JP2010088355A - Primer reagent and method for amplifying nucleic acid by using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for amplifying a nucleic acid by which a target sequence can be amplified in excellent amplification efficiency. <P>SOLUTION: A PCR is carried out by using at least two kinds of primers satisfying the following condition (I): one primer can hybridize to the 3' side from a region to which the other primer can hybridize in a template nucleic acid, and the Tm value of one primer is lower than the Tm value of the other primer. When the primers satisfying the condition are used, the target sequence in the template nucleic acid can be amplified in the excellent efficiency in the same reaction system. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a primer reagent for use in a nucleic acid amplification method of a target sequence by polymerase chain reaction, and a nucleic acid amplification method using the same.

  In various fields such as clinical practice and pathology, polymerase chain reaction (PCR) is widely used for the purpose of gene expression analysis, functional analysis, diagnosis, and the like. PCR is generally performed by (1) dissociation of a double-stranded nucleic acid from a single-stranded nucleic acid by heat treatment, (2) annealing of a primer to a single-stranded nucleic acid, and (3) extension of the primer using a polymerase. This is a method of amplifying a target sequence by setting one step as one cycle and repeating this cycle. And in PCR, in order to improve amplification efficiency, the method (patent document 1) etc. which add glycerol, formaldehyde, etc. to a PCR reaction liquid are proposed, for example. According to such a method, a maximum of twice as many nucleic acids can be obtained per cycle.

However, in recent years, it has been desired to provide improved PCR with excellent amplification efficiency capable of replicating nucleic acid twice or more per cycle.
JP 2003-144169 A

  Accordingly, an object of the present invention is to provide a primer reagent and a nucleic acid amplification method for amplifying a target sequence with excellent amplification efficiency.

In order to achieve the above object, the primer reagent of the present invention amplifies the target sequence in the template nucleic acid by PCR in the same reaction system using a plurality of primers capable of hybridizing on the same strand of the template nucleic acid. A primer reagent for use in the nucleic acid amplification method, comprising at least two kinds of primers, wherein any two kinds of primers satisfy the following condition (I).
(I) One primer can hybridize to the 3 ′ side of the template nucleic acid relative to the region where the other primer of the template nucleic acid can hybridize, and the Tm value of the one primer Lower than the Tm value of the primer

  The nucleic acid amplification reagent of the present invention is a nucleic acid amplification method in which a target sequence in a template nucleic acid is amplified by PCR in the same reaction system using a plurality of primers capable of hybridizing on the same strand of the template nucleic acid. A reagent for nucleic acid amplification for use in the present invention, comprising the primer reagent of the present invention.

  The nucleic acid amplification method of the present invention is a nucleic acid amplification method in which a target sequence in a template nucleic acid is amplified by PCR in the same reaction system using a plurality of primers capable of hybridizing on the same strand of the template nucleic acid. The primer reagent of the present invention is used.

  In the method for producing a nucleic acid amplification product of the present invention, a target sequence in a template nucleic acid is amplified by PCR in the same reaction system using a plurality of primers capable of hybridizing on the same strand of the template nucleic acid. A method for producing a product, wherein the amplification of the target sequence is performed by the nucleic acid amplification method of the present invention.

  According to the primer reagent of the present invention, by using two or more kinds of primers that satisfy the conditions described above, the target sequence can be amplified more efficiently in each stage than in conventional PCR in the same reaction system. Is possible. In addition, since various primers in the primer reagent of the present invention can be designed so that the Tm value satisfies the above condition (I), for example, the primer preparation itself can be performed very simply. For this reason, nucleic acid amplification by PCR becomes possible simply and with excellent amplification efficiency.

<Primer reagent>
As described above, the primer reagent of the present invention uses a plurality of primers capable of hybridizing the target sequence in the template nucleic acid on the same strand of the template nucleic acid, and amplifies the nucleic acid by PCR in the same reaction system. A primer reagent for use in the method, comprising at least two kinds of primers, wherein any two kinds of primers satisfy the following condition (I).
(I) One primer can hybridize to the 3 ′ side of the template nucleic acid relative to the region where the other primer of the template nucleic acid can hybridize, and the Tm value of the one primer Lower than the Tm value of the primer

  The primer reagent of the present invention can be used for the nucleic acid amplification method of the present invention and the method for producing a nucleic acid amplification product as described later. A specific method will be described later. In addition, the higher the Tm value, the higher the binding property to the template nucleic acid.

  The primer reagent of this invention should just contain at least 2 types of primer which satisfy | fills said condition (I), and other conditions and structures are not restrict | limited at all. The primer reagent may include, for example, only two types of primers that satisfy the condition (I) as a primer, or may include three or more types of primers. In the latter case, at least two of the primers may be primers satisfying the condition (I), and two kinds of primers satisfying the condition (I) can be arbitrarily set. Specific examples of such primer reagents include those containing a first primer and a second primer. For example, the first primer and the second primer can hybridize on the same strand of a template nucleic acid, and the second primer has a higher template content than the region of the template nucleic acid to which the first primer can hybridize. Hybridization is possible on the 3 ′ side of the nucleic acid, and the Tm value of the second primer is lower than the Tm value of the first primer. Since each primer only needs to be able to amplify the target sequence of the template nucleic acid, the region to which the first primer can hybridize is, for example, a region containing the entire target sequence in the template nucleic acid, A 3 ′ region, a region including the 3 ′ region, or a region downstream (3 ′ side) from the target sequence is preferable. As described above, the region where the second primer can hybridize is a region 3 ′ side of the template nucleic acid with respect to the region where the first primer can hybridize. It may overlap with the hybridizable region.

  When the primer reagent of the present invention includes three or more kinds of primers, as described above, at least two kinds of primers may satisfy the above-mentioned conditions. ) Is preferably satisfied. That is, it is preferable that each primer has a lower Tm value as the hybridization region to the template nucleic acid is on the 3 ′ side than the primer that hybridizes on the 5 ′ side with respect to the hybridization region. Specific examples of such a primer reagent include those containing a third primer in addition to the first primer and the second primer. The first, second and third primers can be hybridized, for example, on the same strand of a template nucleic acid, and the third primer is more than the region of the template nucleic acid to which the second primer can hybridize, The template nucleic acid can hybridize to the 3 ′ side, and the Tm value of the third primer is preferably lower than the Tm value of the second primer. That is, the first, second, and third primers, for example, hybridize to the template nucleic acid in this order from the 5 ′ side to the 3 ′ side of the template nucleic acid. It is preferable that the relationship of primer> second primer> third primer is satisfied. As described above, the region to which the third primer can hybridize is a region 3 ′ side of the template nucleic acid from the region to which the second primer can hybridize. The primer may overlap with the hybridizable region.

  Further, when the fourth primer is included, the Tm value of the fourth primer can be hybridized to the 3 ′ side of the template nucleic acid with respect to the region where the third primer of the template nucleic acid can hybridize. Is preferably lower than the Tm value of the third primer. As described above, the number of types of primers included in the primer reagent of the present invention may be two or more, and is not limited. However, in the template nucleic acid, the Tm value of the primer that can hybridize to the 3 ′ side is more than 5. It is preferably lower than the Tm value of the primer that can hybridize to the side.

  In the present invention, the template nucleic acid may be, for example, a single-stranded nucleic acid or a double-stranded nucleic acid. In the case of a double-stranded nucleic acid, for example, a primer capable of hybridizing only to either the sense strand or the antisense strand constituting the double-stranded nucleic acid may be used, but a forward primer capable of hybridizing to the antisense It is preferable to use a primer set including a reverse primer capable of hybridizing to the sense strand. Below, the form containing a primer set is demonstrated about the primer reagent of this invention. The forward primer is also called a sense primer, and the reverse primer is also called an antisense primer.

  When the template nucleic acid is a double-stranded nucleic acid comprising a sense strand and an antisense strand, the primer reagent of the present invention comprises a forward primer that can hybridize to the antisense strand and a reverse primer that can hybridize to the sense strand. It is preferable to include at least two types of primer sets. In any two types of primer sets among the primer sets, each forward primer and each reverse primer are preferably primers that satisfy the condition (I).

  The primer reagent of the present invention may include, for example, only two types of primer sets having a forward primer and a reverse primer that satisfy the condition (I), or may include three or more types of primer sets. In the latter case, at least two of the primer sets are preferably primer sets having a forward primer and a reverse primer that satisfy the condition (I), and a primer set that satisfies these conditions can be arbitrarily set. .

  Specific examples of such primer reagents include those containing a first primer set and a second primer set. The first primer set includes, for example, a first forward primer (hereinafter referred to as “F1 primer”) and a first reverse primer (hereinafter referred to as “R1 primer”), and the second primer set includes, for example, a second forward primer. A primer (hereinafter referred to as “F2 primer”) and a second reverse primer (hereinafter referred to as “R2 primer”). The F2 primer of the second primer set can hybridize to the antisense strand 3 'to the region where the F1 primer of the first primer set can hybridize, and the Tm value of the F2 primer is: The Tm value of the R2 primer is lower than the Tm value of the F1 primer, and the R2 primer can hybridize to the 3 ′ side of the region where the R1 primer can hybridize in the sense strand. It is preferably lower than the Tm value of the R1 primer. The F1 primer and the F2 primer and the R1 primer and the R2 primer correspond to the first primer and the second primer, respectively. From the relationship of the hybridizing region to the template nucleic acid, the first primer set can be referred to as, for example, an inner primer set, and the second primer set can be referred to as, for example, an outer primer set.

  When the primer reagent of this invention contains three or more types of primer sets, it is preferable that a forward primer and a reverse primer respectively satisfy | fill the said conditions (I) between each primer sets, for example. Specific examples of such a primer reagent include those containing a third primer set in addition to the first primer set and the second primer set. The third primer set includes, for example, a third forward primer (hereinafter referred to as “F3 primer”) and a third reverse primer (hereinafter referred to as “R3 primer”). The F3 primer of the third primer set can hybridize to the 3 ′ side of the region where the F2 primer can hybridize in the antisense strand, and the Tm value of the F3 primer is the Tm value of the F2 primer. Is preferably lower. In addition, the R3 primer of the third primer set can hybridize 3 ′ to the region where the R2 primer can hybridize in the sense strand, and the Tm value of the R3 primer is the Tm value of the R2 primer. Preferably it is lower than the value. The F3 primer and the R3 primer respectively correspond to the above-described third primer. From the relationship of the hybridizing region with respect to the template nucleic acid, the third primer set can also be referred to as an outer primer set with respect to the second primer set.

  Further, the same applies when the fourth primer set is included. The fourth primer set includes, for example, a fourth forward primer (hereinafter referred to as “F4 primer”) and a fourth reverse primer (hereinafter referred to as “R4 primer”). The F4 primer of the fourth primer set can hybridize 3 ′ to the region where the F3 primer can hybridize in the antisense strand, and the Tm value of the F4 primer is the Tm value of the F3 primer. Is preferably lower. In addition, the R4 primer of the fourth primer set can hybridize 3 ′ to the region where the R3 primer can hybridize in the sense strand, and the Tm value of the R4 primer is the Tm value of the R3 primer. Preferably it is lower than the value. Each of the F4 primer and the R4 primer corresponds to the aforementioned fourth primer. From the relationship of the hybridizing region with the template nucleic acid, the fourth primer set can also be referred to as an outer primer set with respect to the third primer set. As described above, the kind of the primer set included in the primer reagent of the present invention is not particularly limited, but the primer set capable of hybridizing to the 3 ′ side (more outside) in the template nucleic acid (sense strand and antisense strand). The Tm values of the forward primer and the reverse primer are preferably lower than the Tm values of the forward primer and the reverse primer of the primer set capable of hybridizing to the 5 ′ side, respectively.

  In the present invention, the constituent components of each primer are not particularly limited, and examples thereof include DNA, RNA, nucleic acids such as LNA that is an RNA analog, PNA that is a peptide nucleic acid, and artificial nucleic acids such as BNA that is a cross-linked nucleic acid. It is done.

  The Tm value will be described. When a solution containing a double-stranded nucleic acid (for example, double-stranded DNA) is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both strands in the double-stranded nucleic acid are unwound by heating and dissociated (melted) into single-stranded nucleic acid (for example, single-stranded DNA). When all the double-stranded nucleic acids are dissociated into single-stranded nucleic acids, the absorbance is about 1.5 times the absorbance at the start of heating (absorbance of only double-stranded nucleic acids), thereby melting. It can be judged that it has been completed. Based on this phenomenon, the melting temperature Tm is generally defined as the temperature at which the absorbance reaches 50% of the total increase in absorbance.

  The method for setting the Tm value of the primer is not particularly limited, and a conventionally known method can be adopted. As a specific example, it can adjust with the length of each primer, and GC content, for example. When adjusting by length, generally, the shorter the primer, the lower the Tm value can be set. For example, when using the first primer and the second primer as described above, it is preferable to set the length of the second primer to be shorter than the first primer. Thereby, the Tm value of the second primer can be set to a value relatively lower than the Tm value of the first primer. Moreover, when adjusting with GC content, for example, Tm value can be set relatively low, so that GC content is relatively low. For example, when using the first primer and the second primer as described above, it is preferable to set the GC content of the second primer lower than that of the first primer. Moreover, Tm value can also be set with both the length of a primer, and GC content. In addition to these, for example, the first primer is a sequence containing an artificial nucleic acid such as LNA, PNA, or BNA described above, or a sequence containing a larger number than the second primer. By using a sequence that does not contain a nucleic acid or a sequence that contains less than the first primer, the Tm value of the first primer can be set relatively high and the Tm value of the second primer can be set relatively low. it can.

  In addition, the primer sequence can be designed in consideration of the Tm value by, for example, the conventionally known MELTCALC software (http://www.meltcalc.com/) or the nearest base pair method (Nearest Neighbor Method). . For primers designed using such software or the like, for example, it is preferable to confirm the Tm value under the conditions of PCR actually performed.

  The difference in Tm value between primers is not particularly limited, but the difference in Tm value between primers adjacent to the hybridizing region in the template nucleic acid (for example, the difference in Tm value between the first primer and the second primer, the second primer) The difference between the Tm value and the third primer is preferably 0.1 to 20 ° C., more preferably 1 to 15 ° C., and particularly preferably 5 to 10 ° C.

  Although the length in particular of each primer is not restrict | limited, For example, it is 5-50 bases, Preferably it is 10-40 bases, More preferably, it is 15-30 bases. Each primer is preferably complementary to the template nucleic acid so that it can hybridize with the template nucleic acid. Specifically, 80% or more of the primer sequence is preferably complementary, more preferably It is 90% or more, and particularly preferably 100%.

  In the primer reagent of the present invention, the ratio of each primer is not particularly limited. For example, the molar ratio of a primer having a relatively high Tm value and a primer having a relatively low Tm value is 1: 0.01 to A range of 1: 100 is preferable, a range of 1: 0.1 to 1:10 is more preferable, and a range of 1: 0.5 to 1: 2 is particularly preferable. As a specific example, the molar ratio (X: Y) between the first primer (X) and the second primer (Y) is preferably in the range of 1: 0.01 to 1: 100, for example. Preferably, it is in the range of 1: 0.1 to 1:10, and particularly preferably in the range of 1: 0.5 to 1: 2. Further, when the third primer is included, the molar ratio (X: Z) between the first primer (X) and the third primer (Z) is, for example, 1: 0.01 to 1: 100. It is preferably in the range, more preferably in the range of 1: 0.1 to 1:10, and particularly preferably in the range of 1: 0.5 to 1: 2. When the forward primer and the reverse primer are included, the molar ratio (F: R) of the forward primer (F) and the reverse primer (R) is, for example, in the range of 1: 0.01 to 1: 100. Is preferable, more preferably in the range of 1: 0.1 to 1:10, and particularly preferably in the range of 1: 0.5 to 1: 2.

  The form of the primer reagent of the present invention is not particularly limited, and may be, for example, a dry system (dry body) or a wet system (liquid). In the reagent for nucleic acid amplification of the present invention, for example, each primer may be stored separately in a container or the like, but since it is used in the same reaction system, it must be stored in the same container in a mixed state. Is preferred. The primer of the present invention may be, for example, a primer kit, and may further include, for example, instructions for use and other components described later necessary for PCR.

<Reagent for nucleic acid amplification>
The nucleic acid amplification reagent of the present invention is used in a nucleic acid amplification method in which a target sequence in a template nucleic acid is amplified by PCR in the same reaction system using a plurality of primers capable of hybridizing on the same strand of the template nucleic acid. It is a nucleic acid amplification reagent for carrying out, It is characterized by including the primer reagent of this invention. The nucleic acid amplification reagent of the present invention only needs to contain the primer reagent of the present invention, and other configurations are not limited at all, and the usage method is also the same.

  The form of the reagent for nucleic acid amplification of the present invention is not particularly limited, for example, similarly to the primer reagent of the present invention, and may be, for example, a dry system (dry body) or a wet system (liquid). When the nucleic acid amplification reagent of the present invention is a dry system, it is preferable to add an appropriate solvent to the nucleic acid amplification reaction at the time of use. In addition, when the nucleic acid amplification reagent of the present invention is, for example, a wet system and the concentration of the primer is higher than the concentration in the reaction system at the time of use as described later, an appropriate solvent or template nucleic acid is used. It is preferable to use the nucleic acid amplification reaction after adding a solvent or the like. In the nucleic acid amplification reagent of the present invention, each primer may be stored separately in a container or the like, for example, but since it is used in the same reaction system, it is stored in the same container in a mixed state. Preferably it is.

The reagent for nucleic acid amplification of the present invention may further contain, for example, other components necessary for nucleic acid amplification in addition to the primers described above. The other components are not particularly limited and include conventionally known components, and the ratios thereof are not particularly limited. Examples of the other components include polymerases, nucleoside triphosphates (dNTPs), buffers, solvents such as water and buffer solutions, and the like. In addition, for example, glycerol, heparin, betaine, KCl, MgCl ₂ , MgSO ₄ , glycerol and the like may be included.

The polymerase is not particularly limited, and examples thereof include polymerases conventionally used for PCR and the like. As the polymerase, for example, a polymerase derived from a thermostable bacterium is preferable. Specific examples include DNA polymerases derived from Thermus aquaticus (US Pat. Nos. 4,889,818 and 5,079,352). No.) (trade name Taq polymerase), DNA polymerase derived from Thermus thermophilus (WO 91/09950) (rTth DNA polymerase), DNA polymerase derived from Pyrococcus furiosus (WO 92f / 89) polymerase: manufactured by Stratagenes, Thermococcus litoralis lis) -derived DNA polymerase (EP-A 455 430) (Trademark Vent: New England Biolabs). The polymerase is preferably, for example, a polymerase that lacks 5 ′ → 3 ′ exonuclease activity. Thus, according to the polymerase lacking 5 ′ → 3 ′ exonuclease activity, for example, cleavage of the primer by the polymerase can be sufficiently avoided. Examples of the polymerase lacking the 5 ′ → 3 ′ exonuclease activity as described above include, for example, Gene Taq (Nippon Gene), TITANIUM Taq (Clontech), ΔTth (Toyobo) and the like. The polymerase preferably has a strand displacement activity.

  Examples of the nucleoside triphosphate (dNTP) include dATP, dCTP, dGTP, dTTP, and dUTP, and it is preferable to use a mixture thereof.

  Examples of the buffer include buffers such as Tris-HCl, Tricine, MES, MOPS, HEPES, and CAPS. Examples of the solvent include water, a buffer solution containing the buffer, and the like. In the buffer solution, for example, pH and the concentration of the buffering agent to be contained are not particularly limited, and conventionally known conditions can be applied.

  The nucleic acid amplification reagent of the present invention may be, for example, a nucleic acid amplification kit, and may further include instructions for use, for example.

<Nucleic acid amplification method>
The nucleic acid amplification method of the present invention is a nucleic acid amplification method in which a target sequence in a template nucleic acid is amplified by PCR in the same reaction system using a plurality of primers capable of hybridizing on the same strand of the template nucleic acid. The primer reagent of the present invention is used as a primer. In the present invention, the primer reagent of the present invention described above is used as a primer, and other conditions are not limited at all. For example, a nucleic acid can be amplified by conventional PCR.

The nucleic acid amplification method of the present invention preferably includes, for example, the following steps (A) to (C).
(A) heating the reaction system containing the template nucleic acid and the primer reagent to make the template nucleic acid single-stranded (B) lowering the temperature of the reaction system, Step of performing primer annealing and extension reaction from the primer (C) Step of repeating the steps (A) and (B)

  In the present invention, the template nucleic acid at the start of PCR, that is, the template nucleic acid in the first step (A) may be, for example, a single-stranded nucleic acid or a double-stranded nucleic acid. When the template nucleic acid in the first step (A) is double stranded, the double strand is dissociated into single strands by heating. Therefore, in step (B), primer annealing to the single strand is performed. It becomes possible. In addition, when the template nucleic acid at the start of PCR is single-stranded, the first step (A) can be omitted, for example. It is preferable to dissociate the primer annealed to the template nucleic acid of the strand from the template nucleic acid.

  On the other hand, in step (A) after the second time of step (C), the template nucleic acid before heating is usually a double-stranded nucleic acid. Regardless of whether the template nucleic acid in the first step (A) is a single-stranded nucleic acid or a double-stranded nucleic acid, the single-stranded template nucleic acid was annealed to the single-stranded template nucleic acid by the step (B). This is because a double-stranded nucleic acid is formed from the extended strand of the primer. The double-stranded nucleic acid formed in the step (B) subsequently becomes a template nucleic acid in the step (A), and is dissociated into single strands by heat treatment.

  In the step (B), the temperature of the reaction system is lowered, and annealing of the primer to the dissociated single-stranded template nucleic acid and extension reaction from the primer are performed. Various primers in the present invention undergo annealing and extension reactions preferentially in the order of primers having relatively high Tm values. A specific mechanism will be described later.

  In the step (C), the number of repetitions of the step (A) and the step (B) is not particularly limited, but when the step (A) and the step (B) are 1 cycle, a total of 10 to 100 cycles is preferable. More preferably, the total is 30 to 50 cycles. According to the nucleic acid amplification method of the present invention, since the amplification efficiency is excellent as described above, for example, the number of cycles is significantly smaller (for example, 28 cycles) than the conventional cycle number (for example, 37 to 38 cycles). The same level of amplification is possible.

  For the nucleic acid amplification method of the present invention, a primer reagent comprising the first primer set and the second primer set is used as a primer reagent of the present invention, and amplification of a target sequence is performed using a double-stranded nucleic acid as a template. An example will be described. In addition, the primer reagent used by this invention should just be the primer reagent of this invention, and is not restrict | limited to the primer reagent containing a 1st and 2nd primer set. Further, the nucleic acid amplification method of the present invention is not limited to this embodiment.

  First, a first primer set including the aforementioned F1 primer and R1 primer and a second primer set including the F2 primer and R2 primer are prepared. Then, these primer set and double-stranded template nucleic acid are mixed in a solvent to prepare a reaction system. The template nucleic acid is not particularly limited, and examples thereof include double-stranded DNA. As the double-stranded DNA, for example, double-stranded DNA originally contained in a sample such as a biological sample or an environmental sample may be used, or from RNA such as total RNA or mRNA, for example, RT-PCR (Reverse Transcription PCR). )) And the like synthesized by reverse transcription reaction.

  In addition to the first and second primer sets, the reaction system preferably contains a polymerase, dNTP, etc. as described above. As the solvent, water, various buffer solutions, commercially available PCR buffer solutions, commercially available PCR kit buffer solutions and the like can be used as described above.

  The ratio of each primer set in the reaction system is not particularly limited, and examples thereof include the following concentrations. In the reaction system, the final concentration of the F1 primer is preferably, for example, 0.1 to 10 μmol / L, more preferably 0.25 to 2 μmol / L, and the final concentration of the R1 primer is, for example, 0.1 to 10 μmol / L, more preferably 0.25 to 2 μmol / L. In the reaction system, the final concentration of the F2 primer is, for example, preferably 0.1 to 10 μmol / L, more preferably 0.25 to 2 μmol / L, and the final concentration of the R2 primer is, for example, 0.1 to 10 μmol / L, more preferably 0.25 to 2 μmol / L. In the reaction system, the molar ratio between the F1 primer and the F2 primer and the molar ratio between the R1 primer and the R2 primer are the same as the molar ratio between the first primer and the second primer, respectively. is there.

In the reaction system, the addition ratio of the polymerase is not particularly limited, and can be appropriately determined depending on, for example, the type of polymerase used. The final concentration of the polymerase in the reaction system is, for example, 5 to 50 U / mL, preferably 1 to 100 U / mL, and more preferably 20 to 30 U / mL. The activity unit (U) of polymerase generally has an activity of incorporating 10 nmol of all nucleotides into an acid-insoluble precipitate in an activity measurement reaction solution at 74 ° C. for 30 minutes using activated salmon sperm DNA as a template primer. 1U. The composition of the reaction liquid for activity measurement is, for example, 25 mM TAPS buffer (pH 9.3, 25 ° C.), 50 mM KCl, 2 mM MgCl ₂ , 1 mM mercaptoethanol, 200 μM dATP, 200 μM dGTP, 200 μM dTTP, 100 μM “α- ³² P”. DCTP, 0.25 mg / mL activated salmon sperm DNA.

  In the reaction system, the addition rate of dNTP is not particularly limited, but is, for example, 0.01 to 1 mmol / L, preferably 0.05 to 0.5 mmol / L, more preferably 0.1 to 0. .3 mmol / L.

In the reaction system, the addition ratio of the template nucleic acid is not particularly limited, and is, for example, 1 to 10 ⁸ copies / L, preferably 10 to 10 ⁶ copies / L, and more preferably 10 ² to 10 ⁴ copies. / L.

  The total volume of the reaction system is not particularly limited, and can be appropriately determined according to, for example, equipment used such as a thermal cycler. As a specific example, it is 1-500 microliters, for example, Preferably it is 10-100 microliters.

  Subsequently, as the step (A), the reaction system is heat-treated. As a result, the double-stranded template nucleic acid contained in the reaction system is dissociated into single strands.

  The heating condition of the reaction system in the step (A) is not particularly limited, but is preferably a temperature at which double strands can be dissociated into single strands. As a specific example, for example, the heating temperature is preferably 90 to 99 ° C., more preferably 92 to 95 ° C., and the treatment time is preferably 1 to 120 seconds, and more preferably 1 to 60 seconds, for example. .

  Next, as the step (B), the temperature of the reaction system is lowered. Thus, in the reaction system, a primer is annealed to each of the dissociated single-stranded template nucleic acids, and an extended strand complementary to the template nucleic acid is formed from the annealed primers.

  In the step (B), the temperature drop condition of the reaction system is not particularly limited, and can be appropriately determined according to the Tm value of each primer to be used. In the step (B), the temperature of the reaction system is preferably lower than, for example, the lowest Tm value among the Tm values of the primers used. As a specific example, the treatment temperature of the reaction system is, for example, preferably 50 to 80 ° C., more preferably 50 to 70 ° C., and the treatment time is preferably 1 to 300 seconds, and more preferably 5 to 60, for example. Seconds.

  Here, annealing and extension of various primers in the step (B) will be described with reference to FIG. The figure is a schematic diagram schematically showing amplification of a target sequence in the first cycle of PCR using the first primer set and the second primer set. In the figure, the arrow indicates the nucleic acid sequence, the tip on the arrowhead side of the arrow indicates the 3 'end, and the other end of the arrow indicates the 5' end. In the figure, F1 is the F1 primer of the first primer set, R1 is the R1 primer of the first primer set, F2 is the F2 primer of the second primer set, and R2 is the R2 primer of the second primer set. In the same figure, among the single-stranded nucleic acids dissociated by the heat treatment in the step (A), the sense strand is represented by “S”, the antisense strand is represented by “AS”, and F1 primer, R1 primer, F1 Each extended strand from the primer and R2 primer is represented by “F1-e”, “R1-e”, “F2-e” and “F2-e”, respectively.

  When the temperature of the reaction system comprising the two single-stranded nucleic acids dissociated by the heat treatment in the step (A) and the F1 primer and the F2 primer is lowered, the reactions shown in FIGS. proceed. First, as shown in FIG. 5A, the F1 primer is preferentially placed on the antisense strand (AS) and the R1 primer is preferentially placed on the sense strand (S), respectively, over the F2 primer and R2 primer of the second primer set. Annealing. This is because the F1 primer has a higher Tm value than the F2 primer, and the R1 primer has a higher Tm value than the R2 primer. Subsequently, as shown in FIG. 5B, the extended strand (F1-e1) complementary to the antisense strand (AS) from the annealed F1 primer is complementary to the sense strand (S) from the annealed R1 primer. Each of the elongated chains (R1-e1) is formed. Subsequently, as shown in FIG. 3C, in the antisense strand (AS), the F2 primer is outside (3 ′ side) of the annealing region of the F1 primer, and the R1 primer is annealed in the sense strand (S). The R2 primer anneals to the outside (3 ′ side) of the region. Then, as shown in FIG. 4D, the extension strand (F2-e1) complementary to the antisense strand (AS) from the annealed F2 primer is complementary to the sense strand (S) from the annealed R2 primer. Each of the extended chains (R2-e1) is formed. At this time, formation of extended strands (F2-e1 and R2-e1) from the F2 primer and R2 primer resulted in strand displacement, and extension from the previously formed F1 primer and R1 primer shown in FIG. The strands (F1-e1 and R1-e1) are dissociated from the antisense strand (AS) and the sense strand (S), respectively, to become a single strand.

  Then, as shown in FIG. 4E, the dissociated extended strand (F1-e1) is further annealed with new R1 primer and R2 primer in order, and the dissociated extended strand (R1-e1) is dissociated. In addition, new F1 and F2 primers are annealed sequentially. And the reaction similar to the said figure (b)-(d) occurs. That is, as shown in FIG. 5 (f), the extended strand (R1-e2) formed from the new R1 primer annealed to the extended strand (F1-e1) By the extended chain (R2-e2), it is dissociated from the extended chain (F1-e1) as a template to become a single chain. On the other hand, the extended strand (F1-e2) formed from the new F1 primer annealed to the extended strand (R1-e1) is the extended strand (F2-e2) formed from the subsequently formed new F2 primer. To dissociate from the extended strand (R1-e1), which is a template, into a single strand. Then, as shown in FIG. 5G, the F1 primer anneals to the new dissociated extended strand (R1-e2), the R1 primer anneals to the extended strand (F1-e2), and the complementary extended strand. Is formed. Since the extension strand (R1-e2) is formed using the extension strand (F1-e1) from the F1 primer as a template, the annealing region of the F2 primer that can hybridize to the outside (that is, the 3 ′ side) of the F1 primer Does not have. For this reason, only the F1 primer anneals to the extended strand (R1-e2) to form the extended strand (F1-e3), but the F2 primer cannot bind and the single strand cannot be dissociated. The amplification at is finished. Similarly, since the extended strand (F1-e2) is also formed using the extended strand (R1-e1) from the R1 primer as a template, R2 capable of hybridizing to the outside (that is, the 3 ′ side) from the R1 primer. Does not have primer annealing region. For this reason, only the R1 primer anneals to the extended strand (F1-e2) to form the extended strand (R1-e3), but the R2 primer cannot bind and the single strand cannot be dissociated. The amplification at is finished. As described above, according to this embodiment, in the first cycle, for example, a total of 12 amplified products can be obtained as a single strand, including the double-stranded nucleic acid at the start.

  Then, as the step (C), with respect to the amplified product after the step (B), the steps (A) and (B) are repeated to further amplify the target sequence. The conditions for the second and subsequent steps (A) and (B) in step (C) are not particularly limited and are the same as described above.

  The double-stranded amplification product obtained in the step (B) is dissociated into single strands by the subsequent heat treatment in the step (A), and each becomes a template nucleic acid in the step (B). These template nucleic acids include, for example, extension strands from the F1 primer, F2 primer, R2 primer, and R2 primer, in addition to the template at the start of PCR. For this reason, the extension strand used as a template depends on the type of primer involved in the formation of the extension strand, (1) an extension strand that can be annealed by both F1 primer and F2 primer, and (2) both R1 primer and R2 primer Are extension strands that can be annealed, (3) extension strands that can be annealed only by F1 primer, and (4) extension strands that can be annealed only by R1 primer. Since the first primer and the second primer can be annealed with respect to the extended strands of (1) and (2), the extended strand of the first primer (F1, R1) is the second strand as in FIG. Extension from the primers (F2, R2) dissociates from the template and subsequent primer annealing results in subsequent amplification. On the other hand, since the extension strands of (3) and (4) do not have the annealing region of the second primer (F2, R2), the first primer (F1, F2) anneals to form an extension strand. By forming a double strand with the template, amplification in the cycle is completed. Thus, amplification of the target sequence using each extended chain as a template is performed depending on the type of primer involved in the formation of the extended chain. According to this embodiment, in the second cycle, from a total of 12 single-stranded template nucleic acids dissociated from each of the 6 pairs of double-stranded nucleic acids in FIG. A total of 44 amplification products can be obtained. A further cycle results in a similar amplification.

  An outline of amplification in the second cycle is shown in FIG. This figure is a schematic diagram schematically showing amplification of a target sequence in the second cycle of PCR using the amplification product obtained in FIG. 1 as a template. In the figure, only the initial stage amplification in the second cycle is shown.

  In the method for producing a nucleic acid amplification product of the present invention, a target sequence in a template nucleic acid is amplified by PCR in the same reaction system using a plurality of primers capable of hybridizing on the same strand of the template nucleic acid. A method for producing a product, wherein the amplification of the target sequence is performed by the nucleic acid amplification method of the present invention. The present invention is characterized in that the target sequence is amplified by the nucleic acid amplification method using the primer reagent of the present invention, and other conditions and steps are not limited at all. The method for producing the nucleic acid amplification product of the present invention can be performed in the same manner as the nucleic acid amplification method of the present invention described above.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

Amplification of human bilirubin UDP glucuronyltransferase gene The target sequence in the human bilirubin UDP glucuronyltransferase (UGT1A) gene was amplified by PCR using various primer sets. The UGT1A gene sequence information is, for example, NCBI Accession No. It is registered in M84125.

Table 1 below shows the various primers used and the sequences of detection probes used for detection of amplified products described later. “Tm (° C.)” in Table 1 below is a Tm (° C.) when a sequence shown in the following table and a completely complementary sequence are hybridized, and MELTCALC software (http://www.meltcalc.com/ ), The parameters were calculated with the oligonucleotide concentration 0.2 μM and sodium equivalent (Na eq.) 50 mM. In the table below, the annealing region of each primer and detection primer for the UGT1A gene is NCBI Accession No. Described based on the sequence of M84125.

  Among the various primers, the F1 primer and the R1 primer are the first primer set that anneals to the innermost side in the UGT1A gene, and the F2 primer and the R2 primer are the second primer set that anneals to the outside of the first primer set. And the F3 primer and the R3 primer are a third primer set that anneals outside the second primer set. And the Tm value of each primer is set to F1 primer> F2 primer> F3 primer and R1 primer> R2 primer> R3 primer.

  The detection probe is a probe complementary to the sense strand of the UGT1A1 gene, and is a guanine quenching probe in which a fluorescent substance (TAMRA) is bound to the cytosine base at the 3 'end. This guanine quenching probe emits fluorescence independently and has a property of disappearing fluorescence by hybridizing with a complementary strand. For this reason, the amount of amplification of the target sequence of the UGT1A1 gene can be measured by performing RT-PCR in PCR described later and measuring the change in fluorescence intensity over time (decrease in fluorescence intensity).

Genomic DNA was prepared from human blood by a predetermined method using GFX Genomic Blood DNA Purification Kit (manufactured by GE Healthcare Bioscience). PCR was performed with 25 μL of the PCR reaction solution shown in Table 2 below, to which 0.1 μL of the prepared genomic DNA was added. In addition, in each reaction liquid sample, the combination of the added primer is as follows. Since sample A uses only the first primer set, it corresponds to the comparative example, and samples B to D use two or three types of primer sets that satisfy the condition (I). This corresponds to the embodiment.
(Sample A) F1 + R1
(Sample B) F1 + R1 + F2 + R2
(Sample C) F1 + R1 + F3 + R3
(Sample D) F1 + R1 + F2 + R2 + F3 + R3

  The PCR reaction was performed using an iCycler thermal cycler (Bio-Rad Laboratories). The reaction conditions were that each of the reaction liquid samples A to D was treated at 95 ° C. for 60 seconds and then repeated 50 cycles, with 95 ° C. for 5 seconds and 50 ° C. for 30 seconds as one cycle. And between 20 cycles and 40 cycles, the fluorescence intensity change with time was measured about each reaction liquid sample (wavelength 585-700 nm).

  These results are shown in FIG. FIG. 3 is a graph showing changes in relative fluorescence units over time, that is, changes in the amount of increase in target sequence over time. In the figure, the horizontal axis represents the number of PCR cycles, and the vertical axis represents relative fluorescence units (RFU). The RFU value on the vertical axis is a value obtained by removing the measured RFU value from the RFU value of each reaction solution sample before detection of amplification as a background. As shown in the figure, the sample A using only one kind of primer set, which is a comparative example, confirmed a gradual decrease in fluorescence intensity, that is, a gradual increase in amplified product, from about 37 cycles. On the other hand, sample B using the two types of primer sets in the example is from about 32 cycles, sample C is from about 31 cycles, and sample D using the three types of primer sets is about From around 28 cycles, a rapid decrease in fluorescence intensity, that is, a rapid increase in amplified products was confirmed. From the above results, it was found that the target sequence can be amplified with higher efficiency than conventional PCR by using two or more kinds of primers that satisfy the above-mentioned condition (I).

  As described above, according to the primer reagent of the present invention, by using two or more kinds of primers as described above, the target sequence is amplified in each stage more efficiently than conventional PCR in the same reaction system. It is possible. In addition, since various primers in the primer reagent of the present invention can be designed so that the Tm value satisfies the above condition (I), for example, the primer preparation itself can be performed very simply.

FIG. 1 is a schematic diagram showing an outline of amplification in the first cycle of PCR in one embodiment of the nucleic acid amplification method of the present invention. FIG. 2 is a schematic diagram showing an outline of amplification in the second cycle of PCR in the embodiment. FIG. 3 is a graph showing changes in relative fluorescence units over time in Example 1 of the present invention.

Claims (11)

A primer reagent for use in a nucleic acid amplification method in which a target sequence in a template nucleic acid is amplified by polymerase chain reaction in the same reaction system using a plurality of primers capable of hybridizing on the same strand of the template nucleic acid. And
Including at least two types of primers,
Any two kinds of primers among the primers satisfy the following condition (I).
(I) One primer can hybridize to the 3 ′ side of the template nucleic acid relative to the region where the other primer of the template nucleic acid can hybridize, and the Tm value of the one primer Lower than the Tm value of the primer Including a first primer and a second primer;
The second primer can hybridize to the 3 ′ side of the template nucleic acid with respect to the region where the first primer of the template nucleic acid can hybridize;
The primer reagent according to claim 1, wherein the Tm value of the second primer is lower than the Tm value of the first primer. In addition, including a third primer,
The third primer can hybridize to the 3 ′ side of the template nucleic acid with respect to the region where the second primer of the template nucleic acid can hybridize;
The primer reagent according to claim 2, wherein the Tm value of the third primer is lower than the Tm value of the second primer. The template nucleic acid is a double-stranded nucleic acid comprising a sense strand and an antisense strand;
Including at least two kinds of primer sets each having a forward primer capable of hybridizing to the antisense strand and a reverse primer capable of hybridizing to the sense strand;
The primer according to any one of claims 1 to 3, wherein, in any two types of primer sets among the primer sets, each forward primer and each reverse primer are primers that satisfy the condition (I), respectively. reagent. Including a first primer set and a second primer set;
The forward primer of the second primer set can hybridize to the 3 ′ side of the antisense strand with respect to the region where the forward primer of the first primer set in the antisense strand can hybridize, and the second primer set The Tm value of the forward primer of the primer set is lower than the Tm value of the forward primer of the first primer set,
The reverse primer of the second primer set can hybridize to the 3 ′ side of the sense strand with respect to the region where the reverse primer of the first primer set of the sense strand can hybridize, and the second primer set The primer reagent according to claim 4, wherein the Tm value of the reverse primer is lower than the Tm value of the reverse primer of the first primer set. In addition, a third primer set is included,
The forward primer of the third primer set can hybridize to the 3 ′ side of the antisense strand with respect to the region where the forward primer of the second primer set in the antisense strand can hybridize, and the third primer set The Tm value of the forward primer of the primer set is lower than the Tm value of the forward primer of the second primer set,
The reverse primer of the third primer set can hybridize to the 3 ′ side of the sense strand with respect to the region where the reverse primer of the second primer set of the sense strand can hybridize, and the third primer set The primer reagent according to claim 5, wherein the reverse primer has a Tm value lower than that of the reverse primer of the second primer set. A nucleic acid amplification method in which a target sequence in a template nucleic acid is amplified by polymerase chain reaction in the same reaction system using a plurality of primers capable of hybridizing on the same strand of the template nucleic acid,
A nucleic acid amplification method using the primer reagent according to any one of claims 1 to 6 as a primer.   The nucleic acid amplification method according to claim 7, wherein the polymerase used for the polymerase chain reaction is a polymerase lacking 5 ′ → 3 ′ exonuclease activity. The reaction system includes the first primer and the second primer,
The nucleic acid amplification method according to claim 7 or 8, wherein a molar ratio (X: Y) of the first primer (X) to the second primer (Y) is in the range of 1: 0.1 to 1:10. . The reaction system further includes the third primer,
The nucleic acid amplification method according to claim 9, wherein the molar ratio (X: Z) of the first primer (X) to the third primer (Z) is in the range of 1: 0.01 to 1: 100. The nucleic acid amplification method according to any one of claims 7 to 10, comprising the following steps (A) to (C).
(A) heating the reaction system containing the template nucleic acid and the primer reagent to make the template nucleic acid single-stranded (B) lowering the temperature of the reaction system, Step of performing primer annealing and extension reaction from the primer (C) Step of repeating the steps (A) and (B)

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