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WO2006095550A1 - Amorce de pcr, methode mettant en application cette amorce et produit amplifie par pcr, dispositif et complexe adn-proteine mettant en application ce produit amplifie par pcr - Google Patents

Amorce de pcr, methode mettant en application cette amorce et produit amplifie par pcr, dispositif et complexe adn-proteine mettant en application ce produit amplifie par pcr Download PDF

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Publication number
WO2006095550A1
WO2006095550A1 PCT/JP2006/302772 JP2006302772W WO2006095550A1 WO 2006095550 A1 WO2006095550 A1 WO 2006095550A1 JP 2006302772 W JP2006302772 W JP 2006302772W WO 2006095550 A1 WO2006095550 A1 WO 2006095550A1
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dna
pcr
primer
double
amplification product
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Japanese (ja)
Inventor
Kazuhiko Nakatani
Gosuke Hayashi
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Kyoto University NUC
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Kyoto University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • PCR primers PCR methods and PCR amplification products using them, and devices and DNA-protein complexes using PCR amplification products
  • the present invention relates to a primer used in a PCR method, and a gene and protein-related technology using the primer.
  • a DNA microarray is a device in which other types of oligonucleotides are arranged in a minute space, and a target DNA (RNA) labeled (hereinafter “target”) is labeled and hybridized to hybridize.
  • RNA target DNA
  • a detection device By detecting the signal intensity due to the formation with a detection device, it is used as an object to analyze the sequence-specific bound target at once.
  • it is necessary to hybridize the immobilized probe DNA and the target on the microarray (double strand formation). After amplifying the target by the polymerase chain reaction method, it is usually made into a single strand by thermal dissociation so that this single-stranded target is neutralized with the probe DNA (-strand) on the array.
  • hybridization on the microarray can be performed only after the target DNA, which is a PCR amplification product, is made into a single strand, but for that purpose, calo-heat treatment is necessary. From the standpoint that single-stranded DNA tends to spheroidize and has poor detection sensitivity, the target DNA is placed in a part of the double-stranded DNA.
  • a “partial double-stranded DNA” having a single-stranded structure with a structure and other parts, and a production method and a detection method thereof are considered (see Patent Document 1).
  • this partial double-stranded DNA is synthesized by (1) synthesizing one type of oligonucleotide having the same base sequence as a part of the DNA to be produced. Synthesize two kinds of oligonucleotides having a base sequence complementary to the region (3) Make Perform the asymmetric PCR using the target DNA as a saddle, using one of the oligonucleotides synthesized in step (1) and the two oligonucleotides synthesized in step (2) as primers, and (4) prepare the DNA to be prepared. Asymmetric PCR is performed using the other of the two oligonucleotides synthesized in step (1) and the oligonucleotide synthesized in step (2) as a primer.
  • Amplification product and step (3) It can be obtained by mixing the amplification product of 4) and then heating and cooling.
  • the partial double-stranded DNA obtained in this way is obtained by concatenating a single-stranded portion having the same sequence as the DNA sequence continuous to the target portion to the target portion (double-stranded) of the genome. I can say that.
  • Patent Document 1 Japanese Patent Laid-Open No. 11-332566
  • asymmetric PCR is a technique that amplifies one of the DNA strands, and uses one of the two primers in excess. Conditions for starting the adjustment of the amount of each primer Setting is difficult, and asymmetric PCR needs to be performed twice in the method described in the above literature. Moreover, at least five steps including such asymmetric PCR must be performed, which is very complicated. In addition, since the amplification products of the above step (3) and step (4) contain many identical base sequence parts, even if they are mixed, heated and cooled in step (5), the target product is obtained. Since products other than partial double-stranded DNA are also generated with little force, it is difficult to say that the production efficiency of partial double-stranded DNA is very high.
  • step (5) which is the previous stage, it is essential to heat the amplification product of the asymmetric PCR, and thus there is a problem similar to the general method described above.
  • the present invention has an object to solve the above-mentioned conventional problems, and provides and provides a PCR primer obtained by an extremely simple method, and has wide applicability using the primer. For the purpose of providing!
  • the PCR primer of the present invention is used when a target natural DNA region in a sample is amplified by PCR, and is one of the 5 ′ ends of the double strands of the target DNA region. And a primer main body part having a DNA strand strength having a complementary sequence and a tag part which is modified at the 5 ′ end of the primer main body part and has a substance power not involved in PCR.
  • the primer body may be designed as appropriate according to the target region of the target DNA such as the target genome, in the same manner as the primer used in conventional PCR.
  • the tag part does not become a saddle shape nor is amplified by PCR with the primer body part. Therefore, the amplification product by PCR using such a PCR primer is a double-stranded DN having the same sequence as the target DNA region of interest. Takes a form with a tag added to A. Therefore, the PCR primer of the present invention can be used in various applied technologies described in detail later.
  • the primer main body portion and the tag portion may be directly adjacent to each other, or another substance may be interposed between them.
  • substances that can be used as the tag portion include all substances having specific binding ability with another substance.
  • examples of the relationship between the tag portion and the other substance include piotin and avidin, a DNA strand having a base sequence ability that is not present in the sample genome, and a complementary strand thereof.
  • the primer main body portion and the tag portion are not directly adjacent to each other, there is a substance that is not amplified by PCR between the primer main body portion and the tag portion.
  • the thing which intervened the spacer part can be mentioned.
  • the spacer portion due to the presence of the spacer portion, the double-stranded DNA and the tag portion formed by the primer body portion and its complementary strand are separated. The steric congestion between them can be reduced.
  • L-DNA non-natural DNA strand
  • D-DNA double-stranded natural DNA including the genome
  • L-DNA L-type DNA
  • the tag part consisting of L-DNA cannot be a primer in PCR. Therefore, when PCR is performed using this PCR primer, one of the natural double-stranded DNAs has a form in which a single-stranded L-DNA strand is tagged.
  • the base sequence and the number of bases in the L-DNA of the tag part are not particularly limited, and may be determined as appropriate.
  • the term “substance modified at the 5 ′ end of the primer main body and not amplified by PCR” or simply “tag part” in this specification indicates a concept including L-DNA.
  • a non-natural DNA strand can be mentioned as a preferable spacer portion interposed between the primer main body portion and the tag portion.
  • the tag part (L-DNA) of a PCR amplification product is bound to its complementary strand by this spacer part, double-stranded D-DNA and double-stranded L-DNA are present at close positions. Three-dimensional congestion can be eliminated.
  • the base sequence and the number of bases in the L-DNA strand employed as the spacer part are not particularly limited, and may be determined as appropriate.
  • examples of substances applicable to the spacer part include alkyl chains, peptides, peptide nucleic acids, sugar chains and the like.
  • the PCR primer set of the present invention including the PCR primer as described above has the above-described PCR primer as a forward primer, and has a complementary sequence to the other 3 'end of the double strand of the target DNA region. It has the DNA strand strength that it has as a reverse primer.
  • the PCR method according to the present invention includes a double-stranded DNA portion corresponding to a target natural DNA region using the PCR primer set described above, and the PCR primer in one of the double-stranded DNA portions.
  • An amplification product having a tag portion derived from the above-mentioned is obtained, and a region containing the target natural DNA region can be amplified as described above.
  • This pc According to the R method, it is possible to simultaneously perform PCR to obtain multiple types of amplification products in the same tube by approximating the conditions of the primer body, and the equipment, labor, and time required for PCR processing Can be greatly reduced.
  • the amplification product which is a reaction product by this PCR method, includes the double-stranded DNA portion corresponding to the target natural DNA region and the above-mentioned PCR in one of the double-stranded DNA portions. It has the tag part in a primer, It is characterized by the above-mentioned.
  • Such a PCR amplification product has a natural double-stranded DNA part amplified based on the primer body part! / And is therefore very stable. Is available.
  • a DNA carrying device comprises a substance that specifically binds to the tag portion in the PCR primer described above and a carrier that immobilizes and carries the substance.
  • the substance that specifically binds to the tag part is the complementary strand of the tag part L-DNA (of course, non-natural type). It is appropriate to fix the strong L-shaped probe to the carrier.
  • a substrate or a bead used for a general DNA chip can be applied.
  • the substance immobilized on the carrier may be one kind or plural kinds.
  • the following double-stranded DNA modification device can be easily obtained.
  • the double-stranded DNA modification device uses the PCR amplification product and the DNA carrying device described above, and a substance that specifically binds to the tag portion in the DNA carrying device, It is characterized in that the tag part of the PCR amplification product is bound. If such a double-stranded DNA modification device is used, the position of the double-stranded DNA via the tag part and a substance that specifically binds to it is designated on the carrier, and in particular if the carrier is an array. You can specify a fixed address. Therefore, this device can be regarded as a product obtained as a result of the detection process in the DNA detection method described below, and it can also be used for enzymatic reactions on the device as described later. can do.
  • the above-described DNA detection method is a method using the above-described PCR amplification product and a DNA-carrying device, and for a substance that specifically binds to the tag portion in the DNA-carrying device, It is characterized in that it includes a step of binding the tag portion of the PCR amplification product to the single strand DNA of the double-stranded DNA without dissociating it. According to such a detection method, it is not necessary to thermally dissociate the double-stranded DNA portion of the PCR amplification product, so that the PCR amplification product to be detected cannot be degraded.
  • the PCR amplification product can be reliably bound to almost all of the substance on the carrier.
  • the carrier not only one kind of substance that binds to the tag portion of the PCR amplification product but also a plurality of kinds can be immobilized. As a result, it is possible to simultaneously detect multiple types of PCR amplification products having corresponding tag portions.
  • the DNA-protein complex according to the present invention (double-stranded DNA to which a protein is bound) recognizes a predetermined sequence in the double-stranded DNA portion in the double-stranded DNA portion of the PCR amplification product. It is made by binding proteins that bind.
  • the protein is not particularly limited as long as it recognizes and binds to a specific sequence in double-stranded DNA, and a restriction enzyme can be mentioned as an example.
  • Such a DNA-protein complex can be used as a DNA-protein complex modifying device by using it together with a protein-carrying device as described below.
  • the protein-carrying device comprises a substance that specifically binds to the tag portion in either or both of the PCR amplification product and the DNA-protein complex described above, and the substance immobilized thereon. And a carrier to be supported.
  • the double-stranded DNA portion is configured to bind to a protein that recognizes and binds to a predetermined sequence in the double-stranded DNA portion. Since such a device has a substance that binds to the tag part of the PCR amplification product or DNA-protein complex, it can be used as a detection device for the PCR amplification product or DNA-protein complex.
  • the substrate of the DNA-protein complex modification device as described below It can also be used as a part. As a result, it becomes possible to fix the protein at a fixed position on the device.
  • not only one type of substance that binds to the PCR amplification product and the tag portion of the DNA-protein complex but also a plurality of types can be immobilized on the carrier. As a result, it is possible to simultaneously detect multiple types of PCR amplification products and DNA-protein complexes with corresponding tag parts.
  • substrates and beads used for general DNA chips and the like can be applied to the carrier.
  • the DNA-protein complex modifying device refers to the present invention! /, Which uses the above-mentioned DNA-protein complex and the protein-carrying device, and is unique to the tag portion in the protein-carrying device. Characterized in that the tag part of the DNA-protein complex is bound to the substance that binds to the target, or the PCR amplification product and the protein loading device described above, The tag portion of the PCR amplification product is bound to a substance that specifically binds to the tag portion of the device for the device, and the double-stranded DNA portion of the PCR amplification product recognizes the predetermined sequence in the double-stranded DNA portion. It is characterized by being bound to a protein that binds.
  • DNA-protein complex modification devices have the same final form, but the protein may be preliminarily bound to a double-stranded DNA and immobilized on the protein-carrying device, or the PCR amplification product may be immobilized on the protein. It may be bound to the PCR amplification product after being immobilized on the carrier device.
  • a device also referred to as a protein chip in which the protein is immobilized at a fixed position on the carrier via double-stranded DNA is obtained.
  • the complex of protein and double-stranded DNA can be fixed by designating the address on the array, and the latter embodiment
  • the carrier is an array
  • the protein can be immobilized on the double-stranded DNA immobilized at a specified position on the array.
  • immobilizing proteins it is possible to directly identify proteins by the MALDI-TOF method without using a dedicated instrument.
  • a protein transcription factor is immobilized on double-stranded DNA, it is possible to search for proteins that inhibit it, which is also useful for the development of new drugs.
  • an enzyme reaction using a protein immobilized on this device or a reaction with the enzyme can be performed. It is possible to search for a substance to perform.
  • PCR primer of the present invention By using the PCR primer of the present invention, a PCR amplification product having a tag portion not involved in PCR can be obtained by a very simple PCR method.
  • a device that immobilizes DNA on a carrier, a device that immobilizes a protein on double-stranded DNA on the device, and a device that immobilizes a complex of protein and double-stranded DNA are highly efficient. It is possible to obtain.
  • the base sequence of the tag part which cannot be a PCR-type, can be freely designed, so it can be used as a carrier for double-stranded DNA and DNA-protein complexes
  • immobilizing DNA it is possible to specify the position accurately, and it is easy to fix multiple types of double-stranded DNA and DNA-protein complexes.
  • a wide range of applications such as highly efficient detection of DNA, protein, DNA-protein complex, and enzymatic reaction on the device are possible.
  • FIG. 1 is a conceptual diagram showing a PCR step and a step of fixing a PCR amplification product to a DNA chip in one embodiment of the present invention.
  • FIG. 2 is a conceptual diagram showing a PCR amplification product / protein complex generation step, a step of fixing the complex to an array, and a PCR amplification product / protein complex generation step fixed to the array in the same embodiment.
  • FIG. 3 shows a primer set used in Example 1.
  • FIG. 4 shows the denaturing PAGE results of Example 2.
  • FIG. 5 is a partially enlarged conceptual diagram of the L-DNA microarray prepared in Example 3.
  • FIG. 6 is a view showing an SPR difference imaging image of Example 4.
  • FIG. 7 is a conceptual diagram showing a PCR amplification product fixation confirmation process of Example 5 and a graph showing SPR difference imaging results.
  • FIG. 8 is a view showing a test for fixing a tank to a double-stranded DNA portion with an L-DNA tag on the L-DNA microarray of Example 6.
  • FIG. 1 shows, as one embodiment of the present invention, a PCR process using PCR primer 1 and a double-stranded DNA obtained by immobilizing amplification product 3 by PCR to DNA microarray 4 which is a kind of DNA carrying device.
  • 3 is a conceptual diagram showing a process of obtaining a modification device 5.
  • the PCR primer 1 5 of one of the double strands of the target natural DNA region 0 (hereinafter referred to as "target region 0") for amplification by PCR is used. It has a primer main body part 11 composed of a natural (D-type) oligonucleotide having a complementary base sequence on the terminal side, and a tag part 12 connected to the 5 ′ end of the primer main body part 11.
  • the tag portion 12 employs a non-natural (L-type) oligonucleotide as an example of a substance that is not amplified by PCR.
  • the PCR method is performed using these forward primer (PCR primer 1) and reverse primer 2 as primer set 10.
  • the primer set 10 used in the PCR method in the flow (b) compared with the present embodiment as the forward primer 1, the primer body part 11, which is the same as the primer body part 11 of the PCR primer 1, A tag with a D-type oligonucleotide force 12 at the end is used, and the reverse primer 2 is the same oligonucleotide as the reverse primer 2.
  • PCR primer 1 in flow (a) is referred to as “L-DNA tagged primer 1”
  • PCR primer 1 ′ in flow (b) is referred to as “D-DNA tagged primer 1”.
  • a commonly used microarray substrate can be the carrier 41, 41', and the tag 41, 12 'is fixed to the carrier 41, 41'.
  • a DNA probe having a complementary strand strength of the tag parts 12 and 12 ′ is applied. That is, the L-DNA probe, which is the complementary strand of the tag portion 12, is applied to the DNA probe 42 used in the flow (a), and the complementary strand of the tag portion 12 'is applied to the DNA probe 42' used in the flow (b). Apply the D-DNA probe.
  • the L-DNA probe which is the complementary strand of the tag portion 12
  • the complementary strand of the tag portion 12 ' is applied to the DNA probe 42' used in the flow (b).
  • D-DNA-tagged primer 1 'force
  • the extended product has a double-stranded part only in the target region 0, and one of the five ends.
  • the product extended from the reverse primer 2 is a product in which both the target region 0 and the tag portion 12 ′ are double-stranded.
  • the main PCR amplification product 3 ′ finally obtained is normal double-stranded DNA that is longer than the target region 0 by the length corresponding to the tag portion 12 ′.
  • a PCR amplification product 3 ′ is immobilized on a DNA microarray 4 ′, first, the PCR amplification product 3 ′ is made into two single-stranded DNAs by thermal melting, and then this is converted into a DNA microarray 4 By contacting with ', single-stranded DNA on the side having the tag portion 12' is immobilized by annealing the tag portion 12 'and the D-DNA probe 42'.
  • a single-stranded DNA modification device 5 ′ is obtained in which only the tag portion 12 ′ forms a double strand with the DNA probe 42 ′ and the other portion of the single-stranded DNA is immobilized on the DNA microarray 4 ′. .
  • PCR amplification product 3 having a D-DNA double-stranded structure is generated, the step of heat-melting PCR amplification product 3 is not required for immobilization to DNA microarray 4, and the immobilized DNA is also tagged. The whole is double-stranded. Therefore, such a method for immobilizing PCR amplification product 3 on DNA microarray 4 is, for example, a method for detecting DNA as PCR amplification product 3 by applying a fluorescent label to PCR amplification product 3 and the like. It can also be applied as.
  • the double-stranded DNA portion 31 of the PCR amplification product 3 obtained by the PCR method of flow (a) When a specific protein 6 that recognizes a specific base sequence of A is bound, it is possible to generate a DNA-protein complex 7 as shown in FIG. 2 (a).
  • the specific base sequence of DNA and the protein recognizing the sequence are not particularly limited as long as the combination maintains the relationship between the two.
  • This DNA-protein complex 7 has the tag portion 12 that also has L-DNA force in the PCR amplification product 3 as it is. Therefore, as shown in FIG.
  • a device here, an L-DNA probe 82
  • the DNA-protein complex 7 can be immobilized by the hybridization of the tag portion 12 and the probe 82 on the protein-carrying device 8).
  • This immobilization method can also be used as a detection method for the DNA-protein complex 7, and the protein-carrying device 8 on which the DNA-protein complex 7 is immobilized is located at a predetermined position on the array. It can be used as a DNA-tank complex modification device 9 (so-called protein chip).
  • a DNA-protein complex 7 or DNA-protein complex modification device 9 is a single-stranded DNA immobilized on the DNA microarray 4 in flow (b). Stuff) can not get.
  • such a DNA-protein complex modification device 9 is prepared by preparing the above-described double-stranded DNA modification device 5 in advance as shown in FIG. It can also be prepared by binding specific protein 6 to 31 specific base sequences. Any method for producing such a DNA-protein complex modifying device 9 can also be used for screening specific tanks 6. In addition, whether the former or the latter method is used, examples of the use of the DNA-protein complex modification device 9 include an enzyme reaction with a specific protein 6 on the array, identification of the specific protein 6 by the MALDI-TOF method, Search for proteins that interact with specific protein 6 and so on.
  • Example ⁇ 1 Synthesis of L-DNA tagged primers>
  • two types of L-DNA tagged primers were synthesized. This synthesis was performed according to a standard synthesis protocol on an l.Ommol scale using an 392 DNA / RNA automatic synthesizer manufactured by Applied Systems.
  • Figure 3 shows primer sets A and B, both L-DNA tagged primers that are forward primers and their corresponding reverse primers. Since the PCR method using these PCR sets uses the plasmid pUC18 as a saddle type in this example, the primer main body 11 and the reverse primer in each L-DNA tagged primer are pUC18. It corresponds to the base sequence it has.
  • Each primer with L-DNA tag has such a natural (D-type) primer body part 11 that also has DNA chain strength on the 3 ′ end side, and a non-natural type on the 5 ′ end side of the primer body part 11 (L-type) It has a tag portion 12 that also has DNA strand strength, and a spacer portion 13 that also has L-type DNA strand strength is provided between the primer body portion 11 and the tag portion 12.
  • the spacer part 13 is added to eliminate the three-dimensional congestion of the L-type double-stranded DNA based on the tag part 12 and the D-type double-stranded DNA based on the primer main body part 11.
  • the tag part 12 is L-shaped, so it is not a PCR cage based on the primer body part 12.
  • each L-DNA tagged primer is obtained by connecting the tag part 12, the spacer part 13 and the primer main body part 11 in this order from the 5 ′ end side.
  • the tag portion 12 can comprise L-type DNA with an arbitrary base sequence and an arbitrary number of bases. In this embodiment, the number of bases is preferably 15 to 25. Yes.
  • the spacer part 13 can also constitute L-type DNA with any base sequence and any number of bases, but as described above, the L-type double-stranded DNA and the D-type double For the purpose of eliminating the steric congestion of the strand DNA, it is sufficient to set the number of bases to several mer. In this example, three thymines (T) arranged side by side are used.
  • a primer labeled with a biotin label at the end of 5 was also prepared in addition to a normal primer.
  • PCR reaction using primer set containing L-DNA tagged primer> As an example of the present invention, PCR was performed using primer sets A and B prepared in Example 1 above. . PCR reaction conditions were as follows: primer concentration: 500 nM, Taq polymerase Master Mix (Q IAGEN): 100 pg / ml, PCR cycle: 35 cycles (94 ° CX 30 seconds, 55 ° CX 30 seconds, 72 ° CX 1 minute). The PCR reaction using primer sets A and B can be performed simultaneously in one tube, but in this example, it is performed separately.
  • amplification product obtained by such a PCR reaction is such that the tag part 12 and the spacer part 13 of each L-DNA tagged primer should be a type of PCR extension reaction.
  • both PCR amplification products were subjected to heat-melting polyatalylamide gel electrophoresis (Denaturing PAGE) using double-stranded DNA as single strands under the conditions of 8% Polyacrylamide and 7M urea. The results are shown in Fig. 4.
  • Figure (a) is a photograph of Denaturing PAGE for PCR amplification products using Primer Set A. From this result, it can be seen that in this PCR, a 96-mer single strand and a 119-mer single strand hybridized were obtained as amplification products. The 96 mer single strand was extended from the reverse primer, and the 119 mer single strand was also extended from the forward primer (L-DNA tagged primer) force. The difference in the number of bases of 20 mer corresponds to the sum of the 20 mer of the tag part 12 and the 3 mer of the spacer part 13.
  • Figure (b) is a photograph of Denaturing PAG E targeting PCR amplification products using primer set B.
  • L-DNA microarray A DNA microarray was prepared as a DNA-supporting device for hybridizing the PCR amplification product obtained in Example 2 above. To prepare, use an Amino-modified SPR imaging chip manufactured by Toyobo Co., Ltd. with N-hydroxysuccinimide-polyethylene glycol (10 mg / ml, MW 3400, NHS-PEG-MAL, Q
  • the chip After soaking in PEG-thiol and reacting for 1 hour, the chip was thoroughly washed with a phosphate buffer (10 mM phosphate: pH 7.2 and 150 mM NaCl) and used for the experiment.
  • a phosphate buffer (10 mM phosphate: pH 7.2 and 150 mM NaCl)
  • L-DNA strands complementary to the tag portion 12 of each L-DNA tagged primer in primer sets A and B are fixed to an array for surface plasmon resonance difference imaging measurement (manufactured by Toyobo Co., Ltd.). I was ashamed.
  • L-DNA strands with thiols at the 3 ′ end were synthesized and used to react with the above-described maleimide immobilization array.
  • Figure 5 shows a partially enlarged conceptual diagram of the address of each probe (L-DNA probe) fixed on the array.
  • L-DNA microarray this microarray is referred to as “L-DNA microarray”.
  • Fig. 6 (a) shows the SPR imaging image when the PCR amplification product was neutralized with Primer Set A
  • Fig. 6 (b) shows the SPR imaging image when the PCR amplification product was Noiselized with Primer Set B. Indicates.
  • FIG. 7 A conceptual diagram of the test process is shown in Fig. 7 (a).
  • Figure (b) shows the graph of SPR imaging measurement results before and after immobilization of PCR amplification product 3.
  • the SPR signal intensity increased, indicating that avidin was bound to piotin labeled on PCR amplification product 3.
  • the anti-avidin antibody was added, a large signal increase was observed.
  • the reverse primer of primer set B was immobilized on the array, that is, the double-stranded DNA that was the PCR amplification product was immobilized. It was confirmed that this was the case.
  • FIG. 8 shows a probe immobilized on an L-DNA microarray and double-stranded DNA with an L-DNA tag.
  • Figure (b) shows SPR imaging from before immobilization of L-DNA tagged double-stranded DNA on the L-DNA microarray to after EcoRI binding to L-DNA tagged double-stranded DNA. A measurement result is shown with a graph. As is clear from the figure, 1) At the address where the L-DNA-tagged double-stranded DNA tag was immobilized on the probe, the force at which the SPR signal increased due to the immobilization was observed. No increase is observed.
  • a PCR amplification product with a tag part that is not directly involved in PCR can be obtained by ordinary PCR.
  • a device in which double-stranded DNA is immobilized on a carrier is used.
  • a device can be obtained in which a protein is immobilized on double-stranded DNA on the device, and a device in which a complex of protein and double-stranded DNA is immobilized.
  • the present invention can be used in fields such as extremely efficient detection of DNA, protein, DNA-protein complex, enzyme reaction on the device, and the like.

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Abstract

L'invention concerne une amorce de PCR capable d'immobiliser un produit double brin amplifié par PCR sur un réseau dans son état initial sans fusion, ainsi qu'une méthode mettant en application PCR simple au moyen de l'amorce et une technique servant à immobiliser une protéine sur un produit amplifié par PCR obtenu au moyen de ce procédé ou sur un produit amplifié par PCR immobilisé sur le réseau. Cette amorce comporte une région non impliquée dans la réaction par PCR, présente au niveau de la terminaison 5' du corps principal de l'amorce et comprenant une chaîne d'ADN naturel pouvant constituer un gabarit dans la méthode PCR.
PCT/JP2006/302772 2005-03-04 2006-02-16 Amorce de pcr, methode mettant en application cette amorce et produit amplifie par pcr, dispositif et complexe adn-proteine mettant en application ce produit amplifie par pcr Ceased WO2006095550A1 (fr)

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JP2007507025A JPWO2006095550A1 (ja) 2005-03-04 2006-02-16 Ｐｃｒプライマー、それを利用したｐｃｒ法及びｐｃｒ増幅産物、並びにｐｃｒ増幅産物を利用するデバイス及びｄｎａ−タンパク複合体

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JP2005061429 2005-03-04
JP2005-061429 2005-03-04

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009091934A1 (fr) * 2008-01-17 2009-07-23 Sequenom, Inc. Procédés et compositions d'analyse de séquence d'acide nucléique à simple molécule
WO2012070618A1 (fr) 2010-11-24 2012-05-31 株式会社カネカ Procédé de détection d'acide nucléique amplifié et dispositif de détection
JP2013059321A (ja) * 2011-09-14 2013-04-04 Ngk Insulators Ltd 標的核酸の検出方法及びそれに用いるキット
WO2013113965A1 (fr) * 2012-01-30 2013-08-08 Universitat Rovira I Virgili Amorces spéciales pour la réaction en chaîne par polymérase
WO2013162026A1 (fr) 2012-04-27 2013-10-31 株式会社カネカ Procédé d'amplification d'acide nucléique et procédé de détection d'acide nucléique amplifié
WO2014007289A1 (fr) 2012-07-05 2014-01-09 日本碍子株式会社 Méthode de chromatographie d'acide nucléique, composition pour la chromatographie d'acide nucléique et kit la contenant
JP2014057565A (ja) * 2012-09-14 2014-04-03 Ngk Insulators Ltd 標的核酸の検出方法
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WO2015076356A1 (fr) * 2013-11-22 2015-05-28 株式会社カネカ Méthode de détection d'arn à chaîne courte

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03231151A (ja) * 1990-02-07 1991-10-15 Takara Shuzo Co Ltd Ｄｎａの検出方法
JPH05236998A (ja) * 1992-02-27 1993-09-17 Olympus Optical Co Ltd 核酸検出方法
JPH11332566A (ja) * 1997-05-08 1999-12-07 Masao Karube 部分二重鎖ｄｎａを利用したｄｎａの検出方法
JP2001503973A (ja) * 1996-10-04 2001-03-27 ザ パーキン―エルマー コーポレイション 多重性ポリヌクレオチド捕獲方法および組成物
JP2004500850A (ja) * 2000-05-19 2004-01-15 エラジェン　バイオサイエンシズ　インコーポレイテッド 核酸の検出用物質及び検出方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030198980A1 (en) * 2001-12-21 2003-10-23 Applera Corporation Heteroconfigurational polynucleotides and methods of use

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03231151A (ja) * 1990-02-07 1991-10-15 Takara Shuzo Co Ltd Ｄｎａの検出方法
JPH05236998A (ja) * 1992-02-27 1993-09-17 Olympus Optical Co Ltd 核酸検出方法
JP2001503973A (ja) * 1996-10-04 2001-03-27 ザ パーキン―エルマー コーポレイション 多重性ポリヌクレオチド捕獲方法および組成物
JPH11332566A (ja) * 1997-05-08 1999-12-07 Masao Karube 部分二重鎖ｄｎａを利用したｄｎａの検出方法
JP2004500850A (ja) * 2000-05-19 2004-01-15 エラジェン　バイオサイエンシズ　インコーポレイテッド 核酸の検出用物質及び検出方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAYASHI G. ET AL.: "L-tai DNA o Kotei-ka Shita SPR Sensor Chip no Sakusei to Oyo", CSJ: THE CHEMICAL SOCIETY OF JAPAN KOEN YOKOSHU, vol. 85TH, no. 2, 11 March 2005 (2005-03-11), pages 1376, 3G6-11, XP003002256 *

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US9034580B2 (en) 2008-01-17 2015-05-19 Sequenom, Inc. Single molecule nucleic acid sequence analysis processes and compositions
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US11708607B2 (en) 2008-01-17 2023-07-25 Sequenom, Inc. Compositions containing identifier sequences on solid supports for nucleic acid sequence analysis
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