1 to form a single strand by performing a strand displacement elongation reaction [FIG. 1- (2)]. When F2c of the second primer is annealed to F2c to perform complementary strand synthesis [FIG.
(3)], the complementary strand synthesis ends when the 5 ′ end of the first primer is reached. The elongation product starting from the second primer synthesized at this time has R1 at its 3 'end.
[Fig. 1- (4)]. R1 at the 3 'end is the first
This is a region synthesized using the 5'-side R1c of the primer as a template. The extension product starting from the second primer is subjected to a strand displacement extension reaction using, for example, the outer primer F3, whereby the first product having loops at both ends is obtained.
The polynucleotide (basic dumbbel form 1) is obtained [Fig .- (5)]. In this polynucleotide, R1 anneals to its own R1c and becomes a starting point for complementary strand synthesis, and complementary strand synthesis is performed using itself as a template [FIG.
(6)]. The polynucleotide produced through the above reaction has a set of complementary base sequences consisting of the target base sequence and its complementary strand, and when hybridized, forms a loop capable of base pairing when hybridized. In addition,
At the 3 'end, F1 comprising a base sequence complementary to its own F1c is provided. F1 is the second primer F1c
Is a region synthesized using as a template. That is, this product is nothing but the template polynucleotide of the present invention.

The loop of this product is annealed by R2 of the first primer RA, and the complementary strand synthesis accompanied by a strand displacement reaction is performed. Then, the F1 at the 3′-terminal, which has been made into a single strand by strand displacement, anneals to its own F1C to form a loop, and serves as a starting point for complementary strand synthesis, and the complementary strand is synthesized using itself as a template. [FIG. 2- (7)] FIG.
A chain extension product having the loop of (8) is obtained, and the extension product starting from the first primer annealed to the loop is replaced to form a second polynucleotide (basic dumbble) having loops at both ends. form 2) is obtained. [Fig. 2- (5 ')]

In this basic dumbbel form 2, similarly to basic dumbbel form 1, F1 is its own F1.
c as a starting point for complementary strand synthesis, using itself as a template, a complementary strand synthesis reaction [FIG. 2- (5 ′)], annealing of the second primer FA to the loop and strand displacement extension reaction [FIG. (6 ′)], and a single-stranded 3′-terminal R1
Annealing to R1c and a complementary strand synthesis reaction using itself as a template are performed, and the basic dumbbel form 1 in FIG. 2- (5) is obtained together with the chain extension product having a loop in FIG. 2- (8 ′).

The target base sequence in FIG. 2 (8) is a base sequence between F2 and R2c, and a complementary base chain between R2 and F2c. . The reactions (1) to (5) in FIG. 1 actually proceed in parallel also on the complementary strand of the target base sequence, starting from the complementary strand synthesis of the second primer FA,
A second polynucleotide (basic dumbbel form 2) having loops at both ends in FIG. 2- (5 ′) is obtained.

Next, step b) in the reaction for synthesizing the template polynucleotide based on the LAMP method, ie, the second primer in the extension product starting from the first primer synthesized in step a) is used. Use of an outer primer is advantageous for performing a step of bringing a region to be annealed into a state where base pairing is possible. In the present invention, the outer primer is a primer consisting of a base sequence complementary to the first and second primers that anneals to the target base sequence and upstream of the second primer. In the present invention, “upstream” means the 3 ′ side of the template. Therefore, the region where the outer primer anneals is a region on the 5 ′ side when viewed from the first primer and the second primer.

For example, FIG. 1- (2) shows an outer primer R3 that anneals to R3c located on the 3 ′ side of the region R2c to which RA anneals. Similarly, in the complementary strand, 3 ′ of the region where FA anneals
The outer primer F3 can be annealed to F3c located on the side [FIG. 1- (4)]. As the outer primer, an oligonucleotide having a base sequence that functions as a primer on at least the 3 ′ side thereof can be used. The first primer and the second primer can be used as two of the three primers in the present invention. On the other hand, the outer primers described here do not necessarily have to constitute the three kinds of primers of the present invention. The outer primer is used for the synthesis of the template polynucleotide.

While the first primer and the second primer are usually composed of a combination of two primers, the outer primer can be any number. In the present invention, a general outer primer is composed of two outer primers that can provide a starting point for complementary strand synthesis upstream of each of the first primer and the second primer. However, the method of the present invention can be carried out even when the outer primer is disposed only for any one of the first primer and the second primer. Alternatively, a plurality of outer primers can be combined with each or one of the first primer and the second primer. In any case, when a complementary strand is synthesized from a more upstream site, a product of a complementary strand synthesis reaction using the first primer and the second primer as a replication origin can be efficiently generated.

In the present invention, the synthesis of the complementary strand from the outer primer is designed to be started after the synthesis of the complementary strand using the first primer and the second primer as a replication origin. The simplest method for that is to make the concentration of the first primer and the second primer higher than the concentration of the outer primer. Specifically, by using primers at a concentration difference of usually 2 to 50 times, preferably 4 to 10 times, the first primer and the second primer are used.
Of the complementary strand from the primer can be preferentially performed. The timing of synthesis can also be controlled by setting the melting temperature (Tm) of the outer primer to be lower than the Tm of the first primer and the second primer.

That is, (outer primer F3: F
3c) ≦ (R2c / R2) ≦ (R1c / R1). The reason why (R2c / R2) ≦ (R1c / R1) is set here is that annealing between R1c / R1 is performed before R2 anneals to the loop portion. R1
Since annealing between c / R1 is an intramolecular reaction, there is a high possibility that the annealing will proceed preferentially. However, it is significant to consider Tm to give more desirable reaction conditions. It goes without saying that similar conditions should be considered in the design of the reverse primer. With such a relationship, ideal reaction conditions can be stochastically achieved. The melting temperature (Tm) can be theoretically calculated based on the combination of the length of the complementary strand to be annealed and the base constituting the base pairing, if other conditions are constant. Therefore, those skilled in the art can easily derive desirable conditions based on the disclosure of the present specification.

Further, in order to adjust the timing of annealing of the outer primer, a phenomenon called contiguous stacking can be applied. What is Contiguous Stacking?
This is a phenomenon in which an oligonucleotide that cannot be annealed by itself is adjacent to a double-stranded portion, thereby enabling annealing (Chiara Borghesi-Nicoletti et.al.
Bio Techniques 12, 474-477, 1992). That is, the outer primer is designed to be adjacent to the first primer and the second primer, and is designed so that the outer primer alone cannot be annealed under incubation conditions. In this case, the outer primer can be annealed only when the first primer and the second primer have annealed, so that the annealing of the first primer and the second primer is necessarily given priority. . Based on this principle, an example in which an oligonucleotide base sequence required as a primer in a series of reactions is set is described in Examples.

In the above reaction, as the polynucleotide sample containing the target base sequence, any polynucleotide that can serve as a template for complementary strand synthesis can be used. Specifically, DNA and RNA, derivatives thereof, and chimeric molecules can be shown. Furthermore, as the polynucleotide sample, besides natural polynucleotides such as genomic DNA and mRNA, polynucleotides artificially incorporated into plasmids and phages can also be used. The polynucleotide sample can be used not only in a purified state but also in an unpurified state. In situ polynucleotide in cells
Can be used as the object of synthesis.

Next, in the present invention, at least three types of primers capable of providing a starting point for complementary strand synthesis to different regions of the loop are used. A loop in which a primer provides a starting point for complementary strand synthesis is, in addition to the loop provided in the template polynucleotide, a 3′-terminal or a new polynucleotide generated as a result of complementary strand synthesis starting from any primer. Included in the loop. The template polynucleotide contains at least the following 2
With two loops. 1. Loop formed by hybridization of the target base sequence 2. Loop formed by annealing of the 3 'end of the template polynucleotide to itself

When the template polynucleotide is composed of two or more sets of complementary target base sequences and the complementary base sequences are alternately arranged, a loop corresponding to the number is formed on the template polynucleotide. You. For example, assume a polynucleotide composed of a certain base sequence A and its complementary base sequence a. The state in which a plurality of sets of complementary base sequences are alternately arranged can be represented, for example, as follows. 5 '-[A]-(L)-[a]-[A]-(L)-[a]-[A]-
(L)-[a] -3 '

In this example, when hybridization occurs between adjacent [A]-[a], three loops can be formed. A portion where a loop is formed by hybridization of a complementary base sequence is indicated by-(L)-. 3 'more
Addition of a loop formed by annealing the end to itself, the template polynucleotide will form a total of four loops. When the 3 ′ end forms a loop by annealing to itself, a part of the base sequence of [a] is used for annealing. Therefore,
At [a] at the 3 'end, there is a region that is used for annealing to itself without hybridizing with [A], even though the nucleotide sequence is complementary to [A]. The number of complementary base sequences that can be included in the template polynucleotide in the present invention is not limited. Therefore, if a template polynucleotide composed of [A] and [a] is represented by a general formula, it can be represented as follows. In the formula, n means an arbitrary natural number. 5 '-｛[A]-(L)-[a]｝ n-3'

On the other hand, the 3 ′ end of the template polynucleotide,
Alternatively, a loop formed by a new polynucleotide generated as a result of complementary strand synthesis starting from one of the primers is specifically formed as follows. First, the 3 ′ end of the template polynucleotide can be annealed to itself to perform a complementary strand synthesis reaction using itself as a template. This reaction proceeds continuously if the 3 'end and the region to which it anneals can be repeatedly brought into a state capable of base pairing. As a result, the base sequence complementary to the template polynucleotide is repeatedly extended. Therefore, when the template polynucleotide is composed of one set of complementary base sequences, the number of complementary base sequences in a new polynucleotide obtained by complementary strand synthesis doubles every time the complementary strand synthesis reaction is repeated. Become. Here, when the two sets of complementary base sequences in the newly generated polynucleotide hybridize with each other, loops are generated in a number corresponding to the number of sets of the complementary base sequences [for example, FIG. ]. Of these loops,
Some are loops originally possessed by the template polynucleotide. The other loop consists of a complementary nucleotide sequence of the loop of the template polynucleotide. The new loop thus generated is also included in the loop where the primer anneals in the present invention.

Further, the loop in the present invention includes a loop formed in a new polynucleotide generated by the primer annealing to the loop of the template polynucleotide to become a starting point of complementary strand synthesis. For example, a complementary strand can be synthesized starting from a primer that can provide a starting point for complementary strand synthesis to a loop formed by annealing the 3 ′ end of the template polynucleotide to itself. The product is a polynucleotide having a base sequence complementary to the template. This polynucleotide is hybridized by a complementary base sequence similar to the template polynucleotide,
Form a loop. That is, a polynucleotide having a loop consisting of a base sequence complementary to the loop of the template polynucleotide is generated.

The primer of the present invention includes a primer capable of providing a starting point for complementary strand synthesis to the new loop thus formed. Alternatively, as described above, when the template polynucleotide contains a base sequence complementary to an arbitrary region of its own at the 5 ′ end, the 3 ′ end of the polynucleotide synthesized using this as a template is annealed to itself. A loop can be formed. In the present invention, a primer that can provide a starting point for complementary strand synthesis to the loop thus formed can also be used. When the 5 ′ end of the template polynucleotide contains a base sequence complementary to an arbitrary region of the template polynucleotide, the 5 ′ end can hybridize to itself to form a loop. The loop at the 5 'end is also ready for base pairing. However, from a primer that gives a starting point for complementary strand synthesis to this loop, usually, only a small region from the loop to the 5 ′ end can be subjected to complementary strand synthesis.

The primer of the present invention can be a starting point for complementary strand synthesis in at least one, preferably two, or three or more loops selected from several types of loops as described above. Use three or more primers. Three or more means that three primers provide starting points for complementary strand synthesis at mutually different positions.

In the present invention, at least three, preferably four, or five or more primers can be used in combination. The mutually different positions mean that the positions of the 3 'ends of the respective primers when the primers are annealed are different from each other. Therefore, the base sequences required for annealing can be partially overlapped. However, if the regions necessary for annealing overlap, competition occurs between the primers. Therefore, it is desirable to design the base sequence of the primers so that the regions can be annealed as independently as possible. Furthermore, by designing the base sequence of the primer so that the region to be hybridized or annealed in the template polynucleotide does not overlap as much as possible, more rapid complementary strand synthesis can be expected. Therefore, for example, a combination of a template polynucleotide and three or more types of primers that anneal to mutually different loops formed in a complementary strand synthesis product generated using the template polynucleotide as a template,
It can be said that the combination of primers is suitable for the present invention.

The base sequence constituting the primer of the present invention is a base sequence containing a base sequence complementary to the base sequences of the plurality of loops described above. On the 3 'side, it is required that the loop be provided with a starting point for complementary strand synthesis. Therefore, at least its 3 'end should be located within the loop. However, not all of the base sequences required for annealing need be arranged in the loop. Therefore, as long as the primer can provide a starting point for complementary strand synthesis under the required reaction conditions, for example, a part of the base sequence required for annealing may be different from the base sequence constituting the double-stranded polynucleotide adjacent to the loop. Duplication is allowed. Also, an arbitrary base sequence can be added to the 5 ′ side of the primer. In the present invention, the above-described LAMP primer can be used as a primer that gives a starting point of complementary strand synthesis to the loop of the template polynucleotide.

The LAMP method (Nucleic Acid Res. 2000 Vol. 28 N
o.12 e63, WO 00/28082) anneals its own 3 ′ end to the template polynucleotide to serve as a starting point for complementary strand synthesis, and also provides a loop formed at this time with a starting point for complementary strand synthesis. This is a method that enables a complementary strand synthesis reaction at a high isothermal temperature by combining primers that can be used. In the LAMP method, at least the following two primers are used. [1] First primer: The first primer can provide a starting point for complementary strand synthesis to a region defining the 3 ′ side of one of the strands constituting the target base sequence at the 3 ′ end thereof, and A primer having a base sequence complementary to an arbitrary region of a complementary strand synthesis reaction product starting from the primer on the 5 ′ side of the primer [2]. The primer has at its 3 ′ end a base sequence capable of providing a starting point for complementary strand synthesis to a region defining the 3 ′ side of the target base sequence in the extension product starting from the first primer, and 5 'of the primer
On the side, a base sequence complementary to an arbitrary region of a complementary strand synthesis reaction product starting from this primer is provided.

That is, the first primer and the second primer used for the synthesis of the template polynucleotide may constitute a part of at least three kinds of primers in the polynucleotide synthesis method according to the present invention. it can. In other words, in the template polynucleotide and / or the extension product generated by annealing the first or second primer to the template polynucleotide,
The present invention is practiced by combining a third primer having its 5 'end immobilized on an insoluble carrier, which can provide a starting point for complementary strand synthesis at a position different from that of the first and second primers. be able to. In the present invention, in addition to the primers necessary for the synthesis of the target base sequence, the third primer to be combined may be referred to as an immobilized primer.

The insoluble carrier for immobilizing the 5 'end of the immobilized primer is not particularly limited, and any ordinary insoluble carrier can be used. Suitable insoluble carriers include, for example, insoluble agarose, cellulose, natural polymer carriers such as insoluble dextran, polystyrene, styrene-styrene sulfonic acid copolymer, synthetic polymer carrier such as vinyl acetate-acrylate copolymer, and the like. And functional particles such as gold colloids and magnetic particles. Particularly preferred is a latex in which a synthetic polymer carrier is uniformly dispersed in water or the like.

It is not an essential condition that the primer used for the synthesis of the template polynucleotide is also used as a primer for synthesizing the target base sequence. Apart from the primer for synthesizing the template polynucleotide, it is also possible to add a primer for synthesizing the target base sequence. However, it is usually reasonable to make up the reaction with fewer elements.

Preferred immobilized primers in the present invention include a template polynucleotide and / or a loop formed by an extension product formed by annealing the first or second primer to the template polynucleotide.
A primer (fixed loop primer) that can provide a starting point for complementary strand synthesis at a position different from the first or second primer is exemplified. Particularly preferred fixed loop primers are R1-R2 (or F1-F2
Primers capable of providing a starting point for complementary strand synthesis in the region (between) can be shown. When any one of the primers capable of annealing between R1 and R2 or between F1 and F2 is combined with a primer for LAMP, three types of fixed loop primers are used. Further, between R1 and R2 or F1-
If both primers capable of annealing between F2 are combined, two types of fixed loop primers are added to the LAMP primer, so that a total of four types of primers are used. The combination of at least one, preferably two or more of the fixed loop primers according to the present invention can also be expected to have the effect of promoting the polynucleotide synthesis reaction.

This fixed loop primer anneals to a loop containing R2 (for example, FIG. 3- (9)). This loop is composed of a region extending from R1 to R2 in the target base sequence (however, in FIG. 3 (9), R2 is included and R1 is not included). With respect to such a loop, the fixed loop primer of the present invention can use, as the fixed loop primer, a nucleotide having an arbitrary base sequence including a base sequence complementary to the base sequence constituting the loop.

Finally, as a desirable fixed loop primer to be combined when the present invention is applied to the LAMP method,
Primers satisfying the following conditions can be given. That is, of the loop formed in the template polynucleotide and / or the polynucleotide generated by the reaction of the template polynucleotide with the first primer and the second primer, the first primer and the second primer cannot be annealed. In the loop, to provide a starting point for complementary strand synthesis. The fixed loop primer only needs to be able to anneal to the region where the 3 'end serving as the starting point of complementary strand synthesis is located in the loop. Therefore, not only when the base sequence for annealing is completely contained in the loop, but also when a part thereof overlaps a region other than the loop, when the 3 ′ end is located in the loop, It can be said that the desired condition is satisfied. For example, even when a part of the base sequence to be annealed by the fixed loop primer extends to the double-stranded structure adjacent to the loop, the same as when the region to be annealed is completely arranged in the loop A reaction promoting effect can be obtained.

FIG. 5 shows a preferred positional relationship between the fixed loop primer and the nucleotide sequence constituting the template polynucleotide. FIG. 5 shows that complementary strand synthesis is performed starting from the 3 ′ end of F1 that anneals to itself. FIG. 5 shows the same structure as FIG. 2- (5 ′). That is, when the complementary strand synthesis shown in this figure is completed, the template polynucleotide of the present invention is about to be completed.
FIG. 5 shows that the fixed loop primer R is a primer that anneals to the loop containing R2.
FIG. 5 is provided to show a region where the fixed loop primer R anneals. The synthesis of the complementary strand from the fixed loop primer R annealed to the structure of FIG. 5 actually reaches the 5 ′ end immediately. When complementary strand synthesis is performed by annealing to a loop consisting of a similar base sequence formed in a reaction product generated from a template polynucleotide, complementary strand synthesis starting from the fixed loop primer R contributes to an improvement in reaction efficiency. I do.

When the present invention is applied to the LAMP method, it is desirable that the immobilized primer can be annealed under the same conditions as those of the LAMP method. The fact that all reactions can be carried out without temperature changes is a major advantage of the LAMP method. In order not to impair this advantage, when combining the immobilized primers for the present invention, it is ideal to use the immobilized primers under the same temperature conditions as the reaction for the LAMP method. For that purpose, the Tm of the immobilized primer is designed to be at the same level as the primer for the LAMP method. Tm is determined by the type and number of bases constituting the primer, salts contained in the reaction solution, and various components that affect Tm.
Therefore, in the present invention, the Tm of the immobilized primer is adjusted by its base sequence in accordance with the conditions of the reaction solution for the LAMP method. A person skilled in the art can adjust the base sequence of the primer according to various conditions to give an appropriate Tm. Needless to say, these conditions for the fixed loop primer are also applied to the loop constituted by F1c-F2c (or R1c-R2c).

As described above, when the step of annealing the 3 'end to itself and performing complementary strand synthesis shown in FIG. 2- (5) or FIG. 2- (5') is completed, the template of the present invention is obtained. A polynucleotide is obtained. In addition, each of the loops of this template polynucleotide has RA [FIG.
(6)] or FA [FIG. 2- (6 ′)] is annealed to perform complementary strand synthesis, and the 3 ′ end of the template polynucleotide becomes single-stranded again by substitution. 3 'became a single strand
The end anneals to itself and the complementary strand synthesis proceeds (FIG. 2- (7) and FIG. 2- (7 ′)), FIG. 2- (8), and FIG. 2- (8 ′). Thus, annealing of primer RA or FA to the loop, complementary strand synthesis,
A cycle of substitution of the 3 ′ end of the template polynucleotide, annealing to itself and synthesis of the complementary strand is repeated.

At this time, the elongation of the 3 ′ end of the template polynucleotide using the template itself as a template displaces the product of complementary strand synthesis starting from RA or FA, which has annealed to the loop, as a replication origin. [FIG. 2- (7) and FIG. 2- (7 ′)]. This single-stranded polynucleotide has a base sequence complementary to itself at the 3 ′ end and the 5 ′ end. That is, a product having the same structure as that of FIG. 2- (5) or FIG. 2- (5 ′) is obtained. These become new template polynucleotides, and the same reaction repeatedly proceeds. So far, the reaction mechanism has been elucidated as the LAMP method.

In FIG. 2- (8), the primer FA annealed to the loop extends while displacing the double-stranded structure of the template polynucleotide, and eventually reaches its 5 ′ end. At this time, the 3′-terminal side of the substituted template polynucleotide has a single-stranded structure. Therefore, their complementary base sequences hybridize with each other. The structure thus formed is the structure shown in FIG. That is, the 3 ′ of the template polynucleotide anneals to itself,
At the same time, the complementary base sequences hybridize to form a loop. The loop formed by the hybridization of the complementary base sequence is a complementary strand that copies the loop portion of the original template polynucleotide. This loop is a loop including R2 as is clear from the figure, and FA and RA cannot be annealed. However, the fixed loop primer R can anneal to the loop containing R2. Then, the fixed loop primer R anneals to the loop containing R2 to start complementary strand synthesis. On the other hand, the amplification reaction of the polynucleotide based on the known LAMP method continues. That is, the extension reaction using the template polynucleotide itself as a template and the amplification reaction using the primer FA that anneals to the loop continue. This reaction itself is nothing but an amplification reaction of a polynucleotide that continuously produces a new template polynucleotide. In addition, the amplification reaction after FIG. 3- (9) already revealed by the LAMP method is not shown here.

In the present invention, by using an immobilized primer having the 5 ′ end immobilized on an insoluble carrier, the following agglutination reaction occurs as the amplification reaction proceeds from the immobilized primer. The fixed loop primer R (LoopR) anneals to the loop containing the single-stranded structure R2 of the polynucleotide of FIG.
With the progress of the complementary strand elongation reaction starting from pR, the extension product obtained starting from the primer FA in FIG. 2- (8) was displaced, and the single-stranded DNA shown in FIG. A polynucleotide is produced. On the other hand, the extension product of LoopR is the 3′-end F1 of the polynucleotide of FIG.
Is displaced by a chain extension reaction using self as a template,
The single-stranded polynucleotide shown in FIG. 3- (11) is produced.

As shown in FIG. 4, the single-stranded polynucleotide (11) has a loop containing F2, a loop containing R2, and a loop formed by annealing F1 at the 3 ′ end to F1c. There are three loops. F2
And the loop containing R2, the fixed loop primer Lo
opF and LoopR anneal, and a chain extension reaction proceeds, respectively. In addition, a chain elongation reaction using the self as a template proceeds from the 3 ′ terminal F1. The elongation product of LoopF is displaced as the chain elongation reaction proceeds from LoopR as a starting point, and the single-stranded polynucleotide (12)
Is generated. At the 3 ′ end of the polynucleotide (12), there is a sequence LoopRc complementary to LoopR,
When LoopR anneals to LoopRc and the chain elongation reaction proceeds, an aggregation product (13) consisting of a stable double-stranded polynucleotide having fixed loop primers LoopF and LoopR at both ends is generated.

On the other hand, the extension product of LoopR is displaced by a chain extension reaction using the self from the 3 ′ end F1 of the polynucleotide (11) as a template to produce a single-stranded polynucleotide (14). This polynucleotide (1
At the 3 ′ end of 4), a sequence Loop complementary to LoopR
When Rc is present and LoopR anneals to LoopRc and the chain elongation reaction proceeds, an aggregation product (15) consisting of a stable double-stranded polynucleotide having a fixed loop primer LoopR at both ends is generated.

The same aggregated product starts from basic dumbbel form 2 shown in FIG.
It is also produced in the corresponding reaction proceeding via complementary strand synthesis from (5 ′) to FIG. 2- (8 ′). These aggregated products generated as the reaction proceeds can be detected as changes in turbidity in the reaction system using an optical means such as a spectrophotometer or by the naked eye. That is, according to the present invention, the first primer RA and the second primer F used in the basic LAMP method are used.
By using an immobilized primer having at least one 5 ′ end immobilized on an insoluble carrier in addition to A, by simply observing the presence or absence of an agglutination reaction in the reaction system without requiring a new detection step, It is possible to determine whether the target base sequence is a predicted base sequence.

As described above, the agglutination reaction in the present invention is performed by using two types of fixed loop primers (LoopR and LoP).
opF) as well as LoopR or L
Even when any one of the fixed loop primers is used, progress proceeds quickly. The fixed loop primer is contained in the reaction solution from the start of the amplification reaction of the polynucleotide, and does not require any reagent preparation. Therefore, there is no possibility that contamination occurs during the reaction. Furthermore, from the start of amplification to the detection can be performed isothermally as a series of reactions, and the measurement of turbidity after the agglutination reaction can be performed without using an optical analyzer such as a spectrophotometer.
Since it can be carried out with the naked eye, it is a very simple method for detecting a polynucleotide. Also, LAMP
By using the fixed loop primer without affecting the reaction itself, the amplification reaction of the polynucleotide in the basic LAMP method is greatly accelerated.

The template polynucleotide of the present invention can be synthesized using a target base sequence contained in a double-stranded polynucleotide as a starting material. 2
A method for annealing a complementary strand by annealing a primer to a target base sequence in a main strand state is described in Patent Application 2000-111939 filed by the present applicant. In FIG. 1, a template polynucleotide is synthesized using a single-stranded target base sequence as a starting material. However, if the method using the target base sequence contained in the polynucleotide in a double-stranded state as a starting material is applied, the step of denaturation into a single strand is omitted, and the double-stranded polynucleotide is used as a starting material as it is. can do. Examples of the double-stranded polynucleotide include cDNA and genomic DNA. Alternatively, those obtained by inserting these DNAs into various vectors can be used as the double-stranded polynucleotide of the present invention. On the other hand, a polynucleotide in a single-stranded state means that these double-stranded polynucleotides are made into a single strand by denaturation treatment such as heating or alkaline conditions, or originally present as a single strand like mRNA. Can be indicated. The double-stranded polynucleotide of the present invention may be purified or unpurified. In addition, the method of the present invention can be applied in a state where it is present in a cell (in situ). By using a polynucleotide in a cell as a template, in situ analysis of the genome becomes possible.

When cDNA is used as a template in the present invention, the step of synthesizing the cDNA and the method of synthesizing the polynucleotide of the present invention can be performed under the same conditions. When the first strand of cDNA is synthesized using RNA as a template, D
The double-stranded polynucleotide by the NA-RNA hybrid is completed. Using this double-stranded polynucleotide as a template in the present invention, a polynucleotide detection method can be performed. As long as the DNA polymerase used in the method for detecting a polynucleotide of the present invention has reverse transcriptase activity, a single enzyme can be used to synthesize a polynucleotide under the same conditions. For example, Bca DNA
The polymerase is a DNA polymerase having a strand displacement activity and a reverse transcriptase activity. It is needless to say that the method for detecting a polynucleotide according to the present invention can be applied after the second strand is synthesized and converted into a complete double-stranded cDNA.

When a template polynucleotide is synthesized from a target base sequence contained in a double-stranded polynucleotide, first, an arbitrary primer is added to the double-stranded polynucleotide, and a complementary strand synthesis reaction using the primer as a starting point is performed. Are incubated under conditions that can achieve this.
The term "arbitrary primer" used herein is used to bring a region to which the first primer should be annealed into a state capable of base pairing. Therefore, any primer must be able to anneal to the complementary strand to the polynucleotide strand of the target base sequence to be annealed by the first primer. Furthermore,
In the present invention, the complementary strand synthesis using an arbitrary primer as a replication origin should be in a positional relationship such that the first primer proceeds toward the region to be annealed. In other words, it can anneal to any region of the region functioning as a template in the complementary strand synthesis reaction starting from the first primer. Any primer can be selected from any region as long as this condition is satisfied. For example, the second primer can be used as an optional primer. Such an embodiment is one of the desirable embodiments in the present invention because it reduces the components required for the reaction.

Therefore, step a) in the reaction for synthesizing the template polynucleotide based on the LAMP method, ie, annealing the first primer to the target base sequence,
In the step of performing a complementary strand synthesis reaction starting from this, one strand of the double-stranded polynucleotide is displaced by complementary strand synthesis starting from an arbitrary primer, and base pairing by the first primer is possible. It can be. By adopting these conditions, a synthesis reaction requiring no temperature change was realized. The conditions under which an arbitrary primer can be annealed to a double-stranded polynucleotide and a complementary strand synthesis reaction starting from this primer can be achieved are conditions under which the following multiple steps can actually proceed under the same conditions. Can be. i) The primer anneals to the template in a double-stranded polynucleotide state. ii) The complementary strand synthesis with the annealed primer as the replication origin proceeds.

It was thought that a primer would not be able to anneal unless it had at least a single-stranded region to anneal. Therefore, conventionally, in the case where a double-stranded polynucleotide is used as a template, a step of always forming a single strand by denaturation prior to annealing of the primer has been carried out. However, even if it is not necessarily a single strand, a complementary strand synthesis reaction starting from the primer is started by incubating with the primer under the condition that the duplex is destabilized by some means. As a condition for destabilizing the duplex, for example, a method of heating to near the melting temperature (hereinafter abbreviated as Tm) can be shown. Alternatively, the presence of a Tm modifier is also effective.

In the present invention, a series of reactions include a buffer for giving a pH suitable for the enzymatic reaction, salts necessary for maintaining or annealing the catalytic activity of the enzyme, a protective agent for the enzyme, and melting if necessary. It is carried out in the presence of a temperature (Tm) regulator or the like. As the buffering agent, one having a neutral to weak alkaline buffering action such as Tris-HCl is used. The pH is adjusted according to the DNA polymerase used. As salts, KCl, NaCl, (NH ₄ ) ₂ SO _{4 and the} like are appropriately added for maintaining the activity of the enzyme and adjusting the melting temperature (Tm) of the polynucleotide. Bovine serum albumin and saccharides are used as enzyme protecting agents. Further, betaine, proline, dimethyl sulfoxide (hereinafter abbreviated as DMSO), formamide, and trimethylamine N-oxide are generally used as a regulator of the melting temperature (Tm). By utilizing a melting temperature (Tm) modifier, the annealing of the oligonucleotide can be adjusted under limited temperature conditions. Furthermore, betaine (N, N, N, -trimethylglycine) and tetraalkylammonium salts are also effective in improving the efficiency of chain displacement by their isostabilize action. Betaine is present in the reaction mixture at 0.
By the addition of about 2 to 3.0 M, preferably about 0.5 to 1.5 M, the effect of promoting the polynucleotide amplification reaction in the present invention can be expected. Since these melting temperature regulators act in the direction of lowering the melting temperature, conditions for providing appropriate stringency and reactivity are set in consideration of other reaction conditions such as salt concentration and reaction temperature. By using the Tm adjuster, it is possible to easily set a temperature condition suitable for the enzyme reaction. Tm varies depending on the relationship between the primer and the target base sequence. Therefore, it is desirable to adjust the amount of the Tm adjuster used so that the conditions capable of maintaining the enzyme activity coincide with the conditions of the incubation satisfying the conditions of the present invention. Based on the disclosure of the present invention, to set the appropriate amount of Tm adjusting agent according to the base sequence of the primer,
It is obvious to those skilled in the art. For example, Tm can be calculated based on the length of the base sequence to be annealed, its GC content, salt concentration, and the concentration of the Tm modifier.

The annealing of the primer to the double-stranded polynucleotide under these conditions is presumably unstable. However, by incubating with a strand-displacing polymerase, a complementary strand is synthesized with the primer annealed while being unstable as a replication origin. If the complementary strand is synthesized, the annealing of the primer will be gradually stabilized.

In the method for detecting a nucleic acid sequence of the present invention, a polynucleotide amplification reaction can be carried out in the presence of a pyrophosphate formation inhibitor. LAMP
In reaction, DN having a specific base sequence at an accelerated rate
During the process of A amplification, pyrophosphoric acid is produced in large quantities and combines with magnesium contained in the reaction solution to form a white precipitate of magnesium pyrophosphate. The formation of this white precipitate can be visually identified, and can be used as the simplest means for judging the progress of the amplification reaction. When observing the presence or absence of the agglutination reaction accompanying the above, it is an obstacle. Such an obstacle is LAM
It can be suppressed by adding a certain amount of a pyrophosphate formation inhibitor such as the enzyme Pyrophosphatase to the P reaction solution from the start of the reaction. That is, the pyrophosphate formation inhibitor keeps the phosphoric acid generated by the DNA elongation reaction as monophosphate, and can suppress the formation of a white precipitate of magnesium pyrophosphate. When the detection method of the present invention is carried out without adding a pyrophosphate formation inhibitor, when measuring the rise in turbidity in the reaction solution using an optical means such as a spectrophotometer, By subtracting the turbidity increase due to the white precipitation of magnesium pyrophosphate from the measured value of the liquid as a background, the presence or absence of the agglutination reaction by the immobilized primer can be confirmed.

The primers used in the present invention can be chemically synthesized. Methods for synthesizing DNA are known. Alternatively, a natural polynucleotide can be cleaved with a restriction enzyme or the like, and modified or linked so as to be composed of a required base sequence. The various primers used in the present invention can be not only those having the same structure as natural DNA but also artificial mutants. A primer refers to a primer that satisfies the two conditions of being able to form a complementary base pair bond with a target base sequence and providing an —OH group at the 3 ′ end thereof as a starting point for complementary strand synthesis. Furthermore, the primer can desirably be a template for complementary strand synthesis. Therefore, the backbone is not necessarily limited to the phosphodiester bond. For example, it may be composed of a phosphothioate compound or a peptide nucleic acid based on a peptide bond. The base may be any one that enables complementary base pairing. In the natural state, there are generally five types, ACTG and U, but it can also be an analog such as, for example, bromodeoxyuridine. It is desirable that the oligonucleotide used in the present invention not only serves as a starting point of synthesis but also functions as a template for complementary strand synthesis.

The primer used in the present invention can perform base pairing with a complementary strand in a variety of polynucleotide synthesis reactions constituting the present invention while maintaining necessary specificity in a given environment. It has about a chain length. Specifically, it is 5 to 200 base pairs, more preferably 10 to 50 base pairs. Since the length of a primer recognized by a known polymerase that catalyzes a sequence-dependent polynucleotide synthesis reaction is at least about 5 bases, the length of a portion to be annealed needs to be longer. In addition, in order to expect specificity as a base sequence, it is desirable to use a length of 10 bases or more stochastically. On the other hand, an extremely long base sequence is difficult to prepare by chemical synthesis, and thus the above-mentioned chain length is exemplified as a desirable range. The chain length exemplified here is the chain length of the portion that anneals to the complementary strand. For example, RA includes at least two regions R2 and R1c. Therefore, the chain lengths exemplified here should be understood as the chain lengths of the respective regions constituting the primer. The term template used in the present invention means a polynucleotide on the side that serves as a template for complementary strand synthesis. A complementary strand having a base sequence complementary to the template has a meaning as a strand corresponding to the template, but the relationship between the two is merely relative. That is, a chain synthesized as a complementary chain can again function as a template. That is, the complementary strand can serve as a template.

In the polynucleotide synthesis method or amplification method of the present invention, a DNA polymerase capable of catalyzing a complementary strand synthesis reaction involving strand displacement is used. This type of polymerase is used in DNA such as SDA
The same as the polymerase is used. That is, when a complementary strand is synthesized using a primer complementary to the 3 ′ side of a certain base sequence as a synthesis starting point, if there is a double-stranded region on the 5 ′ side, synthesis of the complementary strand is performed while displacing the two strands. Specialized polymerases that perform the above are known. In the present invention, a substrate necessary for complementary strand synthesis is further added.

Supporting the polynucleotide synthesis method of the present invention is a DNA polymerase capable of catalyzing a strand displacement type complementary strand synthesis reaction. This kind of DNA
The following are known as polymerases.
Also, as for various mutants of these enzymes, as long as they have a sequence-dependent complementary strand synthesis activity and a strand displacement activity,
It can be used in the present invention. As used herein, the term “mutant” refers to a product obtained by extracting only a structure that provides the catalytic activity required by an enzyme, or a product obtained by modifying the catalytic activity, stability, or heat resistance by amino acid mutation or 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 excluding exonuclease activity) DeepVent DNA polymerase DeepVent (Exo-) DNA polymerase (Excluding exonuclease activity from DeepVent DNA polymerase) Φ29 phage 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 desirable enzymes because they have some heat resistance and high catalytic activity. In the present invention, particularly when a double-stranded polynucleotide is used as a template, the annealing of the primer and the complementary strand synthesis reaction are performed under the same conditions. Since such reactions often require some degree of heating, it is one of the desirable conditions that the enzyme be thermostable. By using a thermostable enzyme, it is possible to cope with a wide range of reaction conditions.

For example, Vent (Exo-) DNA polymerase
It is an enzyme with high heat resistance as well as strand displacement activity. By the way, a complementary strand synthesis reaction involving strand displacement by DNA polymerase is a single strand binding protein (single strand binding protein).
(Paul M. Lizardi et al, Nature Genetics 19, 225-2)
32, July, 1998). Applying this effect to the present invention, the effect of promoting the complementary strand synthesis can be expected by adding a single-stranded binding protein. For Vent (Exo-) DNA polymerase, T4 gene
32 is valid. For DNA polymerases without 3'-5 'exonuclease activity, complementary strand synthesis requires 5'
A phenomenon is known in which the synthesis proceeds to a state where one base is protruded without stopping at the end. In the present invention, such a phenomenon is not desirable because the sequence at the 3 ′ end when the complementary strand synthesis reaches the end leads to the start of the next complementary strand synthesis. However, the addition of a base to the 3 ′ end by DNA polymerase results in A with a high probability. Therefore, the sequence may be selected so that synthesis from the 3 'end starts at A so that there is no problem even if dATP is added by one base by mistake.
In addition, even if the 3 'end protrudes during synthesis of the complementary strand, the 3' → 5 'exonuclease activity can be utilized by digesting the 3' end and making it a blunt end. For example, natural Vent DNA polymerase has this activity,
This problem can be avoided by using the mixture with Vent (Exo-) DNA polymerase.

In contrast to these DNA polymerases, DNA polymerases such as Taq polymerase generally used in PCR and the like have substantially no strand displacement under ordinary conditions. However, this type of DNA polymerase can be used in the present invention if conditions that allow strand displacement can be provided.

In the LAMP method, a high-level amplification reaction occurs when a region of a target base sequence to which a primer is to be annealed has an appropriate positional relationship and the base sequence is as designed. In other words, when the target base sequence is different from the predicted base sequence in the region that must be used as the starting point for complementary strand synthesis, the LAMP method is significantly inhibited. In particular, synthesis of a complementary strand starting from the 3 'end that anneals to itself is important. In the LAMP method, the 3 'end that anneals to itself corresponds to the 3' end of a complementary strand synthesized using the base sequence located at the 5 'end of the primer as a template. Therefore, at or near the 5 ′ end of the region (R1 or F1) located on the 5 ′ side of the first primer (RA) and / or the second primer (FA),
It is desirable to arrange a complementary base at the mutation site to be detected. Therefore, if this important sequence is designed to correspond to the mutation to be detected, by observing the aggregated products generated as the amplification reaction proceeds by the LAMP method, mutations such as deletion or insertion of a base can be observed. The presence or absence, or gene polymorphisms such as SNPs can be analyzed.

More specifically, the base where the mutation or polymorphism is expected corresponds to the vicinity of the 3 ′ end which is the starting point of the complementary strand synthesis (or the 5 ′ end if the complementary strand is the starting point). Design. That is, when any base is different from the predicted base in the region where the primer or the 3 ′ end complementary to itself anneals, complementary strand synthesis starting from the primer is prevented. Design. Here, the predicted nucleotide sequence may be a wild type or a mutant type. When a mutant type is predicted, complementary strand synthesis starts only when a specific mutation occurs. If there is a mismatch at or near the 3 'end which is the starting point of the complementary strand synthesis, the complementary strand synthesis reaction of the polynucleotide is significantly inhibited. Therefore, when an aggregated product accompanying the amplification reaction is observed, it can be determined that the target base sequence is composed of the predicted base sequence. Conversely, if the agglutination product accompanying amplification is not generated to the same degree as the control, it can be determined that the target nucleotide sequence is different from the predicted nucleotide sequence. In the LAMP method, the terminal structure in the product at the beginning of the reaction does not lead to a high amplification reaction unless the reaction is repeated.
Therefore, even if erroneous synthesis is carried out, the synthesis of the complementary strand constituting the amplification reaction is always hindered at any stage, so that a high degree of amplification does not occur if a mismatch is contained. As a result, the mismatch effectively suppresses the amplification reaction, and ultimately leads to accurate results. That is, it can be said that the amplification reaction of the polynucleotide based on the LAMP method has a more complete base sequence check mechanism. These features are simply 2
This is an advantage that cannot be expected with the PCR method or the like in which amplification reaction is performed in two regions.

Various reagents necessary for the method for detecting a polynucleotide according to the present invention can be packaged in advance and supplied as a kit. That is, the present invention provides a kit for detecting a target base sequence utilizing the presence or absence of an agglutination reaction accompanying a polynucleotide amplification reaction.
Specifically, for the present invention, a first primer and a second primer, various oligonucleotides required as an immobilized primer having a 5 ′ end immobilized on an insoluble carrier,
A kit is provided which comprises reagents such as dNTP as a substrate for complementary strand synthesis, a DNA polymerase for performing strand displacement-type complementary strand synthesis, and a buffer for providing conditions suitable for enzymatic reaction. An outer primer can be combined with the kit of the present invention. By combining the outer primer, a reaction at an isothermal temperature becomes possible. In addition, the kit for detecting a target base sequence according to the present invention includes, in addition to the outer primer, a melting temperature regulator and a pyrophosphate formation inhibitor, and further, if necessary, reagents required for detecting a synthesis reaction product. Types can be combined. In particular, in a desirable embodiment of the present invention, since it is not necessary to add a reagent during the reaction, the reagent necessary for one reaction is supplied in a state of being dispensed to the reaction vessel, and the reaction is performed only by adding the sample. Can be started. In addition, it becomes possible to detect the aggregated product accompanying the amplification reaction in the reaction vessel, and it is possible to completely eliminate the opening of the vessel after the reaction. This is very desirable in preventing contamination. Hereinafter, the present invention will be described more specifically based on examples.

[0082]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Design of Fixed Loop Primers The following primers were respectively designed based on the known LAMP method (WO 00/28082) using λDNA having the following base sequence (SEQ ID NO: 1) as a target base sequence. (ΛDNA: SEQ ID NO: 1) GCTTATCTTT CCCTTTATTT TTGCTGCGGT AAGTCGCATA AAAACCATTC TTCATAATTC AATCCATTTA CTATGTTATG TTCTGAGGGG AGTGAAAATT CCCCTAATTC GATGAAGATT CTTGCTCAAT TGTTATCAGC TATGCGCCGA CCAGAACACC TTGCCGATCA GC GC

Inner primer FA (Inner F: SEQ ID NO: 2): CGTGAGCAAT GGGTATATGC AAATAGAGCT CTGTGTTTG
T CTTCCTGC (48base), inner primer RA (Inner R: SEQ ID NO: 3): ATGTC
CTTGT CGATATAGGG ATGAAGCACA TTATCCTGAA TTTTCGGTG
(49base), outer primer F3 (Outer F: SEQ ID NO: 4): CCTGC
CTCCA AACGATACCT (20base), and outer primer R3 (Outer R: SEQ ID NO: 5): TTGTG
GTGAT ATAGGACAGA CA (22base)

Next, the following loop primers were designed so as to be in the same direction as R1 and F1 defined by the above primers. Where to design the loop primer
The direction other than the R2 or F2 region of the loop structure generated during the LAMP reaction was the same as that of R1 or F1. Note that R1 and F1 mean the same direction as the direction in which the template polynucleotide anneals to itself to perform complementary strand synthesis. The loop primer used was one whose 5 ′ end was modified with an amino group by a known method. Loop primer F (Loop F: SEQ ID NO: 6): (NH ₂ ) GGA
ACTCCGG GTGCTATCAG (20base), and loop primer R (Loop R: SEQ ID NO: 7): (NH ₂ ) TGA
CCTTTCT CTCCCATATT GCAGTCG (27base)

Next, the following primers were similarly designed using M13 DNA having the following base sequence (SEQ ID NO: 8) as a target base sequence. (M13DNA: SEQ ID NO: 8) GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCTTTGGCCG ATTCATTAAT GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TGAGCGCAAC GCAATTAATG TGAGTTAGCT CACTCATTAG GCACCCCAGG CTTTACACTT TATGCTTCCG GCTCGTATGT TGTGTGGAAT TGTGAGCGGA TAACAATTTC ACACAGGAAA CAGCTATGAC CATGATTACG AATTCGAGCT CGGTACCCGG GGATCCTCTA GAGTCGACCT GCAGGAATGC AAGCTTGGCA CTGGCCGTCG TTTTACAACG TCGTGACTGG GAAAACCCTG GCGTTACCCA ACTTAATCGC CTTGCAGCAC ATCCCCCTTT CGCCAGCTGG CGTAATAGCG AAGAGGCCCG CACCGATCGC CCTTCCCAAC AGTTGCGCAG CCTGAATGGC GAATGGCGCT TTGCCTGGTT TCCGGCACCA GAAGCGGTGC CGGAAAGCTG GCTGGAGTGC GATCTTCCTG AGGCCGATAC GGTCGTCGTC CCCTCAAACT GGCAGATGCA CGGTTACGAT GCGCCCATCT ACACCAACGT AACCTATCCC ATTACGGTCA (600base)

Inner primer FA (Inner F: SEQ ID NO: 9): CGACTCTAGA GGATCCCCGG GTACTTTTTG TTGTGTGGAA
TTGTGAGCGG AT (52base) Inner primer RA (Inner R: SEQ ID NO: 10): ACAAC
GTCGT GACTGGGAAA ACCCTTTTTG TGCGGGCCTC TTCGCTATTA
C (51base) Outer primer F3 (Outer F: SEQ ID NO: 11): ACTTT
ATGCT TCCGGCTCGT A (21base) Outer primer R3 (Outer R: SEQ ID NO: 12): GTTGG
GAAGG GCGATCG (17base)

Next, the primer is defined by the above primer.
The following loop primers were set so that R1 and F1 were in the same direction. Also, the loop primer is obtained by a known method.
An amino group-modified 5 ′ end was used. Loop primer F (Loop F: SEQ ID NO: 13): (NH ₂ ) C
ATGGTCATA GCTGTTTCCT GTGT (24base) Loop primer R (Loop R: SEQ ID NO: 14): (NH ₂ ) CA
ACTTAATC GCCTTGCAGC AC (22base)

2. Preparation of fixed loop primers For each of the above loop primers,
The 5 'end was fixed to latex particles to prepare a fixed loop primer. 10mM MES (Morpholinol Ethane sul
fonic acid) (pH6) buffer (Wako Pure Chemical Industries, Ltd.)
100 μl and 2000 pm of a carboxyl group-containing polystyrene latex having an average particle diameter of 103 nm (solid content 1% (w / w), manufactured by JSR)
ol 5'-terminal amino-modified loop primer was mixed (final volume 1 ml), and water-soluble carbodiimide EDC (1-Ethyl-3- [3-di
methylaminopropyl] carbodiimide Hydrochloride) Add the required amount of 10mg / ml aqueous solution. The required amount of the EDC aqueous solution is preferably about five times the total amount of the carboxyl groups on the surface of the carboxy latex to be used. After the addition of EDC, the reaction was carried out in a 65 ° C. warm bath for 4 hours.
The reaction was performed for 1 hour in a 5 ° C warm bath. By this reaction, the carboxyl group on the latex particles and the 5 'of the loop primer
A stable ester bond is formed between the terminal amino groups, and the excess EDC and activated carboxyl groups on the latex particles are further blocked by Glycine.

After completion of the chemical bonding reaction, latex particles were washed by repeating addition of a washing solution, removal of supernatant after ultracentrifugation (80,000 rpm / 10 minutes), and redispersion by ultrasonic treatment. . The following cleaning liquid was used. Wash solution 1: TE buffer containing 0.1% SDS (10 mM Tris / HCl pH7.
5, 1 mM EDTA) Washing solution 2: TE buffer containing 0.25 M NaCl and 0.1% SDS (10
mM Tris / HCl pH 7.5, 1 mM EDTA) Wash solution 3: sterile water Washing was performed at least twice with each wash solution. After washing
Finally, the suspension was redispersed in sterile water to a solid content of 0.25% to obtain a latex solution containing a fixed loop primer.
Stored at ° C.

3. Aggregation reaction using fixed loop primer 2. The immobilized loop primer obtained by fixing the 5 'end to latex particles obtained in the above was added to the reaction solution from the beginning of the amplification reaction of the LAMP method, and an agglutination reaction occurring after the amplification reaction was examined.

(Example 1) (Composition of λ DNA LAMP reaction solution: in 25 μl) 20 mM Tris-HCl pH 8.8 10 mM KCl 10 mM (NH ₄ ) ₂ SO ₄ .4 mM MgSO ₄ .1 M Betaine .0.1% Triton X100 ・ 0.4mM dNTPs ・ 8U Bst polymerase (NEB) ・ 1600nM λDNA Inner F (SEQ ID NO: 2) ・ 1600nM λDNA Inner R (SEQ ID NO: 3) ・ 400nM λDNA Outer F (SEQ ID NO: 4) ・ 400nM λDNA Outer R (SEQ ID NO: 4) 5) Add the following a.) To c.) To the above λDNA LAMP reaction solution,
The agglutination reaction occurring after the reaction was observed. a.) Loop primer solution [λDNA Loop F (SEQ ID NO: 6) and λDNA Loop R (SEQ ID NO: 7)] b.) Loop primer solution (λDNA Loop F and λDNA
Loop R) and unsensitized latex solution (0.25% solids) c.) Latex solution containing immobilized loop primer (λDNA
Loop F and λ DNA LoopR are respectively described in 2. above. Equivalent mixture of those fixed to latex particles by: 0.25% solids)

To the above λDNA LAMP reaction solution, add 4 μl of each of a.) To c.). Therefore, the reaction solution of b.) And c.) Contains 10 μg of latex (0.04% solid content). For comparison of Positive / Nagative, 1 × 10 ⁵ λDNA molecules were used as a Positive sample, and sterile water containing no DNA was used as a Nagative sample. Perform the reaction at 65 ° C for 90 minutes, and measure the turbidity after the
Using rospect2000, the turbidity at a wavelength of 570 nm of visible light was measured (because the amount of the reaction solution was small, each was diluted 4-fold with sterile water and used for measurement). The results are shown in FIGS.

Since the amplification reaction does not proceed in the negative sample, no increase in turbidity is observed (the reaction solution containing latex is only the turbidity of the latex), but in the positive sample containing a specific sequence, An increase in turbidity is observed in the examples (FIG. 6). The increase in turbidity in a.), Which does not contain latex, is due to the white precipitation due to magnesium pyrophosphate generated during the amplification reaction, and the increase in turbidity due to the white precipitation similarly occurs in b.) And c.). . However, when the increase in turbidity due to the white precipitation in a.) Was subtracted, no significant change in turbidity was confirmed between the positive sample and the negative sample in b.) (OD570nm / 0.089 and 0.06).
In 5) and c.), A significant increase in turbidity was confirmed (O
D570nm / 0.064 and 0.329) (Figure 7). From the above results, it can be seen that the agglutination reaction occurs with the progress of the amplification reaction only when the fixed loop primer is added to the specific LAMP reaction solution from the beginning. By the progress of the amplification reaction, a white precipitate is generated by magnesium pyrophosphate.By subtracting the increase in turbidity, it is possible to determine whether specific amplification has progressed based on the increase in turbidity due to aggregation between latexes. There is (corresponding to the white precipitate described above in Example 4).

Example 2 Confirmation of Specificity of Aggregation Reaction (1) In this example, in order to confirm the specificity of the fixed loop primer of the present invention depending on the nucleotide sequence, a single strand loop of a specific amplification product was used as a control. Example 1 using a heterogeneous fixed loop primer that does not anneal to the portion
An agglutination reaction was observed in the same manner as described above. Λ DNA LA of Example 1
4 μl of each of the following a.) To c.) Were added to the MP reaction solution, and an agglutination reaction occurring after the reaction was observed. Therefore, 10 μg of latex is contained in the reaction solution of b.) And c.).
(0.04% solids). a.) Loop primer solution [λDNA Loop F (SEQ ID NO: 6) and λDNA Loop R (SEQ ID NO: 7)] b.) Latex solution containing fixed loop primer (λDNA
Loop F and λ DNA LoopR are respectively described in 2. above. An equal mixture of those fixed to latex particles by means of: 0.25% solids) c.) Latex solution containing fixed loop primer [M13 D
NA Loop F (SEQ ID NO: 13) and M13 DNA Loop R (SEQ ID NO: 14) were each used as described in 2. above. Equivalent mixture of those fixed to latex particles by: 0.25% solids]

For comparison of Positive / Nagative, Positiv
e 1 × 10 ⁵ molecules of λDNA as sample, DNA as Nagative sample
Sterile water containing no was used as a subject. 90 minutes at 65 ° C
The turbidity at the end of the reaction was measured using a Pharmacia spectrophotometer, Ultraspect2000, at a wavelength of visible light of 570 nm. Diluted for measurement). The results are shown in FIGS. According to FIG. 8 and FIG. 9, as in Example 1, the Po
In the case of the sitive sample, the turbidity increases in all cases, including the increase in the turbidity of the white precipitate of magnesium pyrophosphate (FIG. 8). However, the increase in turbidity due to latex alone, after subtracting the increase in turbidity due to the white precipitate, is as follows. It only happens in. As can be seen in FIG. 9, in b.), 0.087 to 0.28
In contrast to c.), There was no significant change from 0.141 to 0.129.

That is, even if another amplification product is amplified at an accelerated rate, the aggregation reaction by the fixed loop primer having the specific base sequence does not occur unless the target amplification product is generated. Although not shown in the data, the reverse, that is, the addition of a fixed loop primer for M13 DNA or λ DNA to the M13 DNA LAMP reaction system, and confirmed the presence or absence of aggregation accompanying the progress of the amplification reaction,
Aggregation was observed only when the fixed loop primer for M13 DNA was added. From the above results, the agglutination reaction proceeds with very specific recognition of only the target amplification reaction. The reaction specificity is strictly defined by the base sequence of the loop primer, and the specificity is greatly affected by the base sequence used.

Example 3 Confirmation of Specificity of Agglutination Reaction 2) Simultaneously with latex agglutination reaction, a fixed loop primer for λDNA was added to λDNA LAMP reaction solution to reconfirm the amplification specificity of LAMP reaction. The following a.) ~
c.) was added to carry out the reaction. a.) Sterile water (DNA free) b.) λ DNA (1 × 10 ⁵ molecules) c.) M13 DNA (6 × 10 ⁵ molecules) In each reaction, add the λ DNA LAMP reaction solution of Example 1 4 μl of a latex solution containing a fixed loop primer for λDNA was added. Therefore, all reactions contain 10 μg of latex (0.04% solids).

That is, in this example, for comparison, Nagat
As a ive sample, sterile water without DNA was used in a.), and M13 was used in c.).
6x10 ⁵ molecules of DNA and λDNA 1x1 as a positive sample
0 ⁵ molecules, were used as subjects, respectively. 90 minutes, 65
C., and the turbidity after the completion of the reaction was measured using a Pharmacia spectrophotometer, Ultraspect2000, at a wavelength of 570 nm of visible light. And used for measurement). FIG. 10 shows the results.
Shown in After completion of the amplification, the reaction solution was electrophoresed on a 2% agarose gel, stained with SYBR-Green, and the amplification was confirmed. The results are shown in FIG.

Due to the specificity of the LAMP reaction, amplification was confirmed only with λDNA containing the target base sequence (FIG. 11).
At the same time, turbidity increase due to latex agglutination
A alone was observed, and the specificity of the agglutination reaction could also be confirmed (FIG. 10). Not shown in the data, but vice versa
That is, the reaction specificity was similarly observed in the reaction system of the M13 DNA LAMP / M13 DNA fixed loop primer. From the above results, the agglutination reaction proceeds with very specific recognition of only the target amplification reaction. In particular, even when a DNA sample containing no target base sequence is contained, neither the amplification reaction nor the agglutination reaction proceeds with the fixed loop primer alone. The reaction specificity is strictly defined by the base sequence of the loop primer, and the specificity is greatly affected by the base sequence used.

Example 4 Corresponding to White Precipitation of Magnesium Pyrophosphate In this example, the enzyme Pyrophosphatase was used as a pyrophosphate formation inhibitor, and amplification and agglutination reactions were performed by the LAMP method. As a λDNA LAMP reaction solution, a solution obtained by adding 5 mU of pyrophosphatase to the λDNA LAMP reaction solution of Example 1 is used, and 4 μl of each of a.) to c.) of Example 1 is added to the λDNA LAMP reaction solution. . Therefore,
b)) and c.) contain 10 μg of latex (0.04
% Solids). Add λDNA (1 × 10 ⁵ molecules), which is a positive sample, to all of the DNA samples,
The reaction was performed at 65 ° C, and the turbidity after the reaction was measured using a Pharmacia spectrophotometer, Ultraspect2000, at a wavelength of 570 nm of visible light. The sample was diluted and used for measurement). Figure 1 shows the results
It is shown in FIG. After completion of the amplification, the reaction solution was electrophoresed on a 2% agarose gel, stained with SYBR-Green, and the amplification was confirmed. FIG. 13 shows the results. Although a specific amplification reaction was performed in all cases (FIG. 13), white precipitate of magnesium pyrophosphate, which had been pending, was not confirmed in a.), And there was no increase in turbidity. Comparison between b.) And c.) Including latex, including fixed loop primer
c.), the turbidity was significantly increased.
It was confirmed that the use of ophosphatase had no effect on the specific agglutination reaction accompanying the progress of the amplification reaction (Fig. 1).
2).

Example 5 4 μl of each of the following a.) To d.) Was added to the λ DNA LAMP reaction solution containing the enzyme Pyrophosphatase as a pyrophosphate formation inhibitor used in Example 4. Therefore, all reactions contain 10 ug of latex (0.04% solids). a.) A latex solution containing fixed Loop F for λDNA (λDNA L
oop F as described in 2. Latex particles immobilized on latex particles: 0.25% solids) b.) Latex solution containing λDNA immobilized Loop R (λDNA L
oop R as described in 2. Immobilized on latex particles by: 0.25% solids) c.) Equivalent mixture of a.) And b.) Above d.) Fixed loop primer immobilized by mixing Loop F for λDNA and Loop R for λDNA Two types of loop primers are simultaneously immobilized on latex particles according to 2 .: 0.25% solids)

For comparison of Positive / Nagative, Positiv
e 1 × 10 ⁵ molecules of λDNA as sample, DNA as Nagative sample
Sterile water containing no was used as a subject. 90 minutes at 65 ° C
The turbidity at the end of the reaction was measured using a Pharmacia spectrophotometer, Ultraspect2000, at a wavelength of visible light of 570 nm. Diluted for measurement). FIG. 14 shows the results. Even when the fixed loop F or the fixed rule R is used alone, the agglutination reaction proceeds, although there is a slight difference in turbidity. Similarly, even when using an equal mixture of fixed loop F and fixed rule R,
The agglutination reaction proceeded without problems, but the increase in turbidity was the largest.

On the other hand, loop on the same latex
When a fixed loop primer in which F and loop R were simultaneously fixed was used, the increase in turbidity was extremely small (FIG. 1).
4). In this case, since the loop primer complementary to the base sequence extended from the loop primer fixed on the latex is present on the same latex, the chance of completing the hybridization on the same latex increases, and the It is considered that the aggregation reaction due to the binding of the latex particles hardly occurs. That is, in the process of hybridizing another fixed loop primer to the base sequence extended from the loop primer fixed on the latex, another latex particle is likely to hybridize with another latex particle to form a matrix and form an aggregate.
Compared with a.) b.) and c.), d.) has more opportunities to complete hybridization on the same latex, which is presumed to be an unsuitable condition for aggregation.

To confirm this, the same LAMP as described above was used.
In the amplification / agglutination reaction, ethidium bromide was added to the reaction solution as an intercalator, and the amplification speed was increased to AB.
Each was confirmed using 7700 company I. FIG. 15 shows the results.
Shown in As is evident from the above results, the amplification efficiency is c.)
>B.)> A.), And d.) The slowest. Probably
In d.), it is presumed that hybridization between the extended strand and the loop primer complementary thereto occurs on the same latex, and the effect of increasing the amplification efficiency inherent in the loop primer is lost in the course of the reaction.

[0105]

According to the present invention, a combination of a template polynucleotide having a specific structure and a primer immobilized on an insoluble carrier capable of providing a starting point for complementary strand synthesis at a specific position of the polynucleotide is provided. Has the following remarkable effects. (1) Since the amplification product by the LAMP reaction has a repeat of a self-complementary sequence, the amplification product causes an agglutination reaction with only one type of immobilized primer. (2) The target nucleic acid sequence in a sample can be detected very simply and quickly by improving the reaction efficiency of complementary strand synthesis and observing the agglutination reaction that occurs as the polynucleotide amplification reaction proceeds. (3) When a pyrophosphate formation inhibitor is added to the reaction system, the formation of magnesium pyrophosphate associated with the amplification reaction of the LAMP method can be prevented. Becomes possible. (4) It is not necessary to add a reagent in the course of the reaction, and it is possible to detect an agglutination product accompanying the amplification reaction in the reaction vessel, and completely abolish the opening of the vessel after the reaction. Therefore, contamination can be easily prevented. (5) Since the LAMP reaction proceeds continuously at an isothermal temperature, the change in absorbance due to the agglutination reaction is measured over time,
The presence or absence of nucleic acid amplification can be monitored in real time. The present invention enables the reaction at an isothermal temperature, suppresses mismatching during the amplification reaction, and realizes all the features of the LAMP method that the target nucleic acid sequence can be detected efficiently. The present invention provides a method for detection and a kit for detection used in the method, and is a highly practical invention.

[0106]

[Sequence List] SEQUENCE LISTING <110> Eiken Chemical Co., Ltd. <120> A Method for Detecting Polynucleotide and a kit therefor <130> <140> <141> <160> 14 <170> PatentIn Ver. 2.1 <210 > 1 <211> 172 <212> DNA <213> Bacteriophage lambda <400> 1 gcttatcttt ccctttattt ttgctgcggt aagtcgcata aaaaccattc ttcataattc 60 aatccattta ctatgttatg ttctgagggg agtgaaatt cccctaattc gatgaagcatgccattg 120cc > DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 2 cgtgagcaat gggtatatgc aaatagagct ctgtgtttgt cttcctgc 48 <210> 3 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 3 atgtccttgt cgatataggg atgaagcaca ttatcctgaa ttttcggtg 49 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer seque nce <400> 4 cctgcctcca aacgatacct 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 5 ttgtggtgat ataggacaga ca 22 < 210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 6 ggaactccgg gtgctatcag 20 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 7 tgacctttct ctcccatatt gcagtcg 27 <210> 8 <211> 600 <212> DNA <213> <400> 8 gcgcccaata cgcaaaccgc ctctccccgc gctttggccg attcattaat gcagctggca 60 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct 240 cggtacccgg ggatcctcta gagtcgacct gcaggaatgc aagcttggca ctggccgtcg 300 ttttacaacg tcgtgactgg gaaaaccc tg gcgttaccca acttaatcgc cttgcagcac 360 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 420 agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc 480 cggaaagctg gctggagtgc gatcttcctg aggccgatac ggtcgtcgtc ccctcaaact 540 ggcagatgca cggttacgat gcgcccatct acaccaacgt aacctatccc attacggtca 600 <210> 9 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 9 cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg at 52 <210> 10 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 10 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta c 51 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 11 actttatgct tccggctcgt a 21 <210> 12 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Descr iption of Artificial Sequence: an artificially synthesized primer sequence <400> 12 gttgggaagg gcgatcg 17 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 13 catggtcata gctgtttcct gtgt 24 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: an artificially synthesized primer sequence <400> 14 caacttaatc gccttgcagc ac 22

[Brief description of the drawings]

FIG. 1 is a diagram showing the reaction principle of a polynucleotide amplification method using the present invention. In the figure, FA: primer for LAMP method FA RA: primer for LAMP method RA

FIG. 2 is a diagram showing a reaction principle of a polynucleotide amplification method using the present invention. The abbreviations in the figure have the same meaning as in FIG.

FIG. 3 is a diagram showing the reaction principle of a polynucleotide amplification method using the present invention. loop F: Fixed loop primer F loop R: Fixed loop primer R

FIG. 4 is a diagram showing a reaction principle of a polynucleotide amplification method using the present invention. The abbreviations in the figure have the same meaning as in FIG.

FIG. 5 is a view showing a preferable positional relationship between a loop primer and a base sequence constituting a template polynucleotide.

FIG. 6 is a view showing the results of measuring the turbidity of a reaction solution obtained by performing a λDNA LAMP reaction in Example 1 at a wavelength of 570 nm of visible light. a.) When an unfixed loop primer is used. b.) When unfixed loop primer and unsensitized latex solution are used. c.) When a latex solution containing a fixed loop primer is used.

FIG. 7 is a view showing the result of measuring the turbidity of a reaction solution obtained by performing a λDNA LAMP reaction in Example 1 at a wavelength of 570 nm of visible light. A value obtained by subtracting an increase in turbidity due to magnesium pyrophosphate formed as the amplification reaction proceeds.

FIG. 8 is a view showing the results of measuring the turbidity of a reaction solution obtained by performing a λDNA LAMP reaction in Example 2 at a wavelength of 570 nm of visible light. a.) When an unfixed loop primer is used. b.) When a latex solution containing a fixed loop primer for λDNA is used. c.) When a latex solution containing a fixed loop primer for M13 DNA is used.

FIG. 9 is a view showing the result of measuring the turbidity of a reaction solution obtained by performing a λDNA LAMP reaction in Example 2 at a wavelength of 570 nm of visible light. A value obtained by subtracting an increase in turbidity due to magnesium pyrophosphate formed as the amplification reaction proceeds.

FIG. 10 is a view showing the result of measuring the turbidity of a reaction solution obtained by performing a λDNA LAMP reaction in Example 3 at a wavelength of 570 nm of visible light. a.) When using sterile water (DNA free) as the sample. b.) When λ DNA (1 × 10 ⁵ molecules) is used as a sample. c.) When M13 DNA (6 × 10 ⁵ molecules) is used as a sample.

FIG. 11 is a diagram showing the results of electrophoresis of a reaction solution after completion of amplification in Example 3 on a 2% agarose gel and staining with SYBR-Green.

FIG. 12 is a graph showing the results of measuring the turbidity of a reaction solution obtained by performing a λDNA LAMP reaction in Example 4 at a wavelength of 570 nm of visible light. a.) When an unfixed loop primer is used. b.) When unfixed loop primer and unsensitized latex solution are used. c.) When a latex solution containing a fixed loop primer is used.

FIG. 13 is a diagram showing the results of electrophoresis of a reaction solution after completion of amplification in Example 4 on a 2% agarose gel and staining with SYBR-Green.

FIG. 14 is a diagram showing the results of measuring the turbidity of a reaction solution obtained by performing a λDNA LAMP reaction in Example 5 at a wavelength of 570 nm of visible light. a.) When a latex solution containing immobilized Loop F for λDNA is used. b.) When a latex solution containing immobilized Loop R for λDNA is used. c.) When an equal mixture of a.) and b.) above is used. d.) When using a fixed loop primer in which Loop F for λDNA and Loop R for λDNA are mixed and fixed.

[FIG. 15] In Example 5, ethidium bromide was added as an intercalator to the reaction solution, and the amplification rate was reduced by ABI 7
The figure which shows the result of having confirmed each using 700.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA11 AA20 CA01 CA11 CA20 HA08 HA14 4B063 QA01 QQ42 QQ52 QR08 QR13 QR32 QR41 QR49 QR62 QR83 QS24 QX01

Claims (21)

[Claims] 1. A method for detecting a target nucleic acid sequence in a sample, comprising mixing and incubating the following elements [1] to [5], and observing the presence or absence of an agglutination reaction accompanying the amplification reaction of the polynucleotide. how to. [1]: a template polynucleotide having the following conditions (a) to (d); (a) a complementary polynucleotide of (b) and (a) having a target base sequence consisting of at least one set of complementary base sequences; When the base sequence hybridizes, it can form a loop capable of base pairing, (c) the 3 'end can anneal to itself to form a loop, (d) anneal to itself The 3 'end can serve as a starting point for complementary strand synthesis using itself as a template [2]: at least two types of primers capable of providing complementary strand synthesis starting points at different positions in a loop of a template polynucleotide, [ 3]: In a template polynucleotide and / or an extension product generated by annealing a primer of [2] to a template polynucleotide, a starting point for complementary strand synthesis can be provided at a position different from that of the primer of [2]. And at least one primer that, the 5 'end of which is immobilized on an insoluble carrier (anchor primer), [4]: DNA polymerase complementary strand synthesis accompanying strand displacement can catalyze,
[5]: a substrate for complementary strand synthesis, 2. The loop according to claim 1, wherein the immobilized primer [3] is a template polynucleotide and / or a loop formed by an extension product generated by annealing the primer [2] to the template polynucleotide. The method according to claim 1, wherein is a primer (fixed loop primer) capable of providing a starting point for complementary strand synthesis at a different position. 3. The method according to claim 1, wherein the insoluble carrier is a natural polymer carrier, a synthetic polymer carrier, a metal colloid, or a magnetic particle. 4. The method according to claim 3, wherein the insoluble carrier is a latex of a synthetic polymer carrier. 5. The method according to claim 1, wherein the template polynucleotide has a base sequence complementary to an arbitrary region of its own base sequence at its 5 ′ end. 6. The method according to claim 1, wherein the template polynucleotide is produced by the following steps. a) annealing a first primer to a target base sequence;
A step of conducting a complementary strand synthesis reaction using this as a starting point; wherein the first primer gives a starting point of complementary strand synthesis to a region defining the 3 ′ side of one of the strands constituting the target base sequence at its 3 ′ end And a base sequence complementary to any region of the complementary strand synthesis reaction product starting from this primer is provided on the 5 ′ side of the first primer. B) Step a) B) making the region to be annealed by the second primer in the extension product starting from the first primer synthesized in the step (1) ready for base pairing; 3 ′ of the target base sequence in the extension product starting from the first primer
A base sequence capable of providing a starting point for complementary strand synthesis in a region defining the side, and a 5 ′ side of the second primer is provided with an optional region of a complementary strand synthesis reaction product starting from this primer. C) a step of annealing a second primer to the region where the base pairing has become possible in step b) and synthesizing a complementary strand starting from the second primer, d) ) Annealing the 3 'end of the extension product starting from the second primer synthesized in step c) to itself,
Step of performing complementary strand synthesis using itself as a template 7. The method according to claim 6, wherein the two kinds of primers [2] are a first primer and a second primer. 8. The method according to claim 1, wherein the immobilized primer [3] comprises a complementary strand synthesized between a region derived from the first primer in the extension product starting from the first primer and the arbitrary region with respect to the first primer. And / or between a region derived from the second primer in the extension product originating from the second primer and said optional region for the second primer. The method according to claim 7, comprising a second immobilized primer capable of providing a starting point for complementary strand synthesis. 9. In the step b) and / or the step c), the first primer or the second primer is subjected to complementary strand synthesis from an outer primer which provides a starting point for complementary strand synthesis upstream thereof. The method according to claim 4, wherein the extension products originating from the primer and / or the second primer are substituted to make each product originating from the first primer or the second primer single-stranded. 10. In step a), the target base sequence is present as a double-stranded polynucleotide, and the region where the first primer anneals is subjected to base pair binding by a complementary strand synthesis reaction starting from an arbitrary primer. 5. The method according to claim 4, wherein the state is enabled. 11. The method according to claim 10, wherein step a) is performed in the presence of a melting temperature modifier. 12. The melting temperature controlling agent is betaine, proline, dimethyl sulfoxide, and trimethylamine N.
The method according to claim 11, which is at least one compound selected from -oxide. 13. The method according to claim 1, wherein the amplification reaction of the polynucleotide is carried out in the presence of a pyrophosphate formation inhibitor. 14. The method of claim 1, wherein the pyrophosphate formation inhibitor is Pyrophosphat.
14. The method according to claim 13, which is ase. 15. A method for detecting a mutation in a target base sequence by the method according to any one of claims 1 to 14, wherein the amplification method is carried out when the target base sequence is not a predicted base sequence. At least one complementary strand synthesis reaction selected from the constituent complementary strand synthesis reactions is hindered, and whether or not an agglutination reaction accompanying the amplification reaction has occurred is observed. Is determined to be not the predicted nucleotide sequence. 16. A method for detecting a mutation in a target base sequence by the method according to any one of claims 1 to 14, wherein an observation is made as to whether an agglutination reaction accompanying the amplification reaction has occurred. And determining that the target base sequence is a predicted base sequence when the target base sequence is detected. 17. A kit for detecting a target nucleotide sequence, comprising the following elements: a) a first primer; wherein the first primer is
A starting point for complementary strand synthesis can be given to a region defining the 3 ′ side of one of the strands constituting the target base sequence at the 3 ′ end, and this primer is used as the starting point on the 5 ′ side of the first primer. B) a second primer; wherein the second primer is the second primer;
At the 3 ′ end, a base sequence capable of providing a starting point for complementary strand synthesis to a region defining the 3 ′ side of the target base sequence in the extension product starting from the first primer,
And the 5 ′ side of the second primer is provided with a base sequence complementary to an arbitrary region of a complementary strand synthesis reaction product starting from the primer. C) At least one type of 5 ′ An immobilized primer having an end immobilized on an insoluble carrier; wherein the immobilized primer provides a starting point for complementary strand synthesis at a position different from each primer in an extension product starting from the first primer or the second primer D) a DN capable of catalyzing complementary strand synthesis with strand displacement
A polymerase, e) substrate for complementary strand synthesis, f) pyrophosphate formation inhibitor, 18. The immobilized primer is located at a position different from each primer in a loop formed by the template polynucleotide and / or an extension product generated by annealing the first or second primer to the template polynucleotide. The kit according to claim 17, which is a primer (fixed loop primer) capable of giving a starting point for complementary strand synthesis to the kit. 19. The method according to claim 19, wherein the immobilized primer c) is used for complementary strand synthesis between a region derived from the first primer in the extension product starting from the first primer and the arbitrary region relative to the first primer. A first fixed primer capable of providing an origin; and complementary strand synthesis between a region derived from the second primer in the extension product originating from the second primer and any of the regions for the second primer. The kit according to claim 17 or 18, comprising a second immobilized primer capable of providing a starting point for the primer. 20. The method according to claim 17, further comprising the following element g):
The kit according to any one of claims 9 to 13. g) outer primer; the outer primer can provide a starting point for complementary strand synthesis upstream of the first primer and / or the second primer. 21. The method according to claim 20, wherein the pyrophosphate formation inhibitor is Pyrophosphat.
The kit according to any one of claims 17 to 20, which is ase.