JP2009296948A - Primer for pcr, method for detecting target nucleic acid and method for detecting target biomolecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a primer for PCR, capable of being synthesized simply, and having excellent multiplicity of labels, and to provide methods for detecting a target nucleic acid and a target biomolecule by using the primer. <P>SOLUTION: The primer usable for the PCR (polymerase chain reaction) has a primer region, a PCR-inhibiting region at the 5' side of the primer region, and an aptamer region at the 5' side of the PCR-inhibiting region, wherein the PCR-inhibiting region has a hairpin structure or a pseudo knot structure. The method for detecting the target nucleic acid having a target base sequence by using the primer, and the method for detecting the target biomolecule in a sample solution by using the primer are also provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a primer for PCR (Polymerase Chain Reaction) labeled with an aptamer, and a method for detecting a target nucleic acid and a target biomolecule using the primer.

  In the field of nucleic acid analysis such as gene analysis, analysis is often performed by amplifying a target nucleic acid having a target base sequence to be analyzed, and PCR (Polymerase Chain Reaction) is used for amplification of the target nucleic acid. The method is most commonly used. The PCR method is a method of amplifying a target nucleic acid several hundred thousand times by repeating a DNA polymerase reaction with two kinds of PCR primers sandwiching a target base sequence and a DNA polymerase. This method basically amplifies a specific target nucleic acid by repeating the cycle 25 to 30 times with three steps of (1) denaturation step, (2) annealing step, and (3) extension step as one cycle. I will do it.

  In order to detect the nucleic acid amplified by PCR in this way, PCR is widely performed using a primer previously labeled at the 5 'end. By using the labeled primer, an amplified nucleic acid (PCR product) labeled at the end is obtained, and the PCR product can be detected by detecting this label. As the label molecule used for primer labeling, for example, a substance capable of binding to a specific antibody or receptor such as hapten is used, and the labeled PCR product can be specifically bound to the label molecule. Detection is performed by various methods such as an agglutination method such as an immunoturbidimetric method, a sedimentation method, an ELISA, and an immunochromatography method using a receptor.

Examples of the hapten include biotin and digoxigenin. As a primer labeled with such a hapten, for example, a nucleic acid in which a terminal phosphate group is labeled with a biotin derivative is disclosed (for example, see Patent Document 1). Since biotin binds strongly to streptavidin, avidin bound to alkaline phosphatase is bound to a primer labeled with a biotin derivative, and the nucleic acid amplified using the primer is detected by measuring the luminescence of alkaline phosphatase, etc. It becomes possible to do. In addition, for example, nucleotides into which a specific functional group such as an amino group or a thiol group has been introduced, various fluorescent substances, proteins such as luminescent enzymes, and the like can be used for labeling primers. In addition, since various labels can be performed, attempts have been made to use antibodies as labels for nucleic acids.
JP-A-5-331178

  An automatic nucleic acid synthesizer is usually used for chemical synthesis of a polynucleotide such as a primer and is automated. For this reason, in general, in order to obtain a primer labeled with a label molecule such as biotin or a fluorescent substance, a labeled molecule is bound to the end of the synthesized oligonucleotide. That is, there is a problem that a chemical modification reaction different from the synthesis of the oligonucleotide is required, resulting in complicated operation and complicated automatic synthesizer. In particular, labeling with an antibody enables various labels, while labeling an antibody with respect to an oligonucleotide requires a very complicated operation.

  On the other hand, by using an aptamer that can specifically bind to a specific ligand as a labeled molecule, in addition to enabling various labels as in the case of antibodies, it is possible to synthesize labeled primers easily. . Since aptamers are functional single-stranded nucleic acids, aptamer-labeled primers are synthesized by synthesizing a polynucleotide having a functional sequence that functions as an aptamer at the end of the primer region using an automatic nucleic acid synthesizer. Obtainable.

  However, when PCR is carried out simply using a primer in which an aptamer and a primer are bound, there is a problem that the ability to bind to the ligand is lost because it is extended to the aptamer region and becomes double-stranded. . An aptamer has a binding ability to a specific ligand in order to form a specific tertiary structure in a single-stranded state, and cannot take a tertiary structure such as a primary structure in a double-stranded state. This is because it cannot bind to the ligand. For this reason, it was difficult to detect a PCR product obtained after performing PCR using an aptamer-labeled primer using an aptamer.

  An object of the present invention is to provide a primer for PCR that can be easily synthesized and has excellent label diversity, and a method for detecting a target nucleic acid and a target biomolecule using the primer.

  As a result of diligent research to solve the above-mentioned problems, the present inventor, in primers used for PCR, PCR that inhibits the progress of DNA polymerase between an aptamer region that functions as an aptamer and a primer region that functions as a primer. By providing an inhibitory region, it becomes possible to perform PCR without making the aptamer region double-stranded, so PCR products obtained using such primers can be detected using aptamers. The present invention was completed.

That is, the present invention
(1) A primer used in PCR, comprising a primer region, a PCR inhibitory region on the 5 ′ side of the primer region, and an aptamer region on the 5 ′ side of the PCR inhibitory region, wherein the PCR inhibitory region is A primer for PCR, characterized by having a hairpin loop structure or a pseudoknot structure,
(2) The PCR primer according to (1), wherein the PCR-inhibiting region has a polynucleotide region represented by the following general formula (I) or (II):

［式中、Ａ１〜Ａａ、Ｂ１〜Ｂｂ、Ｃ１〜Ｃｃ、Ｐ１〜Ｐｍ、Ｑ１〜Ｑｍ、Ｒ１〜Ｒｎ、Ｓ１〜Ｓｎ、Ｔ１〜Ｔｏ、及びＵ１〜Ｕｏは、同一又は異なって、アデニンヌクレオチド、グアニンヌクレオチド、チミンヌクレオチド、又はシトシンヌクレオチドのいずれかを表し、Ｐ１〜ＰｎはＱ１〜Ｑｎに相補的なヌクレオチドを表し、Ｒ１〜ＲｎはＳ１〜Ｓｎに相補的なヌクレオチドを表し、Ｔ１〜ＴｎはＵ１〜Ｕｎに相補的なヌクレオチドを表し、ａ、ｂ、ｃ、ｍ、ｎ、ｏは、同一又は異なって、１〜２５の整数を表す。］

[Wherein, A1 to Aa, B1 to Bb, C1 to Cc, P1 to Pm, Q1 to Qm, R1 to Rn, S1 to Sn, T1 to To, and U1 to Uo are the same or different, and an adenine nucleotide, It represents any one of guanine nucleotide, thymine nucleotide, or cytosine nucleotide, P1-Pn represents a nucleotide complementary to Q1-Qn, R1-Rn represents a nucleotide complementary to S1-Sn, and T1-Tn represents U1 Represents a nucleotide complementary to Un, and a, b, c, m, n and o are the same or different and represent an integer of 1 to 25. ]

(3) In the general formula (I), the Tm value of the polynucleotide region comprising P1 to Pn is higher than the Tm value of the primer region, PCR primer according to (2),
(4) In the general formula (I), the Tm value of the polynucleotide region comprising P1 to Pn is 1 to 20 ° C. higher than the Tm value of the primer region. Primer,
(5) In the general formula (II), the sum of the Tm value of the polynucleotide region composed of R1 to Rn and the Tm value of the polynucleotide region composed of T1 to To is higher than the Tm value of the primer region. The primer for PCR according to (2),
(6) In the general formula (II), the sum of the Tm value of the polynucleotide region consisting of R1 to Rn and the Tm value of the polynucleotide region consisting of T1 to To is 1 to 20 ° C. higher than the Tm value of the primer region. PCR primer according to (2), characterized in that it is high,
(7) The primer for PCR according to any one of (1) to (6), wherein a labeled molecule is bound,
(8) The PCR primer according to (7), wherein the labeling molecule is one or more selected from the group consisting of dispersible fine particles, enzymes, and magnetic substances.
(9) A method for detecting a target nucleic acid having a target base sequence, comprising: (a) a step of performing PCR using PCR primers to obtain a PCR product; and (b) obtained in the step (a). A step of forming a PCR product-ligand complex by binding the PCR product to a ligand capable of specifically binding to the aptamer region of the PCR primer; and (c) the PCR obtained in the step (b). Detecting the product-ligand complex, and the PCR primer is the PCR primer according to any one of (1) to (8),
(10) The PCR primer is the PCR primer according to any one of (2) to (8), and the Tm value of the polynucleotide region consisting of P1 to Pn in the general formula (I), or the general The sum of the Tm value of the polynucleotide region consisting of R1 to Rn and the Tm value of the polynucleotide region consisting of T1 to To in formula (II) is higher than the PCR extension reaction temperature in the step (a). The method for detecting a target nucleic acid according to (9),
(11) The PCR in the step (a) is performed using a nucleotide modified with a modifying molecule, and the PCR product-ligand complex detection in the step (c) detects the modifying molecule. The method for detecting a target nucleic acid according to (9) or (10), wherein
(12) The method for detecting a target nucleic acid according to (11), wherein the modifying molecule is a fluorescent substance or a radioisotope,
(13) The method for detecting a target nucleic acid according to (9) or (10), wherein the ligand is bound to a labeled molecule,
(14) The target nucleic acid detection method according to (13), wherein the labeling molecule is one or more selected from the group consisting of dispersible fine particles, enzymes, fluorescent substances, and radioactive isotopes,
(15) A method for detecting a target biomolecule in a sample solution, wherein (a ′) PCR is performed using a PCR primer having an aptamer region that can specifically bind to the target biomolecule, to obtain a PCR product. (B ′) adding the PCR product obtained in the step (a ′) to the sample solution to form a PCR product-target biomolecule complex; and (c ′) the step (b ′). And a step of detecting the PCR product-target biomolecule complex obtained in (2), wherein the PCR primer is the PCR primer according to any one of (1) to (8) above. A method for detecting a target biomolecule,
(16) The PCR primer is the PCR primer according to any one of (2) to (8), and the Tm value of the polynucleotide region consisting of P1 to Pn in the general formula (I), or the general The sum of the Tm value of the polynucleotide region comprising R1 to Rn and the Tm value of the polynucleotide region comprising T1 to To in formula (II) is higher than the PCR extension reaction temperature in the step (a ′). The method for detecting a target biomolecule according to (15),
(17) The PCR in the step (a ′) is performed using a nucleotide modified with a modifying molecule, and the PCR product-target biomolecule complex detection in the step (c ′) The method for detecting a target biomolecule according to (15) or (16), wherein the method is performed by detection,
(18) The method for detecting a target biomolecule according to (17), wherein the modifying molecule is a fluorescent substance or a radioisotope,
Is provided.

  Since the primer for PCR of the present invention uses an aptamer that is a single-stranded nucleic acid as a label, an automatic nucleic acid synthesizer or the like does not require another modification reaction different from polynucleotide synthesis for modification of the labeled molecule. Can be easily synthesized. In addition, aptamers can form various tertiary structures by arbitrarily designing the nucleotide sequence, and thus have excellent label diversity as in the case of antibody labeling. Furthermore, by using the PCR primer of the present invention, a biomolecule such as a nucleic acid can be easily detected using an aptamer.

  The target base sequence in the present invention is a base sequence to be amplified by PCR, and is not particularly limited as long as the base sequence has been clarified to such an extent that it can be amplified by PCR. . For example, it may be a base sequence present in animal or plant chromosomes, bacterial or viral genes, or a base sequence present in biological RNA such as mRNA.

  The target nucleic acid in the present invention is not particularly limited as long as it is a nucleic acid having a target base sequence and can be a template in PCR. For example, it may be a nucleic acid contained in a sample prepared from a biological sample (specimen) such as blood or body fluid or a cultured cell solution, or may be a nucleic acid extracted from these samples. It may be a nucleic acid amplified using a nucleic acid as a template. Alternatively, it may be cDNA synthesized using reverse transcriptase from RNA contained in a biological sample or the like.

  The PCR primer of the present invention is characterized by having a primer region, a PCR inhibitory region 5 'to the primer region, and an aptamer region 5' to the PCR inhibitory region. By having a PCR inhibition region between the aptamer region and the primer region, the DNA polymerase extension reaction does not proceed to the aptamer region, and therefore, binding to the ligand is caused on the 5 ′ end side from the PCR primer of the present invention. A PCR product having a single-stranded aptamer having the ability can be obtained.

  FIG. 1 is a diagram schematically showing a case where PCR is performed using the PCR primer of the present invention and a case where PCR is simply performed using an aptamer-labeled control primer. The PCR primer 2 of the present invention having the primer region 1a, the PCR inhibition region 1b, and the aptamer region 1c, and the control primer 3 having only the primer region 1a and the aptamer region 1c are both aptamer region 1c and specific to ligand 8. It has a three-dimensional structure that can be bonded. When these primers are used as forward primers, the target nucleic acid 4 as a template, and a reverse primer 5 as a pair, PCR is performed, and when the PCR primer 2 of the present invention is used, the obtained PCR product 6, the aptamer region 1c remains single-stranded and can specifically bind to the ligand 8. On the other hand, when the control primer 3 is used, the resulting PCR product 7 becomes an aptamer region 1c duplex, and cannot bind to the ligand 8.

  In the present invention, the primer region means a polynucleotide region having a base sequence that can function as a primer in PCR among primers for PCR. Specifically, it is a polynucleotide region having a base sequence that can be paired (hybridized) with the 5 'end side or 3' end side in the target base sequence of the target nucleic acid. Generally, it is a base sequence that is homologous or complementary to a partial base sequence on the 5 'end side or 3' end side of the target base sequence, but may have a mismatch site.

  The base sequence of such a primer region may be any of methods well known in the art depending on the type of target base sequence, the type of DNA polymerase used in PCR, PCR reaction conditions, and the like. Can be designed. For example, it can be designed easily using base sequence information obtained from known genome sequence data and the like and a widely used primer design tool. As such a primer design tool, for example, Primer 3 (Rozen, S., H. J. Skalsky, 1996, http: //www-genome.wi.mit.edu/genome_software/other/ primer 3.html), Visual OMP (manufactured by DNA Software), and the like. Also, known genome sequence data can usually be obtained from NCBI (National Center for Biotechnology Information), DDBJ (DNA Data Bank of Japan), and the like, which are international base sequence databases.

  In the present invention, the PCR-inhibiting region means a polynucleotide region having a three-dimensional structure in which the progress of the DNA polymerase used in PCR is inhibited. As such a three-dimensional structure, a stable loop structure formed by pairing with another single-stranded region in the same molecule, such as a hairpin loop structure or a pseudoknot structure. Is mentioned. Specifically, it has a polynucleotide region represented by the following general formula (I) or (II).

［式中、Ａ１〜Ａａ、Ｂ１〜Ｂｂ、Ｃ１〜Ｃｃ、Ｐ１〜Ｐｍ、Ｑ１〜Ｑｍ、Ｒ１〜Ｒｎ、Ｓ１〜Ｓｎ、Ｔ１〜Ｔｏ、及びＵ１〜Ｕｏは、同一又は異なって、アデニンヌクレオチド、グアニンヌクレオチド、チミンヌクレオチド、又はシトシンヌクレオチドのいずれかを表し、Ｐ１〜ＰｎはＱ１〜Ｑｎに相補的なヌクレオチドを表し、Ｒ１〜ＲｎはＳ１〜Ｓｎに相補的なヌクレオチドを表し、Ｔ１〜ＴｎはＵ１〜Ｕｎに相補的なヌクレオチドを表し、ａ、ｂ、ｃ、ｍ、ｎ、ｏは、同一又は異なって、１〜２５の整数を表す。］

[Wherein, A1 to Aa, B1 to Bb, C1 to Cc, P1 to Pm, Q1 to Qm, R1 to Rn, S1 to Sn, T1 to To, and U1 to Uo are the same or different, and an adenine nucleotide, It represents any one of guanine nucleotide, thymine nucleotide, or cytosine nucleotide, P1-Pn represents a nucleotide complementary to Q1-Qn, R1-Rn represents a nucleotide complementary to S1-Sn, and T1-Tn represents U1 Represents a nucleotide complementary to Un, and a, b, c, m, n and o are the same or different and represent an integer of 1 to 25. ]

In the general formula (I), A1 to Aa are the same or different and each represents an adenine nucleotide, a guanine nucleotide, a thymine nucleotide, or a cytosine nucleotide. The nucleotide sequence of the polynucleotide region comprising A1 to Aa (A1 to Aa region, the same shall apply hereinafter) is not particularly limited as long as it does not inhibit the functions of the primer region and aptamer region in the PCR primer. Can be set.
In the general formula (I), a represents an integer of 1 to 25. In the PCR primer of the present invention, a is preferably 1 to 12, more preferably 1 to 10, and further preferably 3 to 7.

In the general formula (I), P1 to Pm and Q1 to Qm are the same or different and represent any of adenine nucleotide, guanine nucleotide, thymine nucleotide, or cytosine nucleotide, and P1 to Pn are complementary to Q1 to Qn. Represents a nucleotide. That is, the base sequence of the P1-Pn region is a base sequence complementary to the base sequence of the Q1-Qn region, and a hairpin loop is formed when the P1-Pn region and the Q1-Qn region are paired. .
In the said general formula (I), m represents the integer of 1-25. In the PCR primer of the present invention, m is preferably from 3 to 25, more preferably from 5 to 25, and even more preferably from 10 to 25.

  In order for the PCR inhibition region to prevent the progress of the extension reaction by DNA polymerase, it is necessary that this hairpin loop structure is stably formed during the extension reaction by DNA polymerase in PCR. The stability of the hairpin loop structure depends on the stability of the pairing between the P1 to Pn region and the Q1 to Qn region, and the stability of the pairing can be determined using the Tm value as an index.

  For this reason, in the primer for PCR of this invention, it is preferable that Tm value of P1-Pn area | region is higher than Tm value of a primer area | region. Since the stability of the hairpin loop structure is higher than the stability of the pairing between the primer and the target nucleic acid as a template in PCR, the hairpin loop structure is more stably formed even during the extension reaction by DNA polymerase. As a result, the double strand formation of the aptamer region can be effectively inhibited. In the present invention, the Tm value of the P1 to Pn region is more preferably 1 to 20 ° C higher than the Tm value of the primer region, and more preferably 5 to 10 ° C.

On the other hand, in said general formula (II), B1-Bb and C1-Cc are the same or different, and represent either an adenine nucleotide, a guanine nucleotide, a thymine nucleotide, or a cytosine nucleotide. Each base sequence of the B1 to Bb region and the C1 to Cc region is not particularly limited as long as the functions of the primer region and the aptamer region in the PCR primer are not inhibited, and can be arbitrarily set.
In the said general formula (I), b and c are the same or different and represent the integer of 1-25. In the PCR primer of the present invention, b and c are the same or different and are preferably 1 to 12, more preferably 1 to 10, and further preferably 3 to 7.

In the general formula (II), R1 to Rn, S1 to Sn, T1 to To, and U1 to Uo are the same or different and each represents an adenine nucleotide, guanine nucleotide, thymine nucleotide, or cytosine nucleotide, and R1 ~ Rn represents a nucleotide complementary to S1 to Sn, and T1 to Tn represents a nucleotide complementary to U1 to Un. That is, the base sequence of the R1-Rn region is a base sequence complementary to the base sequence of the S1-Sn region, the base sequence of the T1-Tn region is a base sequence complementary to the base sequence of the U1-Un region, A pseudoknot is formed by the R1 to Rn region and the S1 to Sn region, and the T1 to Tn region and the U1 to Un region, respectively.
In the general formula (II), n and o are the same or different and each represents an integer of 1 to 25. In the PCR primer of the present invention, n and o are the same or different and are preferably 3 to 25, more preferably 5 to 25, and still more preferably 10 to 25.

  Similar to the case represented by the general formula (I), in the case represented by the general formula (II), this pseudo-knot structure is stably formed during the extension reaction by DNA polymerase in PCR. It is necessary. The stability of the pseudoknot structure depends on both the stability of the pairing of the R1 to Rn region and the S1 to Sn region and the pairing stability of the T1 to Tn region and the U1 to Un region. The stability of the pairing can be determined using the Tm value as an index.

  For this reason, in the primer for PCR of this invention, it is preferable that the sum of Tm value of R1-Rn area | region and Tm value of T1-To area | region is higher than Tm value of a primer area | region. In the present invention, the sum of the Tm value of the R1 to Rn region and the Tm value of the T1 to To region is more preferably 1 to 20 ° C higher than the Tm value of the primer region, and further preferably 5 to 10 ° C higher. .

  In the PCR primer of the present invention, the Tm value of the P1 to Pn region in the general formula (I), the Tm value of the R1 to Rn region and the Tm value of the T1 to To region in the general formula (II). It is also preferable to make the sum higher than the elongation reaction temperature of PCR. This is because the hairpin loop structure and the pseudoknot structure of the PCR inhibition region can be more stably formed during the PCR extension reaction.

  In the present invention, the aptamer region means a polynucleotide region having a base sequence that can function as an aptamer that is a functional nucleic acid, among PCR primers. An aptamer means a single-stranded nucleic acid having a three-dimensional structure that can specifically bind to a specific ligand. However, in the present invention, specifically binding means that it can be specifically bound to such an extent that it can be normally used for detection or purification of the ligand, and crosses with other substances. Also good.

  The base sequence of the aptamer region may have the same base sequence as a known aptamer, or may be arbitrarily designed so as to have a binding ability with a desired ligand. The base sequence of a single-stranded nucleic acid having a specific tertiary structure that can bind to a specific ligand can be designed by a conventional method. In the present invention, the type of aptamer ligand is not particularly limited. For example, fluorescent substance, biotin, glutathione, DNP (dinitrophenol), digoxigenin, digoxin, sugar chain composed of two or more sugars, six or more amino acids Polypeptide, auxin, gibberellin, steroid, protein, and analogs thereof.

  The aptamer region in the present invention may be a polynucleotide composed of DNA, a polynucleotide composed of RNA, or a chimeric polynucleotide composed of DNA and RNA. In addition, the polynucleotide may be an artificial nucleic acid or a modified nucleic acid modified with an amino group or the like. A polynucleotide comprising DNA and / or RNA is preferable because it can be synthesized more inexpensively and easily.

  The PCR primer of the present invention thus designed can be synthesized using any method well known in the art. For example, it may be synthesized by requesting an oligo synthesis manufacturer, or may be synthesized independently using a commercially available automatic nucleic acid synthesizer.

  In addition, the base sequence of the PCR primer of the present invention can be arbitrarily designed. In addition to the primer region, the PCR inhibition region, and the aptamer region, an additional sequence is provided as long as the function of each region is not inhibited. Various functions can be added. Examples of the additional sequence include a restriction enzyme recognition sequence and a sequence that can function as a linker.

  Furthermore, the PCR primer of the present invention may be labeled with various target molecules in order to facilitate detection and analysis of PCR products obtained using the primer. The label molecule is not particularly limited as long as it is generally used for labeling polynucleotides, and can be appropriately selected and used in consideration of a PCR product detection method and the like. Examples of such labeling molecules include dispersible fine particles, enzymes, magnetic substances, fluorescent substances, radioactive isotopes, chemiluminescent substances, and the like. In addition, it may be a ruthenium complex that can be detected electrochemically. The chemiluminescent substance may be a substance that emits light by an inorganic reaction such as luminol, or a luminescent substance that is catalyzed by an enzyme such as luciferin or coelenterazine. The labeled molecule of the PCR primer of the present invention is preferably a dispersible fine particle, an enzyme, a magnetic substance or the like, and more preferably a magnetic particle (magnetic dispersible fine particle) or an enzyme.

  In the present invention, the dispersible fine particles mean fine particles dispersed in a solution. Examples of such dispersible fine particles include particles made of silica, glass, ceramics, plastics, latex and the like, magnetic particles, metal particles made of gold, aluminum, nickel, alumina, titania, zirconia, and agarose resin. It is done. Since the PCR product can be easily recovered and purified, the dispersible fine particles used for labeling the PCR primer of the present invention are preferably magnetic particles.

In the present invention, the magnetic particles are not particularly limited as long as they are magnetic particles. Examples of the magnetic particles include particles made of a metal such as triiron tetroxide (Fe ₃ O ₄ ), iron sesquioxide (γ-Fe ₂ O ₃ ), various ferrites, iron, manganese, nickel, cobalt, and chromium, There are particles made of an alloy containing cobalt, nickel, manganese, and the like. The magnetic particles may be particles composed of only a magnetic material, or may be particles in which fine particles of a magnetic material are contained inside nonmagnetic particles such as latex particles.

  In the present invention, the average particle size of the dispersible fine particles used as the labeling molecule is not particularly limited, and is a size usually used in the fields of molecular biology and biochemistry, for example, about 0.1 to 100 μm. Can be used. In order to measure the turbidity using a fully automatic immunoturbidimetric apparatus or the like as detection of the dispersible fine particles, the particle diameter of the dispersible fine particles is preferably 0.3 μm or less.

  The enzyme used for labeling the PCR primer of the present invention is not particularly limited as long as it does not inhibit the binding between the PCR and the aptamer region in the PCR primer of the present invention and a ligand, The enzyme can be appropriately selected from enzymes usually used in detection of a target substance using an enzyme reaction. Examples of such enzymes include alkaline phosphatase, peroxidase, luciferase and the like.

  The fluorescent substance used for labeling the PCR primer of the present invention is not particularly limited, and can be appropriately selected from fluorescent substances usually used for labeling nucleic acids and the like. Examples of such fluorescent substances include FITC (fluorescein isothiocyanate), fluorescein, rhodamine, TAMRA, NBD, TMR (tetramethylrhodamine), Alexa dye series (manufactured by Molecular Probes), Cy dye series (GE Healthcare). Bioscience).

  The target nucleic acid can be detected using the PCR primer of the present invention. Since the primer for PCR of the present invention is aptamer-labeled, it has excellent label diversity such as antibody labeling. For example, in the case of a fluorescent label, the types of fluorescent substances used for the label are limited, and the detection accuracy decreases when the detected fluorescence wavelengths overlap or there is leakage of fluorescence. . For this reason, there is a tendency that the types of target nucleic acids that can be detected from one sample are limited to about 1 to 3 types. On the other hand, in the case of the PCR primer of the present invention, it may be designed to have an aptamer region for a different ligand for each type of target nucleic acid, and various labels are possible. Further, the binding between the ligand and the aptamer has high specificity as in the antigen-antibody reaction. Therefore, by using the PCR primer of the present invention, a target nucleic acid can be easily and accurately detected from a sample containing a plurality of types of PCR products such as a multiplex PCR product.

Specifically, the target nucleic acid detection method of the present invention was obtained by (a) performing PCR using the PCR primer of the present invention to obtain a PCR product, and (b) obtained in the step (a). A step of forming a PCR product-ligand complex by binding the PCR product to a ligand capable of specifically binding to the aptamer region of the PCR primer; and (c) the PCR obtained in the step (b). Detecting the product-ligand complex.
Hereinafter, it demonstrates for every process.

  First, in step (a), PCR is performed using the PCR primer of the present invention to obtain a PCR product. Reagents used for PCR, such as DNA polymerase, nucleotides, reaction buffer, etc. are not particularly limited, and those usually used for PCR can be used in the amounts usually used. The DNA polymerase is preferably a thermostable polymerase. Furthermore, a polymerase having a calibration function may be used.

  PCR reaction conditions can be appropriately determined in consideration of the type of DNA polymerase used, the length of the target base sequence, the stability of the loop structure of the PCR inhibition region, and the like. When the PCR-inhibiting region has the polynucleotide represented by the general formula (I), the Tm values of the P1 to Pn regions in the general formula (I) may be higher than the PCR extension reaction temperature. preferable. Further, when the PCR-inhibiting region has the polynucleotide represented by the general formula (II), the sum of the Tm value of the R1 to Rn region and the Tm value of the T1 to To region in the general formula (II) Is preferably higher than the elongation reaction temperature of PCR.

  Next, in step (b), the PCR product obtained in step (a) is bound to a ligand that can specifically bind to the aptamer region of the PCR primer, thereby forming a PCR product-ligand complex. . Specifically, a ligand is added to a PCR solution containing the PCR product and mixed as appropriate to bring the PCR product into contact with the ligand to form a PCR product-ligand complex.

  The ligand to be contacted with the PCR product may be labeled with a labeled molecule as long as it does not inhibit the formation of the PCR product-ligand complex. By preliminarily labeling the ligand, the PCR product-ligand complex can be detected more easily. As the labeling molecule used for labeling of the ligand, the same as those mentioned as the labeling molecules that can be used for labeling the above-mentioned PCR primer can be used. The ligand labeling molecule used in the target nucleic acid detection method of the present invention is preferably a dispersible fine particle, an enzyme, a fluorescent substance, a radioisotope, and the like. Magnetic particles (magnetic dispersible fine particles), latex More preferred are particles, gold particles, and enzymes.

  Thereafter, as the step (c), the target nucleic acid amplified by PCR can be detected by detecting the obtained PCR product-ligand complex. The method for detecting the PCR product-ligand complex is not particularly limited, and the PCR product-ligand complex can be detected using a method usually used in the detection of nucleic acids, proteins and the like.

  For example, when a ligand labeled with a labeled molecule is used in step (b), the PCR product-ligand complex can be easily detected using the labeled molecule. More specifically, for example, when a fluorescent substance or a radioisotope is used as a labeling molecule, a PCR product-ligand complex is detected by detecting fluorescence or radiation emitted by the labeling molecule. Can do. When an enzyme is used as the labeling molecule, the PCR product-ligand complex can be detected by adding the enzyme substrate and measuring the enzyme activity. Furthermore, when dispersible fine particles are used as the labeling molecule, the PCR product-ligand complex can be recovered and detected by collecting the dispersible fine particles by a centrifugal method or the like. When magnetic particles are used as labeling molecules, the PCR product-ligand complex can be recovered and detected by using a magnetic field. In addition, the PCR product-ligand complex can also be detected by measuring the turbidity (light scattering degree) of the solution after adding a ligand labeled with dispersible microparticles using an immunoturbidimetric method. Can do.

  In the method for detecting a target nucleic acid of the present invention, when a nucleotide modified with a modifying molecule such as a fluorescent substance or a radioisotope is used in the PCR in the step (a), fluorescence or radiation emitted from the modifying molecule is used. By detecting the PCR product-ligand complex can also be detected. For example, in the case where a ligand modified with a fluorescent substance or the like and a ligand labeled with a dispersible fine particle in step (b) is used, the dispersible fine particle recovered using centrifugation or a magnetic field is used. By measuring fluorescence or the like, the PCR product-ligand complex can be detected.

  In the target nucleic acid detection method of the present invention, one of the two primers sandwiching the target base sequence used in the PCR in step (a) is used as the PCR primer of the present invention, and the other is used for the PCR of the present invention. Primers other than the primer may be used, and both types may be used as the PCR primers of the present invention. The primer other than the primer for PCR of the present invention is not particularly limited as long as it is a primer usually used in PCR, and may be labeled or may not be labeled.

  In the method for detecting a target nucleic acid of the present invention, when both the forward primer and the reverse primer used in the PCR in the step (a) are the PCR primers of the present invention, for example, the aptamer region of both primers is the same ligand ( For example, when an aptamer for ligand a) is used (for example, aptamer A), a PCR product labeled at both ends with the same aptamer A can be obtained. On the other hand, when the aptamer region of the forward primer is aptamer A for ligand a and the aptamer region of the reverse primer is aptamer B for ligand b, aptamer A is labeled at one end and aptamer B is labeled at the other end. PCR products can be obtained.

  FIG. 2 is a diagram schematically showing a method for detecting a PCR product labeled at both ends with different aptamers. In the figure, “A” indicates aptamer A, “a” indicates ligand a, “B” indicates aptamer B, and “b” indicates ligand b. When the dispersible particles 10 having the ligand b bound thereto are brought into contact with the PCR product 9, a PCR product-ligand b-dispersible particle complex is formed by the binding of the ligand b and the aptamer B. Thus, the PCR product can be easily recovered and purified by recovering the dispersible fine particles. When the recovered PCR product is brought into contact with ligand a, the PCR product can be detected with higher accuracy by utilizing the binding between ligand a and aptamer A.

  In addition, a dispersive particle-ligand a-PCR product-ligand b-dispersible particle complex is obtained by bringing a dispersible particle having ligand b bound thereto and a dispersible particle having ligand a bound thereto into contact with the PCR product. Is formed. By forming such large particles, the change in the turbidity (light scattering degree) of the solution can be further increased. Therefore, it is preferable to detect a PCR product using an immunoturbidimetric method.

  The PCR product obtained using the PCR primer of the present invention can also be used as a detection probe for a ligand. That is, a target biomolecule is detected as a ligand, PCR is performed using a PCR primer having an aptamer region that can function as an aptamer for the ligand, and the obtained PCR product is used as a detection probe. can do.

  In addition, the target biomolecule in this invention means the biomolecule which is object of detection etc. The biomolecule means a molecule constituting the living body such as a low molecular compound such as protein, lipid, saccharide, nucleic acid, vitamins and the like.

  Specifically, the method for detecting a target biomolecule of the present invention is a method for detecting a target biomolecule in a sample solution, and (a ′) a book having an aptamer region that can specifically bind to the target biomolecule. A step of performing PCR using the PCR primer of the invention to obtain a PCR product; and (b ′) adding the PCR product obtained in the step (a ′) to the sample solution, and combining the PCR product and the target biomolecule complex And (c ′) detecting the PCR product-target biomolecule complex obtained in the step (b ′).

  For example, in the PCR in the step (a ′), as in the step (a) in the method for detecting a target nucleic acid of the present invention, when a nucleotide modified with a modifying molecule such as a fluorescent substance or a radioisotope is used, The obtained PCR product can function as an aptamer modified by fluorescence or the like. Therefore, in step (c ′), the target biomolecule in the sample solution can be detected by measuring fluorescence emitted from the modified nucleotide.

  FIG. 3 is a diagram schematically showing a method of detecting a target biomolecule when PCR is performed using a modified nucleotide. In the figure, “A” and “a” are the same as those in FIG. When PCR is performed using a nucleotide dGTP modified with the fluorescent substance 11, a PCR product 12 labeled with the fluorescent substance 11 is obtained. Since the PCR product 12 binds to the ligand a which is a target biomolecule, the target biomolecule can be detected by measuring the fluorescence emitted from the fluorescent substance 11.

  As in the target nucleic acid detection method of the present invention, both the forward primer and the reverse primer used in the PCR in the step (a ′) are the PCR primers of the present invention, so that the target biomolecule and the specific primer are specific at both ends. PCR product having an aptamer region that can bind to the target, or an aptamer region that can specifically bind to the target biomolecule on one side and an aptamer region that can specifically bind to a ligand other than the target biomolecule on the other side The product can be obtained.

  For example, a PCR product having an aptamer region that can specifically bind to a target biomolecule on one side and an aptamer region that can specifically bind to a ligand other than the target biomolecule (eg, ligand b) was obtained. In step (b ′), in the same manner as in step (b) in the method for detecting a target nucleic acid of the present invention, when ligand b labeled with a labeled molecule is used, target biomolecule-PCR product-ligand Since the b-label molecule complex is formed, the target biomolecule in the sample solution can be easily detected using the label molecule. In addition, as a labeled molecule used for labeling of the ligand b, the same as those mentioned as the labeled molecules that can be used for labeling the above-mentioned PCR primer can be used.

  Further, when ligand b labeled with dispersible fine particles is used, it is combined with the dispersible fine particles by using a centrifugal method or the like, and particularly when ligand b labeled with magnetic particles is used, a magnetic field is used. The target biomolecule forming the body can be recovered and purified and detected. In addition, when ligand b labeled with gold particles is used, it is also possible to quantify the target biomolecule by surface plasmon resonance (SPR; Surface Plasmon Resonance).

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1]
The target nucleic acid was detected using the PCR primer of the present invention having an aptamer region capable of specifically binding to streptavidin. The target base sequence was the base sequence (SEQ ID NO: 7) in the plasmid (pBlueScript II SK +).
<Primer design>
As primers for PCR amplification of the target nucleic acid, an aptamer region that can specifically bind to streptavidin (Anti-StAv aptamer region), a PCR inhibition region having a hairpin loop structure, and a forward primer (PSA-) having a primer region Primer-F, SEQ ID NO: 1) and reverse primer (PSA-Primer-R, SEQ ID NO: 2), forward primer having only the primer region (P-Primer-F, SEQ ID NO: 3) and reverse primer (P-Primer-R) , SEQ ID NO: 4), a forward primer (PA-Primer-F, SEQ ID NO: 5) having an Anti-StAv aptamer region and a primer region and a reverse primer (PA-Primer-R, SEQ ID NO: 6) were designed, respectively.
The base sequence of each primer is shown in Table 1. In each base sequence of Table 1, the capital letter is the primer region, the black and white inverted capital letter is the PCR inhibition region, and the underlined capital letter is the Anti-StAv aptamer region. FIG. 4 shows the hairpin loop structure of the PCR inhibition region obtained by simulation. In FIG. 4, “A” (CATG) corresponds to the A1 to Aa region (a = 4) in the general formula (I), “P” corresponds to the P1 to Pm region (m = 19), “ “Q” corresponds to the Q1 to Qm region (m = 19).
Among these primers, the Tm value of the primer region was 55 ° C., and the Tm value of the PCR inhibition region (Tm value of the P1 to P19 region) was 69 ° C.
Moreover, each primer was obtained by carrying out chemical synthesis based on these designs.

<PCR>
Using a plasmid (pBlueScript II SK +) as a template, a primer set consisting of PSA-Primer-F and PSA-Primer-R, a primer set consisting of P-Primer-F and P-Primer-R, PA-Primer-F and PA- PCR was performed using each primer set consisting of Primer-R to obtain a PCR product. The primer primer solution was prepared by mixing the forward primer and the reverse primer of each primer set so as to be 10 μM each.
Specifically, for 5 μL of 10 × KOD-plus- PCR buffer (manufactured by Toyobo Co., Ltd.), 5 μL of 2 mM dNTPs, 2 μL of 25 mM magnesium sulfate, 1.5 μL of primer mix solution, 1 μL of plasmid solution, 10 pg 1 μL of / μL KOD-plus- (manufactured by Toyobo Co., Ltd.) was added, and an appropriate amount of sterilized ultrapure water was added to prepare 50 μL of a PCR solution. The PCR solution was treated at 94 ° C. for 2 minutes, and then subjected to PCR by performing 30 cycles of thermal cycles of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute.

<Detection of PCR products>
The resulting PCR product was detected by performing a gel shift assay with streptavidin.
First, 5 μL of the PCR solution after PCR was mixed with 500 ng of streptavidin. Thereafter, this mixture was separated by Native-PAGE and stained with CYBR Green I and SYPRO Ruby to detect PCR products.
As a result, as compared with the case where the solution containing only streptavidin was separated, gel shift was observed only in the PCR product obtained using the primer set consisting of PSA-Primer-F and PSA-Primer-R, and streptavidin and Binding was confirmed.
From this result, when the PCR primer of the present invention having a PCR inhibitory region is used, a PCR product maintaining the ligand binding ability of the aptamer region can be obtained, so that the PCR product can be detected using the ligand. It is clear.

[Example 2]
The target biomolecule was detected using the PCR primer of the present invention. Streptavidin was used as a target biomolecule, PSA-Primer-F synthesized in Example 1 was used as a forward primer, and P-Primer-R was used as a reverse primer. In addition, PSA-Primer-F and P-Primer-R were mixed in advance so as to be 10 μM, respectively, thereby preparing a primer mix solution.

<PCR>
Using a plasmid (pBlueScript II SK +) as a template, PCR was performed using a nucleotide (Cy3 conjugated dGTP) modified with Cy3 (manufactured by GE Healthcare Bioscience), which is a fluorescent substance, to obtain a PCR product.
Specifically, 2 μL of 5 mM dATP, 2 μL of 5 mM dTTP, 2 μL of 5 mM dCTP, 2 μL of 5 mM Cy3 conjugated dGTP, 25 mM magnesium sulfate with respect to 5 μL of 10 × KOD-plus- PCR buffer (manufactured by Toyobo) 2 μL, primer mix solution 1.5 μL, plasmid solution 1 μL, 10 pg / μL KOD-plus- (manufactured by Toyobo Co., Ltd.) 1 μL respectively, an appropriate amount of sterilized ultrapure water was added, and 50 μL PCR was added. A solution was prepared. The PCR solution was treated at 94 ° C. for 2 minutes, and then subjected to PCR by performing 30 cycles of thermal cycles of 94 ° C. for 15 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 1 minute.

<Detection of streptavidin>
Streptavidin was detected by performing Western blotting on the transfer membrane to which streptavidin was transferred.
First, two transfer films to which streptavidin was transferred were prepared. Specifically, 1, 5, 10, 50, 100, 500, and 1000 μg of streptavidin were developed by PAGE, transferred to a nitrocellulose membrane, and blocked with skim milk.
PCR product is added to one of these two transfer membranes, and Cy3-labeled anti-streptavidin antibody (GE Healthcare Bioscience) is added to the other one, and each is reacted at room temperature for 30 minutes. I let you. Thereafter, a Cy3-labeled substance bound to streptavidin in the transfer film was detected using a fluorescence imager.
As a result, by using the PCR product, streptavidin could be detected with higher sensitivity than when the Cy3-labeled anti-streptavidin antibody was used. That is, it is clear that a biomolecule can be detected by using a PCR product obtained using the PCR primer of the present invention as a detection probe.

  Since the PCR primer of the present invention can be easily synthesized and has excellent diversity of labels, it can be used in fields such as biochemistry, molecular biology, and clinical testing that detect biomolecules such as nucleic acids. It can be used.

It is the figure which showed typically the case where PCR was performed using the primer for PCR of this invention, and the case where PCR was performed only using the aptamer-labeled control primer. In the detection method of the target nucleic acid of this invention, it is the figure which showed typically the detection method of the PCR product labeled with the aptamer from which both ends differ, respectively. In the detection method of the target biomolecule of this invention, it is the figure which showed typically the detection method of the target biomolecule at the time of performing PCR using the modified nucleotide. It is the figure which showed the hairpin loop structure calculated | required by simulation of the PCR inhibition area | region of the primer used in the Example.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1a ... Primer area | region, 1b ... PCR inhibition area | region, 1c ... Aptamer area | region, 2 ... PCR primer of this invention, 3 ... Control primer, 4 ... Target nucleic acid, 5 ... Reverse primer, 6 ... PCR product, 7 ... PCR product, 8 ... Ligand, 9 ... PCR product, 10 ... Dispersible particles, 11 ... Fluorescent substance, 12 ... PCR product.

Claims (18)

Primers used for PCR (Polymerase Chain Reaction),
A primer region, a PCR inhibitory region on the 5 ′ side of the primer region, and an aptamer region on the 5 ′ side of the PCR inhibitory region, wherein the PCR inhibitory region has a hairpin loop structure or a pseudoknot structure. Characteristic primer for PCR. The PCR primer according to claim 1, wherein the PCR inhibition region has a polynucleotide region represented by the following general formula (I) or (II).

[Wherein, A1 to Aa, B1 to Bb, C1 to Cc, P1 to Pm, Q1 to Qm, R1 to Rn, S1 to Sn, T1 to To, and U1 to Uo are the same or different, and an adenine nucleotide, It represents any one of guanine nucleotide, thymine nucleotide, or cytosine nucleotide, P1-Pn represents a nucleotide complementary to Q1-Qn, R1-Rn represents a nucleotide complementary to S1-Sn, and T1-Tn represents U1 Represents a nucleotide complementary to Un, and a, b, c, m, n and o are the same or different and represent an integer of 1 to 25. ]   The PCR primer according to claim 2, wherein in the general formula (I), the Tm value of the polynucleotide region comprising P1 to Pn is higher than the Tm value of the primer region.   The PCR primer according to claim 2, wherein in the general formula (I), the Tm value of the polynucleotide region comprising P1 to Pn is 1 to 20 ° C higher than the Tm value of the primer region.   In the general formula (II), the sum of the Tm value of the polynucleotide region composed of R1 to Rn and the Tm value of the polynucleotide region composed of T1 to To is higher than the Tm value of the primer region. Item 3. A PCR primer according to Item 2.   In the general formula (II), the sum of the Tm value of the polynucleotide region consisting of R1 to Rn and the Tm value of the polynucleotide region consisting of T1 to To is 1 to 20 ° C. higher than the Tm value of the primer region. The primer for PCR according to claim 2.   The primer for PCR according to any one of claims 1 to 6, wherein a labeled molecule is bound thereto.   The PCR primer according to claim 7, wherein the labeling molecule is at least one selected from the group consisting of dispersible fine particles, enzymes, and magnetic substances. A method for detecting a target nucleic acid having a target base sequence,
(A) performing PCR using PCR primers to obtain a PCR product;
(B) forming the PCR product-ligand complex by binding the PCR product obtained in the step (a) with a ligand capable of specifically binding to the aptamer region of the PCR primer;
(C) detecting the PCR product-ligand complex obtained in the step (b);
A method for detecting a target nucleic acid, characterized in that the PCR primer is the PCR primer according to any one of claims 1 to 8. The PCR primer is the PCR primer according to any one of claims 2 to 8,
Tm value of the polynucleotide region consisting of P1 to Pn in the general formula (I), or Tm value of the polynucleotide region consisting of R1 to Rn and the Tm of the polynucleotide region consisting of T1 to To in the general formula (II) The method for detecting a target nucleic acid according to claim 9, wherein the sum of the values is higher than the PCR extension reaction temperature in the step (a).   The PCR in the step (a) is performed using a nucleotide modified with a modifying molecule, and the PCR product-ligand complex detection in the step (c) is performed by detecting the modifying molecule. The method for detecting a target nucleic acid according to claim 9 or 10, wherein the target nucleic acid is present.   The method for detecting a target nucleic acid according to claim 11, wherein the modifying molecule is a fluorescent substance or a radioisotope.   The method for detecting a target nucleic acid according to claim 9 or 10, wherein the ligand is bound to a labeled molecule.   The method for detecting a target nucleic acid according to claim 13, wherein the labeling molecule is one or more selected from the group consisting of dispersible fine particles, enzymes, fluorescent substances, and radioisotopes. A method for detecting a target biomolecule in a sample solution,
(A ′) performing PCR using a primer for PCR having an aptamer region capable of specifically binding to a target biomolecule to obtain a PCR product;
(B ′) adding the PCR product obtained in the step (a ′) to a sample solution to form a PCR product-target biomolecule complex;
(C ′) detecting the PCR product-target biomolecule complex obtained in the step (b ′);
A method for detecting a target biomolecule, characterized in that the PCR primer is the PCR primer according to any one of claims 1 to 8. The PCR primer is the PCR primer according to any one of claims 2 to 8,
Tm value of the polynucleotide region consisting of P1 to Pn in the general formula (I), or Tm value of the polynucleotide region consisting of R1 to Rn and the Tm of the polynucleotide region consisting of T1 to To in the general formula (II) 16. The method for detecting a target biomolecule according to claim 15, wherein the sum of the values is higher than the PCR extension reaction temperature in the step (a ′).   PCR in the step (a ′) is performed using a nucleotide modified with a modifying molecule, and the PCR product-target biomolecule complex detection in the step (c ′) detects the modifying molecule. The method for detecting a target biomolecule according to claim 15 or 16, wherein   The method for detecting a target biomolecule according to claim 17, wherein the modifying molecule is a fluorescent substance or a radioisotope.

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