WO2008007571A1 - Procédé de détection d'acide nucléique - Google Patents

Procédé de détection d'acide nucléique Download PDF

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Publication number
WO2008007571A1
WO2008007571A1 PCT/JP2007/063162 JP2007063162W WO2008007571A1 WO 2008007571 A1 WO2008007571 A1 WO 2008007571A1 JP 2007063162 W JP2007063162 W JP 2007063162W WO 2008007571 A1 WO2008007571 A1 WO 2008007571A1
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molecular weight
nucleic acid
primer
bound
detecting
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PCT/JP2007/063162
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English (en)
Japanese (ja)
Inventor
Mari Nakamoto
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Olympus Corporation
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Publication of WO2008007571A1 publication Critical patent/WO2008007571A1/fr

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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

  • the present invention relates to a method for efficiently and easily detecting a nucleic acid, which is an amplification product obtained by a polymerase chain reaction method, with high accuracy.
  • DNA is prepared by using two kinds of primers, which are oligonucleotides each having a base sequence complementary to a specific site of each single strand constituting double-stranded DNA.
  • a method for amplifying a target region containing a base sequence to be detected is known (see Patent Document 1).
  • This method is well known as a polymerase chain reaction method (hereinafter sometimes abbreviated as a PCR method). That is, first, a sample double-stranded DNA is prepared, two primers are hybridized to each single-stranded DNA, and then DNA polymerase is allowed to act. Using the single-stranded DNA as a cage, each primer chain extension strand is generated.
  • the base sequence to be detected sandwiched between the primers in the double-stranded DNA can be obtained by amplifying a target region containing.
  • a DNA amplification product obtained by PCR is a mixture of a plurality of types of nucleic acids. Therefore, by electrophoresis using a gel such as polyacrylamide Nyagarose as a carrier for separation, the plurality of types of DNA amplification products are obtained. Amplified products are separated based on differences in molecular weight, and then stained with a dye such as ethidium bromide. Thereby, the amplification product of the target region can be detected by detecting the fluorescent signal emitted by the dye. Alternatively, when the base sequence of the target region can be predicted, a so-called blotting operation can be applied.
  • a radioisotope-labeled probe having a base sequence complementary to the expected base sequence is hybridized to the amplified product after transfer to autoradiography.
  • the amplification product of the target region can be detected.
  • various DNA sequences can be selected by appropriately selecting the base sequence of the primer. This region can be specifically amplified. For this reason, applications such as detecting the genomes of pathogenic bacteria and viruses by PCR and diagnosing infectious diseases have been made.
  • the target DNA when the target DNA is very small, it may be necessary to amplify multiple regions in the DNA simultaneously by multiplex PCR.
  • the molecular weights of the amplification products are similar to each other, primers having different base sequences are labeled with different fluorescent dyes for each region, and the obtained amplification products are electrophoresed, and then the respective fluorescence signals.
  • a method is disclosed that detects the detection of! (See Patent Document 2).
  • the target region is amplified by PCR in two steps using DNA immobilized on a carrier. That is, pre-amplification is performed using a pair of primers corresponding to one target region in the DNA, with the immobilized DNA as a cage.
  • pre-amplification means the amount of amplification product normally required.
  • PCR Perform PCR with fewer thermal cycles than is possible. Specifically, the number of thermal cycles of pre-amplification is preferably 1 to 6, more preferably 1 to 4, and particularly preferably 3. However, the number may be larger as long as the object is achieved.
  • the temperature conditions and the number of cycles for pre-amplification are selected in consideration of the length of the target sequence, the Tm of the primer, the number of samples, the specifications of the amplification device, the operation time, etc. Transfer the resulting pre-amplification product to a separate container for temporary storage.
  • preamplification is performed using the same immobilized DNA as a template and a primer pair corresponding to another target region, and this preamplified product is further transferred to another container and temporarily stored. Repeat this procedure to prepare pre-amplification products for all target regions, then subject all of these pre-amplification products to normal thermal cycling at the same time until the desired amount of amplification product is obtained. Perform PCR. In this way, by performing amplification by PCR in two steps, a plurality of target regions in a small amount of DNA can be amplified in separate containers.
  • This method is useful for simultaneously amplifying multiple single nucleotide polymorphism (SNP) sites using a small amount of genomic DNA sample.
  • SNP single nucleotide polymorphism
  • the amplification region in the PCR method is set so that the migration distances of the amplification products at the time of separation are not the same as each other rather than detecting a plurality of types of amplification products having the same molecular weight.
  • a method for adjusting the position of a primer to be designed and subjected to hybridization is also disclosed (see Patent Document 4).
  • Patent Document 1 Japanese Patent Laid-Open No. 62-217161
  • Patent Document 2 Japanese Patent No. 3322894
  • Patent Document 3 Japanese Patent Laid-Open No. 2005-198581
  • Patent Document 4 Japanese Patent Laid-Open No. 2005-95025
  • Patent Document 2 requires a primer labeled with a fluorescent dye.
  • a primer is not only expensive, but also the fluorescent dye is unstable. There is a problem that the intensity of the signal decreases.
  • the fluorescent dye There is also a problem that it is unstable to light and requires attention for its storage and operation.
  • a detector for detecting a fluorescent signal is also expensive, and there is a problem that detection of a nucleic acid is expensive. The current detector can only detect fluorescent signals of 4 to 5 colors. Excluding the amount of fluorescent signal used as a detection size marker, the multiplex PCR is limited to 3 to 4 multiplexes. There is a problem.
  • Patent Document 3 has a problem in that throughput is reduced because operation is very complicated and time-consuming and detection cannot be performed at one time.
  • the present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for efficiently and easily detecting a target nucleic acid with high accuracy using a PCR method.
  • the present inventor performs PCR using a primer in which a substance that adjusts the molecular weight of the amplification product is previously bound, and adjusts the molecular weight of the obtained amplification product, thereby allowing the amplification product to migrate during electrophoresis. It has been found that the distance can be easily adjusted. And, by using a sucrose chain preferably having a large molecular weight as the molecular weight adjusting substance, and applying this technique, a plurality of amplification products can be obtained during electrophoresis even when a nucleic acid sample having a plurality of target regions is used. In order to complete the present invention, it was found that even if the target region can be clearly separated and the target region size is small, the migration distance of the amplification product can be reduced, and nucleic acid can be easily detected. It came.
  • the present invention relates to a plurality of target regions obtained by simultaneously amplifying a plurality of target regions in a nucleic acid sample by a polymerase chain reaction method using a pair of primers for each target region.
  • a method for detecting a nucleic acid by electrophoresis wherein all the primers of the primer pair have different base sequence powers, and at least one primer of at least one primer pair has a molecular weight modifier bound thereto, The sum of the molecular weights of the molecular weight adjusting substances to be bound is different for each primer pair, and the electrophoresis distances of the plurality of amplified products are made different from each other. It is a detection method.
  • the present invention also relates to a nucleic acid for amplifying a target region in a nucleic acid sample by a polymerase chain reaction method using a pair of primers, and detecting an amplification product of the obtained target region by electrophoresis.
  • a molecular weight adjusting substance is bound to at least one primer of the primer pair, and the electrophoresis distance of the amplification product is coupled with the molecular weight adjusting substance. It is a method for detecting a nucleic acid, characterized in that it is reduced as compared with the case of using a pair.
  • the present invention relates to the above (1) or (2), characterized in that the total molecular weight of the molecular weight adjusting substance coupled to the primer pair is different by at least 33,000 or more for each primer pair. It is a detection method of the nucleic acid as described.
  • the present invention provides the nucleic acid detection method according to any one of the above (1) to (3), wherein the molecular weight adjusting substance is a sugar chain.
  • the present invention is the method for detecting a nucleic acid according to the above (4), wherein sugar chains having the same molecular weight are bound to the primer by a different number for each primer pair.
  • the present invention provides the nucleic acid detection method according to (4) above, wherein sugar chains having different molecular weights are bound to the same number of primers for each primer pair.
  • the present invention provides the nucleic acid according to (4) above, wherein a plurality of sugar chains having different molecular weights are combined and bound to the primer so that the sum of the molecular weights differs for each primer pair. This is a detection method.
  • the present invention provides the nucleic acid detection method according to any one of (4) to (7) above, wherein the sugar chain has a molecular weight of 540 to 20000.
  • the present invention provides the nucleic acid detection method according to any one of (4) to (8) above, wherein the sugar chain is composed of subunits of maltotriose units. is there.
  • the invention's effect is to be applied to any one of (4) to (8) above, wherein the sugar chain is composed of subunits of maltotriose units. is there. The invention's effect
  • the obtained amplification product is based on the difference in the sum of the molecular weights of the molecular weight regulators bound thereto. Since the molecular weights are different from each other, individual amplification products can be clearly separated by electrophoresis. In addition, when the size of the target region is small, it can be adjusted so that the migration distance of the amplification product is reduced. Thereby, the target nucleic acid can be easily detected with high accuracy.
  • nucleic acid even if a small amount of nucleic acid is used, it can be multiplexed without changing the PCR conditions, which enables high-throughput analysis of a large number of nucleic acid samples at a time, enabling efficient analysis. Furthermore, since a fluorescent dye and a fluorescent signal detector are unnecessary, the target nucleic acid can be detected efficiently at low cost.
  • FIG. 1A is a conceptual diagram illustrating a primer to which a sugar chain is used, used in the present invention.
  • FIG. 1B is a conceptual diagram illustrating a primer to which a sugar chain is bound used in the present invention.
  • FIG. 1C is a conceptual diagram illustrating a primer with a sugar chain used in the present invention.
  • FIG. 1D is a conceptual diagram illustrating a primer with a sugar chain used in the present invention.
  • FIG. 2 shows the results of electrophoresis in Example 1 and Comparative Example 1.
  • FIG. 3 shows the results of electrophoresis in Example 2 and Comparative Example 2.
  • FIG. 4 is a diagram showing the results of electrophoresis in Example 3 and Comparative Example 3.
  • FIG. 5 shows the results of electrophoresis in Example 4 and Comparative Example 4.
  • a target region is a region that contains a base sequence to be detected in a nucleic acid sample and is amplified by a PCR method.
  • a plurality of target regions in a nucleic acid sample are simultaneously amplified by a polymerase chain reaction method using a pair of primers for each target region.
  • a nucleic acid detection method for detecting an amplification product of a target region by electrophoresis wherein the primers of the primer pair all have different base sequences, and at least one primer of at least one primer pair has a molecular weight adjusting substance.
  • the total molecular weight of the molecular weight adjusting substances is different for each primer pair, and the electrophoresis distances of the plurality of amplification products are made different from each other! It is characterized by.
  • the nucleic acid sample is not particularly limited as long as it is DNA containing the base sequence to be detected.
  • DNA including cDNA, genomic DNA, synthetic DNA, and the like, and analogs thereof may be derived from living organisms or artificially synthesized.
  • Primers having different base sequence capabilities are used. That is, since two types of primers, a forward primer and a reverse primer, are used for each target region, all primer pairs are different for each target region in the present invention.
  • any substance can be used as long as it can be bound to the primer by a conventionally known method and does not affect the PCR.
  • examples include sugar chains and heat resistant proteins. Of these, sugar chains are particularly preferred.
  • the molecular weight adjusting substance bound to one primer is not particularly limited as long as it does not impair the effect of the present invention that does not affect the good PCR.
  • the plurality of molecular weight adjusting substances may be the same or different.
  • the type and number of molecular weight adjusting substances to be bound to the primer may be appropriately selected so as to obtain an amplification product having a desired molecular weight in consideration of the mass of the molecular weight adjusting substance to be used and the solubility in water.
  • the binding of the molecular weight adjusting substance to the primer pair may be such that the migration distances of the plurality of amplification products during electrophoresis differ from each other. Therefore, if the molecular weight of the amplification product is set to be different !, it is all right! Therefore, when the molecular weight regulator is bound to the primer pair, the forward primer and the reverse primer of the primer pair are used. It may be bonded to at least one of the rimers.
  • the binding position of the molecular weight adjusting substance to the primer is not particularly limited.
  • the primer pairs to which the molecular weight modifier is bound need not be all of the primer pairs used for PCR amplification.
  • the molecular weight adjusting substance is not bound to the primer pair used for amplification of one target region of the plurality of target regions. Then, the molecular weight adjusting substance is bound to the primer pairs used for amplification of the remaining target regions, and the sum of the molecular weights of the molecular weight adjusting substances is different for each primer pair to which these molecular weight adjusting substances are bound (hereinafter, Abbreviated as Method A).
  • molecular weight adjusting substances may be bound to all pairs of primers used for amplification of a plurality of target regions, and the total molecular weight of the molecular weight adjusting substances may be different for each primer pair (hereinafter referred to as method B).
  • method B the total molecular weight of the molecular weight adjusting substances may be different for each primer pair.
  • the method A method is preferable because it can be used more easily because a primer pair that does not require binding of a molecular weight adjusting substance is used in combination.
  • the detection method of the present invention and the conventional detection method not using a molecular weight adjusting substance may be performed in parallel using the same nucleic acid sample.
  • the method A or the method B may be applied to the partial target regions.
  • the molecular weight of the amplification product obtained using the primer pair to which the molecular weight adjusting substance is bound is originally different from the size of the target region. Therefore, the molecular weight adjusting substance is bound, and the primer is used. It is necessary to ensure that the molecular weight of the amplification product obtained is comparable. As described above, it is also necessary to ensure that the molecular weight is not the same as the molecular weight of other amplification products mixed as impurities.
  • the molecular weight adjusting substance bound to the primer only needs to have a different total molecular weight for each primer pair.
  • sugar chains are used as molecular weight regulators, (i) a method in which sugar chains of the same molecular weight are bound to different numbers for each primer pair, and (ii) sugar chains of different molecular weights are used as primers. The same number of primers per pair And (iii) a method in which a plurality of sugar chains having different molecular weights are combined in such a way that the sum of molecular weights is different for each primer pair and bonded to the primer.
  • sugar chains of the same molecular weight refer to sugar chains of the same type that have almost no molecular weight distribution, or sugar chains that are of different types but have the same molecular weight and almost no molecular weight distribution.
  • the above methods (i) to (iii) can also be applied to molecular weight adjusting substances other than sugar chains.
  • FIGS. 1A to 1D are conceptual diagrams illustrating a polymer to which a sugar chain is bound as a molecular weight adjusting substance used in the present invention.
  • primer 1 is all of the same type.
  • each sugar chain is composed of the same type of unit 20
  • the first sugar chain 2 is composed of three units 20
  • the second sugar chain 3 is composed of twelve units 20.
  • the third sugar chain 4 consists of six units 20. That is, the first sugar chain 2, the second sugar chain 3, and the third sugar chain 4 have different molecular weights depending on the number of units 20.
  • the first sugar chain 2 is bound to primer 1 but the number of bound is different.
  • the total number of force units 20 with different types and numbers of sugar chains bound to primer 1 is the same, the sum of the molecular weights of the bound sugar chains is the same.
  • the combination of the types of linked sugar chains is different from any of FIG. 1A, FIG. IB and FIG. ID, and the total number of units 20 is also different.
  • the power explained using three types of sugar chains with three, six and twelve units is limited to the number of units in the present invention as long as the effects of the present invention are not impaired. There is no. And the number of units is not necessarily a multiple of 3.
  • the migration distance in the gel decreases as the molecular weight increases, mainly due to the molecular sieving effect, and the migration distance decreases.
  • the nucleic acid fragments subjected to electrophoresis usually need to have a size difference of about 15 to 20 base pairs. It is.
  • the molecular weight of a single base pair of nucleic acid is approximately 660, although there are some differences depending on the type of nucleobase.
  • nucleic acid fragments subjected to electrophoresis have a molecular weight difference of about 10,000 to 14,000, these nucleic acid fragments can be detected as bands that can be clearly distinguished. You can. In other words, if the molecular weight of the amplified product is stable without fluctuation due to PCR, a plurality of target regions of the same size can be obtained if the difference in the molecular weight of these amplified products is about 10000-14000. The migration distances during electrophoresis differ from each other, and can be clearly distinguished and detected.
  • the molecular weight adjusting substance bound to the primer pair has a sum of molecular weights. It is sufficient that the primer pairs differ by at least about 10,000 to 14,000. That is, it is preferable that the sum of the molecular weights of the molecular weight adjusting substances bound to the primer pair is different by at least 10,000 or more for each primer pair.
  • some target regions vary in the molecular weight of the amplification product depending on individual differences in the organism from which they are derived.
  • the amplification product may cause individual differences of about 20 to 30 base pairs depending on the individual from which it is derived.
  • the molecular weight adjusting substance is used as a primer so that the molecular weight of the amplification product is preferably 50 base pairs in terms of base pair, more preferably 60 base pairs, in other words, preferably about 33000, more preferably about 40,000. If they are bound in pairs, these amplification products can be clearly distinguished and detected.
  • the sum of the molecular weights of the molecular weight adjusting substances bound to the primer pairs is preferable for each primer pair. Should be different by about 3300, more preferably about 40000. That is, it is preferable that the sum of the molecular weights of the molecular weight modifiers bound to the primer pair differs by at least 33,000 for each primer pair.
  • the type of sugar chain is not particularly limited as long as the effect of the present invention that does not affect PCR is impaired. Therefore, any of naturally-derived materials, naturally-derived materials obtained by chemical modification and Z or chemical treatment, artificially synthesized materials, and the like can be used.
  • chemical modification refers to conversion of functional groups, introduction of substituents, and the like
  • chemical treatment refers to decomposition by enzymes and the like.
  • a sugar chain having higher solubility in water is more preferable. Examples of such preferred sugar chains include amylose.
  • the molecular weight of the product can be easily adjusted, those having a molecular weight of 540 to 20000 are preferred.
  • these sugar chains those prepared by a conventionally known method can be used. For example, when a natural product is prepared by chemical treatment, the method described in Japanese Patent No. 3012917 can be applied. The sugar chain to be used is more preferable as the molecular weight distribution is uniform.
  • particularly preferred sugar chains include those composed of three units as shown in FIG. 1A, and specifically, for example, those composed of subunits of maltotriose units.
  • a sugar chain having no molecular weight distribution when the molecular weight is about 7000 or more.
  • a sugar chain composed of subunits of maltotriose units can be obtained as having no molecular weight distribution, even if it has a molecular weight of about 10,000 to 22,000, by the method described in Japanese Patent No. 301 2917.
  • Such a high molecular weight sugar chain having no molecular weight distribution is suitable for giving a large difference in molecular weight between PCR amplification products.
  • the sugar chain can be bound to the primer by a conventionally known method.
  • a conventionally known method For example, the technique described in Japanese Patent Publication No. 2001-247596 can be applied.
  • any conventionally known method applied to nucleic acids can be applied.
  • the agarose gel electrophoresis method and the polyacrylamide gel electrophoresis method which are easy to make a gel and are inexpensive, are preferable.
  • the molecular weight of the amplified product can be determined by comparing the migration distance of the amplified product with the marker, and the molecular weight of the amplified product can be determined by subtracting the mass of the molecular weight adjusting substance bound to the primer. Can be calculated.
  • any method for detecting a band of an amplified product after electrophoresis can be applied as long as it is a conventionally known method.
  • a nucleic acid is stained with ethidium bromide and fluorescence is detected when UV irradiation is performed.
  • capillary electrophoresis is also suitable.
  • Agilent 2100 Bioanal yzer (trade name) has a small device, which is used to migrate amplification products in a gel packed in a single column, and use an intercalator at specific locations to move the amplification products. It can be easily detected sequentially.
  • the obtained amplification products contain molecular weight adjusting substances such that the sum of molecular weights is different. Are combined.
  • the target nucleic acid can be detected efficiently and easily with high accuracy.
  • one target region in a nucleic acid sample is amplified by a polymerase chain reaction method using a pair of primers, and the amplification product of the obtained target region is electrically converted.
  • a method for detecting a nucleic acid detected by electrophoresis wherein a molecular weight adjusting substance is bound to at least one primer of the primer pair, and an electrophoresis distance of the amplification product is bound to a molecular weight adjusting substance! It is characterized by being reduced compared to the case of using a small ⁇ primer pair.
  • the migration distance becomes too large, a band appears outside the marker, or other amplification mixed as an impurity after PCR amplification.
  • such a small target region is adjusted so that the migration distance of the amplification product of the target region is reduced by performing PCR amplification using a primer pair to which a molecular weight adjusting substance is bound.
  • the amplification product can be detected with high accuracy, efficiency, and simplicity.
  • the sum of the molecular weights of the molecular weight adjusting substances bound to the primer may be appropriately adjusted according to the size of the target region.
  • Human genomic DNA consisting of the base sequence shown in SEQ ID NO: 1 including the target region D3S 1293 shown in SEQ ID NO: 2, human genomic DNA consisting of the base sequence shown in SEQ ID NO: 3 containing the target region D3S 1234 shown in SEQ ID NO: 4, SEQ ID NO: 6
  • the primer pairs shown below were prepared in order to amplify these five target regions simultaneously by multiplex PCR using the human genomic DNA consisting of the base sequence shown in SEQ ID NO: 9 containing the region 13 actin.
  • target region D5S346 a forward primer in which four sugar chains having a molecular weight of 20000 are bound to the base sequence shown in SEQ ID NO: 15, and a reverse primer consisting of the base sequence shown in SEQ ID NO: 16.
  • Amplification of target region ⁇ -actin a forward primer in which eight sugar chains having a molecular weight of 20000 are bound to the base sequence shown in SEQ ID NO: 19, and a reverse primer consisting of the base sequence shown in SEQ ID NO: 20.
  • the human genomic DNA (about 5 ng), each primer pair (20 pmol) and 5 ⁇ L of 10 ⁇ Ex Taq buffer were mixed, and dNTPmix was mixed to a concentration of 0.2 mM to make a total of 50 ⁇ m.
  • dNTPmix was mixed to a concentration of 0.2 mM to make a total of 50 ⁇ m.
  • TaKaRa Ex Taq (Takara Bio Inc.) 1.25units
  • the PCR amplification was performed by repeating the thermal cycle of 94 ° CZ for 30 seconds, 55 ° CZ for 30 seconds, and 74 ° C / 30 seconds 30 times.
  • amplification products were separated by 4% agarose gel electrophoresis according to a conventionally known method, stained with ethidium bromide, and amplification products were detected at UV302 nm.
  • the result is shown in figure 2.
  • the numbers attached to the markers indicate the number of base pairs.
  • FIGS. As is clear from Fig. 2, the amplification products of five target regions of the same size can be clearly separated by changing the number of sugar chains of the same molecular weight to the forward primer of each primer pair. could be detected.
  • PCR amplification and amplification product detection were carried out in the same manner as in Example 1 except that no sugar chain was used. The result is shown in figure 2. As is clear from Fig. 2, the amplification products of the five target regions had almost the same mobility and were unable to be separated clearly.
  • Amplification of five target regions was performed by PCR in the same manner as in Example 1 except that the primer pairs shown below were used, and the amplification products obtained were detected by electrophoresis.
  • Amplification of target region ⁇ -actin a forward primer in which four sugar chains with a molecular weight of 20000 are bound to the base sequence shown in SEQ ID NO: 19, and a base sequence shown in SEQ ID NO: 20
  • a reverse primer in which four sugar chains with a molecular weight of 20000 are bound.
  • Target region shown in SEQ ID NO: 10 Amplification of the target region was performed by PCR using human genomic DNA having the nucleotide sequence shown in SEQ ID NO: 9 containing 8-actin.
  • a primer pair a foreprimer primer in which two sugar chains having a molecular weight of 10,000 were bonded to the base sequence shown in SEQ ID NO: 19, and one sugar chain having a molecular weight of 10,000 was connected to the base sequence shown in SEQ ID NO: 20.
  • a reverse primer was used.
  • PCR amplification and amplification product detection were performed in the same manner as in Example 1. The results are shown in Fig. 4. As is clear from Fig.
  • PCR amplification and amplification product detection were carried out in the same manner as in Example 3 except that no sugar chain was used. The results are shown in Fig. 4. As is clear from FIG. 4, the amplification product of the target region having a small size has a too long migration distance, and it is difficult to detect with high accuracy.
  • Example 4 As a primer pair, a forward primer obtained by binding a sugar chain having a molecular weight of 20000 and a sugar chain having a molecular weight of 14000 to the base sequence shown in SEQ ID NO: 19 one by one, and a reverse primer having a base sequence ability shown in SEQ ID NO: 20 PCR amplification and amplification product detection were performed in the same manner as in Example 3 except that was used. The results are shown in FIG. As is apparent from Fig. 5, by combining multiple sugar chains with different molecular weights with the forward primer, a target region with a small size can be obtained as an amplification product with a large molecular weight, and the migration distance in electrophoresis can be reduced. , Can be easy to detect.
  • PCR amplification and amplification product detection were performed in the same manner as in Example 4 except that no sugar chain was used. The results are shown in FIG. As is clear from FIG. 5, the amplification product of the target region with a small size has a too long migration distance, and it is difficult to detect with high accuracy.
  • the present invention is useful in the medical-related field in which a large number of nucleic acid samples are analyzed, and is suitable for simplification of clinical examinations, low cost, and the like.

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Abstract

La présente invention concerne un procédé de détection simple et efficace, avec une grande précision, d'un acide nucléique cible au moyen d'une réaction d'amplification en chaîne par polymérase. Le procédé de détection d'un acide nucléique consiste à amplifier simultanément de multiples régions cibles d'un échantillon d'acide nucléique par la réaction d'amplification en chaîne par polymérase en utilisant une paire d'amorces pour chacune des régions cibles, et à détecter par électrophorèse les produits amplifiés des multiples régions cibles ainsi obtenus. Le procédé est caractérisé en ce que les amorces des paires d'amorces sont toutes constituées par différentes séquences de nucléotides, un régulateur de la masse moléculaire est lié à au moins l'une des amorces d'au moins l'une des paires d'amorces, la masse moléculaire totale du régulateur de masse moléculaire lié est différente entre les paires d'amorces respectives, et la distance de migration des multiples produits amplifiés dans l'électrophorèse étant différente de l'un à l'autre. En tant que régulateur de la masse moléculaire, on préfère une chaîne de sucre.
PCT/JP2007/063162 2006-07-10 2007-06-29 Procédé de détection d'acide nucléique WO2008007571A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09285300A (ja) * 1996-04-23 1997-11-04 Gotaro Watanabe 遺伝子の塩基置換の検査法
JPH1060005A (ja) * 1996-08-14 1998-03-03 Agency Of Ind Science & Technol 糖鎖分子量マーカーおよびその製造方法
JP2000513202A (ja) * 1995-09-15 2000-10-10 ジェンザイム・コーポレーション 核酸の塩基配列決定または遺伝的置換の大量スクリーニング法
JP2001247596A (ja) * 2000-03-07 2001-09-11 Univ Nagoya オリゴヌクレオチド糖コンジュゲート、オリゴヌクレオチド糖コンジュゲートの製造方法
JP2005192498A (ja) * 2004-01-08 2005-07-21 Casio Comput Co Ltd 検査装置、検査方法及び合成装置

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000513202A (ja) * 1995-09-15 2000-10-10 ジェンザイム・コーポレーション 核酸の塩基配列決定または遺伝的置換の大量スクリーニング法
JPH09285300A (ja) * 1996-04-23 1997-11-04 Gotaro Watanabe 遺伝子の塩基置換の検査法
JPH1060005A (ja) * 1996-08-14 1998-03-03 Agency Of Ind Science & Technol 糖鎖分子量マーカーおよびその製造方法
JP2001247596A (ja) * 2000-03-07 2001-09-11 Univ Nagoya オリゴヌクレオチド糖コンジュゲート、オリゴヌクレオチド糖コンジュゲートの製造方法
JP2005192498A (ja) * 2004-01-08 2005-07-21 Casio Comput Co Ltd 検査装置、検査方法及び合成装置

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