WO2011001496A1 - 検体解析方法およびそれに用いるアッセイキット - Google Patents
検体解析方法およびそれに用いるアッセイキット Download PDFInfo
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- WO2011001496A1 WO2011001496A1 PCT/JP2009/061878 JP2009061878W WO2011001496A1 WO 2011001496 A1 WO2011001496 A1 WO 2011001496A1 JP 2009061878 W JP2009061878 W JP 2009061878W WO 2011001496 A1 WO2011001496 A1 WO 2011001496A1
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- 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
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
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- 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
- C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
Definitions
- the present invention relates to a nucleic acid analysis method.
- a DNA chip is a device in which 10 to 10 5 types of DNA nucleic acid chains are immobilized as probes on a slide glass or silicon substrate of several centimeters.
- the contained nucleic acid is first labeled with a fluorescent dye or a radioisotope, and then reacted with a probe on the chip.
- Hybridization occurs if the nucleic acid in the sample contains a nucleic acid complementary to the probe on the chip.
- the arrangement and fixing position of the probe fixed on the chip are clear. Therefore, by specifying the position on the chip where the signal derived from the label is obtained, the sequence of the nucleic acid contained in the sample can be determined (Non-Patent Document 1).
- the DNA chip is a very useful device (testing instrument) for analyzing many genes at one time for one sample. Usually, one chip is used for one sample. When looking at the difference in the expression level of two samples, two chips are used on one chip.
- the number of genes examined is small, but the number of samples may be large.
- the inspection cost including the chip, labor, and time is high.
- an object of the present invention is to provide a method for analyzing a plurality of specimens quickly and simply by reducing the test cost per specimen.
- the present invention provides: A method for analyzing multiple samples, (A) preparing a primer set including a first primer including a tag sequence having a different sequence for each specimen and a second primer used in a pair with the first primer for each specimen; Wherein the tag sequence is designed to loop out when the first primer hybridizes to a template sequence in the template nucleic acid for each specimen; (B) a step of amplifying a template nucleic acid for each specimen using the primer set corresponding to each specimen in an independent reaction system for each specimen to obtain an amplification product into which the tag sequence has been introduced; (C) mixing the amplification products obtained for each specimen into one; (D) reacting a nucleic acid probe having a sequence for detecting a target sequence containing the tag sequence and immobilized on a substrate, with the amplification product mixed in step (c); (E) detecting the presence and / or amount of the target nucleic acid for each specimen by detecting the amount of hybridization produced in step (d); It is
- sequence introduction primer and a nucleic acid probe The scheme figure which shows an amplification process.
- the figure which shows a primer The figure which shows the intermediate product of LAMP amplification.
- the figure which shows a primer The scheme figure which shows an amplification process.
- nucleic acid is a generic term for substances capable of representing a part of their structure by nucleotide sequence, such as DNA, RNA, PNA, LNA, S-oligo, and methylphosphonate oligo. It is shown.
- the “specimen” is a target to be subjected to the analysis method according to the present invention, and may be any sample that may contain a nucleic acid.
- the specimen is preferably in a state that does not interfere with the amplification reaction and / or the hybridization reaction.
- pretreatment may be performed by any means known per se.
- the specimen may be a liquid, in which case it may be referred to as a “test liquid”. Therefore, the “test solution” may be understood as a solution in which a nucleic acid or a template nucleic acid may exist.
- multiple samples and “plural samples” indicate two or more samples and can be used interchangeably.
- sample nucleic acid The nucleic acid contained in the sample is referred to as “sample nucleic acid”.
- sample nucleic acid the sequence to be amplified by the primer according to the present invention is referred to as “template sequence”.
- a nucleic acid containing a template sequence is referred to as a “template nucleic acid” or “template”.
- a partial sequence contained in the template nucleic acid is referred to as a “partial nucleic acid sequence”.
- the partial nucleic acid sequence is the sequence or base to be analyzed, and the primer according to the present invention is designed to amplify the region containing the partial nucleic acid sequence.
- the partial nucleic acid sequence may be equal to or included in the template sequence.
- Target nucleic acid refers to an amplification product obtained by amplifying a template nucleic acid or a template sequence using a forward primer and a reverse primer according to the present invention.
- the target nucleic acid includes a target sequence in a part thereof.
- Target sequence consists of a tag sequence and a partial sequence of a template sequence. The target sequence is used to detect a target nucleic acid using a nucleic acid probe.
- Nucleic acid probe is a nucleic acid containing a sequence complementary to a target sequence.
- the nucleic acid probe is used by being immobilized on a solid phase such as a substrate and forms a hybrid with an amplification product containing a region derived from the template sequence.
- a region derived from the template sequence means a region in which the template sequence is reflected in the region amplified by the primer other than the region to which the primer binds. When detecting gene polymorphisms or gene mutations, the sites are designed to fit within this region.
- a “DNA chip” is an apparatus that analyzes a nucleic acid using a hybridization reaction between a nucleic acid probe having a sequence complementary to the nucleic acid to be detected and the nucleic acid to be detected.
- the term DNA chip is synonymous with commonly used terms such as “nucleic acid chip”, “microarray” and “DNA array”, and is used interchangeably.
- “Analyzing multiple samples” means analyzing a plurality of samples simultaneously. “Analyzing a plurality of nucleic acid sequences in a specimen” refers to simultaneously analyzing a plurality of partial nucleic acid sequences contained in one specimen. A plurality of partial nucleic acid sequences to be analyzed simultaneously may be included in one template nucleic acid, or may be included in different types of sample nucleic acids included in the sample.
- the analyzed items include, for example, detection of specific nucleic acids such as genes derived from viruses and / or bacteria, measurement of gene expression levels, identification of genotypes for polymorphisms and / or detection of the presence or absence of mutations. And so on.
- the present invention is not limited to this.
- FIG. 1 shows a tag sequence introduction primer and a nucleic acid probe.
- ⁇ Primer> As exemplified by the tag introduction F primer in FIG. 1, a sequence that binds to the primer binding site of the template nucleic acid and a tag sequence for analyzing multiple samples are introduced into the primer.
- tag sequences are used depending on the specimen. For example, in the case of preparing a tag sequence 5 bases, and A, T, C, when composed of four bases G, the tag sequence of 4 5 next 1024 are candidates. However, when a tag sequence having a difference of one base is used, there is a high risk of mismatch hybridization, and therefore, tag sequences having different sequences of two or more bases are preferably prepared.
- the number of tag sequences to be prepared may be as many as the number of samples constituting the sample group to be analyzed at a time. Thereby, it is possible to identify all the samples constituting the sample group.
- the tag sequence is designed so that when the primer containing it binds to the template nucleic acid, the tag sequence portion loops out without binding to the template nucleic acid.
- the length of the tag sequence may be within a range that allows loop-out and that includes a primer that can amplify the template nucleic acid. Although not limited thereto, it may be 20 bases or less, preferably 10 bases or less. For example, it may be 1-20 bases, 2-20 bases, 1-10 bases, 2-10 bases.
- the length of the primer may be 13 to 40 bases, for example, 15 to 30 bases.
- the place where the tag sequence is inserted into the primer may be at least one base from the 3 'end of the primer, but the primer must loop out of the tag sequence and bind to the template. It is preferable that the position is 3 bases or more from the 3 ′ end.
- the nucleic acid probe needs to contain both a tag sequence and a sequence derived from the template sequence. In this case, if the insertion site is positioned on the 5 'end side, the probe becomes longer and the specificity is lowered. From this, the insertion site is preferably closer to the 3 'end as long as the primer can be amplified by looping out, and is within 25 bases, more preferably within 15 bases from the 3' end.
- the primer into which the tag sequence to be used is introduced may be either a Forward primer (F primer) or a Reverse primer (R primer).
- F primer Forward primer
- R primer Reverse primer
- a tag sequence may be introduced.
- the type of nucleic acid constituting the tag sequence is not particularly limited as long as it is a substance capable of expressing a partial structure by a base sequence, such as DNA, RNA, PNA, LNA, S-oligo, and methylphosphonate oligo.
- the nucleic acid probe is designed to include a sequence complementary to the tag sequence in order to detect the tag sequence contained in the target nucleic acid. If necessary, it is designed to include a sequence complementary to the region derived from the template sequence as shown in FIG.
- the gene mutation and / or gene polymorphism site is located in a region derived from the template sequence in the vicinity of the primer binding site.
- a wild type detection nucleic acid probe and a wild type detection nucleic acid probe, each having a tag sequence detection sequence and a gene mutation and / or polymorphism detection sequence, are used.
- the target genotype can be determined by comparing the amount of hybridization between the target nucleic acid and the nucleic acid probe for detecting the mutant type and the target nucleic acid.
- the tag introduction primer that amplifies in common with the genus is prepared for each sample and amplified.
- a region characteristic of each species and specific to other species is located in a region derived from the template sequence in the vicinity of the primer binding site.
- the amount of hybridization between the nucleic acid probe for identifying the species and the target nucleic acid having the sequence for detecting the tag sequence, the sequence characteristic to each species, and the nucleic acid probe for the negative control indicating the sequence unrelated to the species and the target nucleic acid The species can be identified by comparing.
- nucleic acid amplification when detecting the presence or absence of nucleic acid amplification in a specimen, it is not always necessary to include a sequence complementary to the region derived from the template sequence, but it may be included.
- the chain length of the nucleic acid probe according to the present invention is not particularly limited, but is preferably in the range of 5 to 50 bases, more preferably in the range of 10 to 40 bases, and still more preferably in the range of 15 to 35 bases.
- the nucleic acid probe may be modified with a reactive functional group such as an amino group, a carboxyl group, a hydrosyl group, a thiol group or a sulfone group, or a substance such as avidin or biotin in order to be immobilized on the substrate. It is also possible to introduce a spacer between the functional group and the nucleotide. As the spacer, for example, an alkane skeleton, an ethylene glycol skeleton or the like may be used.
- the solid phase for immobilizing the nucleic acid probe may be any substrate generally used as a solid phase for a DNA chip.
- a substrate may be composed of glass, silicon, nitrocellulose film, nylon film, microtiter plate, electrode, magnet, bead, plastic, latex, synthetic resin, natural resin, or optical fiber, but is not limited thereto.
- a DNA chip may be constructed by immobilizing a plurality of types of nucleic acid probes on these substrates.
- primers (sample 1 primer, sample sequence 1, tag 2 and tag 3) introduced with tag sequences (tag sequence 1, tag sequence 2, tag sequence 3) having different sequences for each sample (sample 1, sample 2, sample 3).
- sample 2 primer, Sample 3 primer are prepared, and amplification is performed in an independent reaction system for each sample.
- the reaction system independent for each sample may be a reaction system in which each sample does not mix.
- amplification may be performed in a separate tube for each specimen.
- the “reaction system” refers to a space in which a reaction is performed, and may be a container such as a tube and a well.
- amplification products with partially different sequences depending on the specimen are obtained.
- the template nucleic acid does not exist (specimen 2)
- amplification does not occur and an amplification product is not obtained.
- the amplification product obtained in each reaction system includes a tag sequence and a region derived from the template nucleic acid that are different for each sample (sample 1, sample 3). Therefore, it is possible to specify the specimen by identifying the tag sequence contained in the amplification product.
- This amplification product is mixed and hybridized with a nucleic acid probe containing a sequence complementary to each tag sequence immobilized on the substrate as shown in FIG. Thereafter, hybridization between the amplification product and the nucleic acid probe is detected by an appropriate detection method.
- a primer is designed so that a mutation or polymorphic site is located in a region derived from the template sequence of the amplification product, the tag sequence and these mutations and / or polymorphisms are detected or identified. Mutation and / or polymorphism analysis can be performed by using a nucleic acid probe having a sequence and containing a sequence derived from a template sequence.
- FIG. 4 it is possible to multi-amplify a plurality of template sequences having different sequences in one reaction system.
- a plurality of template sequences can be detected by detecting the DNA chip on the amplification product of the specimen. Detection is performed by hybridization with a nucleic acid probe containing a sequence complementary to each tag sequence immobilized on the substrate. Thereafter, hybridization between the amplification product and the nucleic acid probe is detected by an appropriate detection method.
- FIG. 4 shows an example of three template sequences, it is clear that the number of template nucleic acids is not limited to this.
- a primer is designed in the region derived from the template sequence of the amplification product so that a site characteristic of the species to be mutated, polymorphic and / or identified and specific to other species can be located, the tag sequence and
- the detection target in the present invention includes, for example, individual genomic DNA, genomic RNA, mRNA, and the like.
- Individuals include, but are not limited to, humans, non-human animals, plants, and microorganisms such as viruses, bacteria, bacteria, yeasts and mycoplasmas.
- nucleic acids are extracted from samples collected from individuals, such as blood, serum, leukocytes, urine, stool, semen, saliva, tissue, biopsy, oral mucosa, cultured cells, sputum and the like. Alternatively, it is extracted directly from the microorganism. Nucleic acid extraction can be performed using, but not limited to, commercially available nucleic acid extraction kits such as QIAamp (manufactured by QIAGEN), Sumitest (manufactured by Sumitomo Metals), and the like. A solution containing nucleic acid extracted from an individual sample or a microorganism is used as a test solution.
- QIAamp manufactured by QIAGEN
- Sumitest manufactured by Sumitomo Metals
- the specimen is amplified by the amplification method according to the method of the present invention.
- the detection target is RNA
- it can be converted into complementary strand DNA by, for example, reverse transcriptase before amplification. Both reverse transcriptase and DNA polymerase may be added in the same tube, and reverse transcription and DNA amplification may be performed simultaneously.
- amplification methods include Polymerase chain reaction method (PCR method), Loop mediated isothermal amplification method (LAMP method), Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN method), Nucleic acid sequence-based amplification method (NASBA) Methods), Strand displacement amplification (SDA method), Ligase chain reaction (LCR method), Rolling Circle Amplification method (RCA method), and the like.
- PCR method Polymerase chain reaction method
- LAMP method Loop mediated isothermal amplification method
- ICAN method Isothermal and chimeric primer-initiated amplification of nucleic acids
- NASBA Nucleic acid sequence-based amplification method
- SDA method Strand displacement amplification
- LCR method Ligase chain reaction
- RCA method Rolling Circle Amplification method
- the obtained amplification product is fragmented or single-stranded as necessary.
- means for forming a single strand include heat denaturation, a method using beads and enzyme
- the target nucleic acid obtained by amplification preferably has a stem-loop structure.
- the amplification product having a stem-loop structure can conveniently use the sequence of the single-stranded loop portion for the reaction with the probe.
- the LAMP method for amplification of the target nucleic acid, the LAMP method (for example, refer to Japanese Patent No. 3313358) is preferably used.
- the LAMP method is a rapid and simple gene amplification method, and the amplification product has a stem-loop structure.
- FIG. 5 is a diagram showing an example of basic primer design used in the LAMP method.
- the principle of the LAMP method will be briefly described with reference to the schematic diagram of FIG.
- six types of primers that recognize a maximum of eight regions of a template nucleic acid and a strand displacement type DNA synthase are used.
- the template nucleic acid is amplified under isothermal (60-65 ° C.) conditions.
- the eight regions are defined as F3 region, F2 region, LF region, F1 region, B3c region, B2c region, LBc region, and B1c region in this order from the 5 'end side of the template nucleic acid.
- the F1c, F2c, F3c, B1, B2, and B3 regions indicate regions in the complementary strands of the F1, F2, F3, B1c, B2c, and B3c regions, respectively.
- the eight types of primers shown in FIG. 5 have a FIP inner primer having a sequence complementary to F1 on the 5 ′ end side and the same sequence as F2 on the 3 ′ end side, and the same sequence as B1c on the 5 ′ end side.
- 'BIP inner primer with sequence complementary to B2c on the terminal side F3 primer with the same sequence as F3 region, B3 primer with sequence complementary to B3c region, LFc primer with sequence complementary to LF region, LBc primer having the same sequence as the LBc region.
- Indispensable for the amplification reaction are the FIP inner primer and the BIP inner primer, and the F3 primer, B3 primer, LF primer and LB primer are added to increase the amplification efficiency.
- a loop structure as shown in Fig. 6 is formed in the amplified product by the LAMP method, between F2 region and F1 region, between F2c region and F1c region, between B2 region and B1 region, and between B2c region and B1c region. Is a single-stranded region. Therefore, if a target sequence is designed in this region, the target nucleic acid can be detected easily and with high sensitivity (see, for example, JP-A-2005-143492). When the target sequence overlaps with the LF primer and / or LB primer, it is preferable not to add the LF primer and / or LB primer.
- a primer having the tag sequence introduced into the F2 region and / or B2 region is prepared for each sample.
- amplification is performed in a reaction system independent for each specimen using the primer. For example, amplification may be performed in a separate tube for each specimen. After amplification, amplification products having partial sequences that differ depending on the specimen are obtained in the single-stranded loop portion. If no template nucleic acid is present, amplification does not occur and no amplification product is obtained. This amplified product is mixed and hybridized with a nucleic acid probe containing a sequence complementary to each tag sequence immobilized on a substrate as shown in FIG. Thereafter, hybridization between the amplification product and the nucleic acid probe is detected by an appropriate detection method.
- the region derived from the template sequence detected by the nucleic acid probe that is, between the F2 region and the F1 region, between the F2c region and the F1c region, between the B2 region and the B1 region, and between the B2c region and the B1c region. If mutations or polymorphic sites are located between them, detection can be performed by using a nucleic acid probe that detects these mutations or polymorphisms.
- FIG. 10 it is also possible to multi-amplify a plurality of types of template sequences consisting of different sequences within one reaction system.
- a DNA chip is detected for the amplification product of the specimen, it is possible to analyze a plurality of specimens for a plurality of target sequences. Detection is performed by hybridization with a nucleic acid probe containing a sequence complementary to each tag sequence immobilized on the substrate. Thereafter, hybridization between the amplification product and the nucleic acid probe is detected by an appropriate detection method.
- FIG. 10 shows an example of three template nucleic acids, but it is clear that the number of template nucleic acids is not limited to this.
- a primer is designed in the region derived from the template sequence of the amplification product so that a mutation or polymorphic site, and / or a site characteristic of the species to be identified and specific to other species can be located
- the tag sequence And analysis of mutations, polymorphisms, biological species, etc. by using nucleic acid probes comprising sequences derived from template sequences having sequences for detecting or identifying these mutations, polymorphisms and / or biological species It can be carried out.
- the DNA chip used in the present invention may be provided with a substrate and a nucleic acid probe immobilized on the substrate.
- the substrate of the DNA chip may be any conventionally known microarray substrate such as an electrochemical detection type represented by a current detection type, a fluorescence detection type, a chemical color development type, and a radioactivity detection type.
- any type of microarray can be produced by a method known per se.
- the negative control probe immobilization region and the detection probe immobilization region may be arranged on different electrodes.
- FIG. 11 An example of a DNA chip is shown in FIG. 11 as a schematic diagram, but is not limited thereto.
- the DNA chip has an immobilization region 2 on a substrate 1.
- the nucleic acid probe is immobilized on the immobilization region 2.
- Such a DNA chip can be manufactured by a method well known in the art. A person skilled in the art can appropriately change the design of the number and the arrangement of the immobilization regions 2 arranged on the substrate 1 as necessary.
- Such a DNA chip may be suitably used for a detection method using fluorescence.
- the DNA chip of FIG. 12 includes an electrode 12 on a base 11.
- the nucleic acid probe is immobilized on the electrode 12.
- the electrode 12 is connected to the pad 13. Electrical information from the electrode 12 is acquired via the pad 13.
- Such a DNA chip can be manufactured by a method well known in the art. A person skilled in the art can appropriately change the design of the number and arrangement of the electrodes 12 arranged on the substrate 11 as necessary.
- the DNA chip of this example may include a reference electrode and a counter electrode as necessary.
- the electrode is composed of a simple metal such as gold, gold alloy, silver, platinum, mercury, nickel, palladium, silicon, germanium, gallium or tungsten, or an alloy thereof, or carbon such as graphite or glassy carbon, or a material thereof. Although an oxide or a compound can be used, it is not limited to them.
- the DNA chip as in this example may be suitably used for an electrochemical detection method.
- Hybridization may be performed under appropriate conditions that allow sufficient hybridization. Appropriate conditions vary depending on the type and structure of the target nucleic acid, the type of base contained in the target sequence, and the type of nucleic acid probe.
- hybridization may be performed in a buffer solution having an ionic strength in the range of 0.01 to 5 and a pH in the range of 5 to 9.
- the reaction temperature may range from 10 ° C to 90 ° C.
- the reaction efficiency may be increased by stirring or shaking.
- a hybridization accelerator such as dextran sulfate, salmon sperm DNA, and bovine thymus DNA, EDTA, or a surfactant may be added.
- a washing solution for washing the DNA chip after hybridization has an ionic strength in the range of 0.01 to 5, and a buffer in the range of pH 5 to 9 is preferably used.
- the cleaning liquid preferably contains a salt and a surfactant.
- an SSC solution, Tris-HCl solution, Tween 20 solution, or SDS solution prepared using sodium chloride or sodium citrate is preferably used.
- the washing temperature is, for example, in the range of 10 ° C to 70 ° C.
- the washing solution passes or stays on the surface of the probe-immobilized substrate or the region where the nucleic acid probe is immobilized.
- the DNA chip may be immersed in a cleaning solution.
- the cleaning liquid is preferably stored in a temperature-controllable container.
- the detection of the hybrid produced by the hybridization process can utilize a fluorescence detection method and an electrochemical detection method.
- Primers used in the nucleic acid amplification step may be labeled with a fluorescently active substance such as a fluorescent dye such as FITC, Cy3, Cy5, or rhodamine.
- a second probe labeled with these substances may be used.
- a plurality of labeling substances may be used simultaneously.
- the detection device detects the labeled sequence or the label in the secondary probe. Use an appropriate detection device depending on the label used. For example, when a fluorescent substance is used as a label, it is detected using a fluorescence detector.
- Electrochemical detection method A double-stranded recognition substance well known in the art is used.
- the double-stranded recognition substance may be selected from Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator and polyintercalator. Further, these double strand recognition substances may be modified with an electrochemically active metal complex such as ferrocene or viologen.
- Double-strand recognition substances vary depending on the type, but are generally used at concentrations ranging from 1 ng / mL to 1 mg / mL.
- a buffer solution having an ionic strength in the range of 0.001 to 5 and a pH in the range of 5 to 10 can be used.
- the measurement is performed by applying a potential higher than the potential at which the double-stranded recognition substance reacts electrochemically and measuring the reaction current value derived from the double-stranded recognition substance.
- the potential may be applied at a constant speed, may be applied in pulses, or a constant potential may be applied.
- the current and voltage may be controlled using devices such as a potentiostat, a digital multimeter, and a function generator.
- Electrochemical detection can be performed by methods well known in the art. For example, the method described in JP-A-10-146183 can be used.
- the present invention also provides an assay kit for use in the nucleic acid analysis method described above.
- an assay kit is A tag sequence having a different sequence for each specimen, wherein the tag sequence is designed to loop out when hybridized to a template sequence in a template nucleic acid for each specimen; A primer set comprising a second primer used in pairs and a primer; and a DNA chip comprising a substrate and a nucleic acid probe complementary to a target sequence comprising the tag sequence immobilized on the substrate; What is necessary is just to comprise.
- the nucleic acid probe may be a nucleic acid probe complementary to a target sequence including a tag sequence and at least a part of a sequence derived from a template sequence in a specimen.
- the assay kit is A tag sequence having a different sequence for each nucleic acid partial sequence, wherein the tag sequence is designed to loop out when hybridized to a template sequence for each nucleic acid partial sequence;
- a primer set comprising a second primer used in pairs and a primer; and a DNA chip comprising a substrate and a nucleic acid probe complementary to a target sequence comprising the tag sequence immobilized on the substrate; What is necessary is just to comprise.
- the nucleic acid probe may be a nucleic acid probe complementary to a target sequence including a tag sequence and at least a part of a sequence derived from a template sequence for each nucleic acid partial sequence.
- the first primer included in the assay kit contains at least one kind and amount of primer necessary for use in one analysis.
- n types of first primers are used.
- the sequences may be identical except for the tag sequence.
- the second primer included in the assay kit contains an amount of primer necessary for use in at least one analysis.
- the second primer may include a tag sequence.
- the second primer may comprise the type and amount of primer necessary for use in at least one analysis.
- n types of second primers are used.
- the sequences may be the same except for the tag sequence.
- the first primer includes, for example, a tag sequence associated with at least n analytes that can contain at least a template nucleic acid, and a partial sequence of the template nucleic acid.
- the second primer contains the amount of primer necessary for use in at least one analysis.
- Such a second primer may be at least one reverse primer or forward primer used in pairs with the first primer. If the first primer is a forward primer, the second primer is a reverse primer, and if the first primer is a reverse primer, the second primer may be a forward primer.
- An assay kit for analytical methods utilizing the LAMP method is also provided.
- the F3, F2, LF, and F1 regions are designed from the 5 'end of the template sequence, and the B3c, B2c, LBc, and B1c regions are designed from the 3' end, the following 1 to 9 A primer set selected from at least one selected from the group consisting of: 1.
- FIP primer (first primer) having a sequence complementary to F1 on the 5 ′ end side, the same sequence as F2 on the 3 ′ end side, and a tag sequence that differs depending on the specimen in the F2 sequence, and 5 ′ BIP primer (second primer) having the same sequence as B1c on the terminal side and a sequence complementary to B2c on the 3 'terminal side; 2.
- FIP primer (second primer) having the same sequence as F2 on the 3 'end side with the sequence complementary to F1 on the 5' end side, and the same sequence as B1c on the 5 'end side, on the 3' end side
- FIP primer (first primer) that has a sequence complementary to F1 on the 5 'end, the same sequence as F2 on the 3' end, and a tag sequence that differs depending on the sample in the F2 sequence (first primer), 5 'end A BIP primer (second primer) having the same sequence as B1c on the side and a sequence complementary to B2c on the 3 'end side, an F3 primer (third primer) having the same sequence as the F3 region, and a B3c region B3 primer with a complementary sequence (fourth primer); Five.
- FIP primer (second primer) that has the same sequence as F2 on the 3 'end with the sequence complementary to F1 on the 5' end, the same sequence as B1c on the 5 'end, and B2c on the 3' end
- a BIP primer (first primer) having a sequence complementary to the B2c sequence and a tag sequence that differs depending on the sample
- an F3 primer (third primer) having the same sequence as the F3 region, and the B3c region B3 primer with a complementary sequence (fourth primer); 6.
- FIP primer (first primer) that has a sequence complementary to F1 on the 5 'end, the same sequence as F2 on the 3' end, and a tag sequence that differs depending on the sample in the F2 sequence (first primer), 5 'end BIP primer (second primer) that has the same sequence as B1c on the side, a sequence complementary to B2c on the 3 'end side, and a tag sequence that differs depending on the sample in the B2c sequence, the same sequence as the F3
- the assay kit may include an enzyme and / or container for performing an amplification reaction, a washing solution, a buffer solution, salts for preparing a buffer solution, and the like.
- the DNA chip may be included in a state where the nucleic acid probe and the substrate are not integrated.
- Such an assay kit makes it possible to analyze nucleic acids more easily.
- Example A specific example of detection by the method of the present invention will be shown.
- the following example is an example of a method for analyzing a plurality of samples using a primer and a DNA chip in which different tag sequences are inserted for each sample.
- FIP primer without base insertion (FIP-1; SEQ ID NO: 1), AC (FIP-2; SEQ ID NO: 2), ACAC (FIP-3; SEQ ID NO: 3) at the 4th base from the 3 ′ end side of the FIP primer, ACACAC (FIP-4; SEQ ID NO: 4), TGTG (FIP-5; SEQ ID NO: 5), TCTC (FIP-6; SEQ ID NO: 6) inserted, AC (6th base from FIP primer 3 'end) FIP-7 SEQ ID NO: 7), ACAC (FIP-8; SEQ ID NO: 8), ACACAC (FIP-9; SEQ ID NO: 9), TGTG (FIP-10; SEQ ID NO: 10), TCTC (FIP-11; SEQ ID NO: 11) 11 types of FIP primers were prepared.
- the BIP primer (SEQ ID NO: 12), F3 primer (SEQ ID NO: 13), B3 primer (SEQ ID NO: 14), and LBc primer (SEQ ID NO: 15) were
- Nucleic acids were amplified by the LAMP method at 63 ° C. for 1 hour. In the negative control, sterilized water was used instead of the template nucleic acid. The rise time of amplification was determined by detecting the white turbidity of pyrophosphoric acid produced in the amplification reaction and magnesium in the solution using a Loopamp real-time turbidity measuring device.
- the insert containing AC (FIP-7), ACAC (FIP-8), and ACACAC (FIP-9) at the 6th base from the 3 'end side of the FIP primer was cleaved with CviQI.
- FIP primer without base insertion (FIP-1) is not cleaved by CviQI.
- AC (FIP-7), ACAC (FIP-8), or ACACAC (FIP-9) is inserted, the primer insert loops out and anneals to the template nucleic acid. Therefore, if the target product is produced, the amplified product is cleaved with CviQI.
- nucleic acid probe The synthetic DNA oligonucleic acid probes used for detection are shown in Table 3.
- a nucleic acid probe having ACAC inserted at the 4th base from the 3 ′ end of the FIP primer (probe 2; SEQ ID NO: 17), TGTG inserted (probe 3: SEQ ID NO: 18), or TCTC inserted (Probe 4; SEQ ID NO: 19) and a single-base mutation (probe 5; SEQ ID NO: 20) were prepared in a portion that becomes a complementary strand of the region derived from the template sequence.
- a negative control a negative probe (probe 1; SEQ ID NO: 16) having a sequence unrelated to the MTHFR gene sequence was used.
- the above 5 types of probes were thiol-modified at the 3 ′ end for immobilization on the gold electrode.
- the nucleic acid probe was immobilized on a gold electrode.
- the nucleic acid probe was immobilized using the strong covalent bond between thiol and gold.
- the nucleic acid probe solution was spotted on a gold electrode and allowed to stand at 25 ° C. for 1 hour. Then, it was immersed in a 1 mM mercaptohexanol solution and washed with a 0.2 ⁇ SSC solution. The same probe was spotted on each of the four electrodes. The position of the electrode of each nucleic acid probe is shown below. After washing, it was washed with ultrapure water and air-dried to obtain a DNA chip for electrochemical detection.
- Electrode arrangement The correspondence between the electrode and the nucleic acid probe immobilized thereon is as follows.
- the DNA chip for electrochemical detection was immersed in a phosphate buffer containing 50 ⁇ M Hoechst 33258 solution as an intercalating agent for 1 minute. Thereafter, the oxidation current response of Hoechst 33258 solution was measured.
- FIG. 14A shows the amplification of the TGTG (FIP-5) and TCTC (FIP-6) insertion primer sets amplified by adding the ACAC (FIP-3) insertion primer set amplified by adding the human genome and sterilized water. It is a mixture of products.
- FIG. 14B shows amplification of the TGTG (FIP-5) insertion primer set amplified by adding the human genome and the ACAC (FIP-3) and TCTC (FIP-6) insertion primer set amplified by adding sterile water. It is a mixture of products.
- FIG. 14A shows the amplification of the TGTG (FIP-5) and TCTC (FIP-6) insertion primer sets amplified by adding the human genome and sterilized water. It is a mixture of products.
- FIG. 14B shows amplification of the TGTG (FIP-5) insertion primer set amplified by adding the human genome and the ACAC (FIP-3) and TCTC (FIP-6) insertion primer set amp
- FIG. 14C shows the amplification product of the TCTC (FIP-6) insertion primer set amplified by adding the human genome and the ACAC (FIP-3) and TGTG (FIP-5) insertion primer set amplified by adding sterile water.
- TCTC TCTC
- FIG. 14B a high signal was obtained from the nucleic acid probe 3 for detecting TGTG.
- FIG. 14C a high signal was obtained from the nucleic acid probe 4 for detecting TCTC.
- the present invention is useful for analyzing nucleic acids such as genes in the fields of medicine, medicine, food, agriculture, fishery, livestock industry and horticulture.
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Abstract
Description
複数検体の解析方法であって、
(a)前記検体毎に異なる配列を有するタグ配列を含む第1のプライマーと、前記第1のプライマーと対で使用される第2のプライマーとを含むプライマーセットを検体毎に準備する工程と、ここで、前記タグ配列は、前記第1のプライマーが、前記検体毎の鋳型核酸中の鋳型配列にハイブリダイズしたときにループアウトするように設計されている;
(b)前記検体毎に独立した反応系で、前記検体毎に対応する前記プライマーセットを用いて、前記検体毎の鋳型核酸を増幅し、前記タグ配列が導入された増幅産物を得る工程と;
(c)前記検体毎に得られた前記増幅産物を1つに混合する工程と;
(d)前記当該タグ配列を含む標的配列を検出する配列を有し、基体上に固定化された核酸プローブと、工程(c)で混合された前記増幅産物とを反応させる工程と;
(e)工程(d)において生じたハイブリダイゼーション量を検出することにより、各検体について前記標的核酸の有無および/または量を検出する工程と;
を具備する解析方法
である。
本明細書で使用される「核酸」という用語は、DNA、RNA、PNA、LNA、S-オリゴ、メチルホスホネートオリゴなど、その一部の構造を塩基配列によって表すことが可能な物質を総括的に示すものである。
以下、本発明の実施態様について説明する。
図1のタグ導入Fプライマーに例示されるように、プライマーには、鋳型核酸のプライマー結合部位に結合する配列と、多検体を解析するためのタグ配列を導入する。
核酸プローブは、標的核酸中に含まれるタグ配列を検出するため、タグ配列に相補的な配列を含むように設計される。また必要に応じて図1に示すように鋳型配列由来の領域と相補的な配列を含むように設計される。
次にタグ配列を導入したプライマーとDNAチップを用いた多検体の解析方法について説明する。
本発明において使用されるDNAチップは、基体と基体上に固定化された核酸プローブとを具備すればよい。DNAチップの基体は、電流検出型を代表とする電気化学的検出型、蛍光検出型、化学発色型および放射能検出型など、従来公知の何れの種類のマイクロアレイ用の基体であってよい。
ハイブリダイゼーションは、ハイブリッドが十分に形成される適切な条件下で行えばよい。適切な条件は、標的核酸の種類及び構造、標的配列に含まれる塩基の種類、核酸プローブの種類によって異なる。例えば、イオン強度が0.01~5の範囲であり、pH5~9の範囲の緩衝液中でハイブリダイゼーションを行えばよい。反応温度は10℃~90℃の範囲であってよい。攪拌や振盪などにより、反応効率を高めても良い。反応溶液中には、硫酸デキストラン、サケ精子DNA、及び牛胸腺DNAのようなハイブリダーゼション促進剤、EDTA、又は界面活性剤などを添加しても良い。
ハイブリダイゼーション後、DNAチップを洗浄するための洗浄液は、イオン強度が0.01~5の範囲であり、pH5~9の範囲の緩衝液が好適に用いられる。洗浄液は塩及び界面活性剤などを含むことが好ましい。例えば、塩化ナトリウム又はクエン酸ナトリウムを用いて調製したSSC溶液、Tris-HCl溶液、Tween20溶液、又はSDS溶液などが好適に用いられる。洗浄温度は、例えば10℃~70℃の範囲で行う。洗浄液は、プローブ固定化基体の表面又は核酸プローブを固定化した領域に通過又は滞留させる。或いは、洗浄液中にDNAチップを浸漬させてもよい。この場合、洗浄液は温度制御可能な容器中に収容されることが好ましい。
ハイブリダイゼーション工程により生じたハイブリッドの検出は、蛍光検出方式及び電気化学的検出方式を利用することができる。
蛍光標識物質を用いて検出する。核酸の増幅工程で用いるプライマーをFITC、Cy3、Cy5、又はローダミンなどの蛍光色素のような、蛍光学的に活性な物質で標識してもよい。或いは、それらの物質で標識したセカンドプローブを用いてもよい。複数の標識物質を同時に使用してもよい。検出装置により、標識された配列または2次プローブ中の標識を検出する。使用する標識に応じて適切な検出装置を用いる。例えば、蛍光物質を標識として用いた場合、蛍光検出器を用いて検出する。
当該分野で周知の2本鎖認識物質を用いる。2本鎖認識物質は、ヘキスト33258、アクリジンオレンジ、キナクリン、ドウノマイシン、メタロインターカレーター、ビスアクリジン等のビスインターカレーター、トリスインターカレーター及びポリインターカレーターから選択してよい。更に、これらの2本鎖認識物質を電気化学的に活性な金属錯体、例えばフェロセン、ビオロゲンなどで修飾してもよい。
また本発明は、上述した核酸の解析方法において使用するためのアッセイキットも提供する。そのようなアッセイキットは、
検体毎に異なる配列を有するタグ配列を含み、前記タグ配列は、前記検体毎の鋳型核酸中の鋳型配列にハイブリダイズしたときにループアウトするように設計された第1のプライマーと、前記第1のプライマーと対で使用される第2のプライマーとを含むプライマーセット;および
基体と、前記基体上に固定化された前記タグ配列を含む標的配列に相補的な核酸プローブとを具備するDNAチップ;
を具備すればよい。
核酸部分配列毎に異なる配列を有するタグ配列を含み、前記タグ配列は、前記核酸部分配列毎の鋳型配列にハイブリダイズしたときにループアウトするように設計された第1のプライマーと、前記第1のプライマーと対で使用される第2のプライマーとを含むプライマーセット;および
基体と、前記基体上に固定化された前記タグ配列を含む標的配列に相補的な核酸プローブとを具備するDNAチップ;
を具備すればよい。
1. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー(第1のプライマー)、および5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもつBIPプライマー(第2のプライマー);
2. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもつFIPプライマー(第2のプライマー)、および5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー(第1のプライマー);
3. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー(第1のプライマー)、および5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー(第2のプライマー);
4. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー(第1のプライマー)、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもつBIPプライマー(第2のプライマー)、F3領域と同じ配列をもつF3プライマー(第3のプライマー)、およびB3c領域と相補的な配列をもつB3プライマー(第4のプライマー);
5. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもつFIPプライマー(第2のプライマー)、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー(第1のプライマー)、F3領域と同じ配列をもつF3プライマー(第3のプライマー)、およびB3c領域と相補的な配列をもつB3プライマー(第4のプライマー);
6. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー(第1のプライマー)、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー(第2のプライマー)、F3領域と同じ配列をもつF3プライマー(第3のプライマー)、およびB3c領域と相補的な配列をもつB3プライマー(第4のプライマー);
7. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー(第1のプライマー)、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもつBIPプライマー(第2のプライマー)、F3領域と同じ配列をもつF3プライマー(第3のプライマー)、B3c領域と相補的な配列をもつB3プライマー(第4のプライマー)、LF領域と相補的な配列をもつLFcプライマー(第5のプライマー)、およびLBc領域と同じ配列をもつLBcプライマー(第6のプライマー);
8. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもつFIPプライマー(第2のプライマー)、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー(第1のプライマー)、F3領域と同じ配列をもつF3プライマー(第3のプライマー)、B3c領域と相補的な配列をもつB3プライマー(第4のプライマー)、LF領域と相補的な配列をもつLFcプライマー(第5のプライマー)、およびLBc領域と同じ配列をもつLBcプライマー(第6のプライマー);
9. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー(第1のプライマー)、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー(第2のプライマー)、F3領域と同じ配列をもつF3プライマー(第3のプライマー)、B3c領域と相補的な配列をもつB3プライマー(第4のプライマー)、LF領域と相補的な配列をもつLFcプライマー(第5のプライマー)、およびLBc領域と同じ配列をもつLBcプライマー(第6のプライマー)。
本発明の方法による検出の具体例を示す。以下の例は、検体ごとに異なるタグ配列を挿入したプライマーとDNAチップを用いて複数検体を解析する方法の例である。
はじめにタグ配列を挿入したプライマーを用いて遺伝子増幅を行い、目的の遺伝子産物が生成されているかを制限酵素により確認した。配列番号21のヒトのMTHFR遺伝子を鋳型核酸として使用し、これをLAMP法により増幅した。
標的核酸の増幅に用いた合成DNAオリゴプライマーを表1に示した。
LAMP法により、63℃で1時間、核酸を増幅させた。ネガティブコントロールでは、鋳型核酸の代わりに滅菌水を用いた。増幅の立ち上がり時間はLoopampリアルタイム濁度測定装置を用い、増幅反応に伴い生成されるピロリン酸と溶液中のマグネシウムの白濁を検出することで行った。
FIPプライマーの3’末端側から4塩基目にAC(FIP-2)、ACAC(FIP-3)、ACACAC(FIP-4)を導入したものをAccIで切断した。塩基挿入なしのFIPプライマー(FIP-1)はAccIで切断されないが、AC(FIP-2)、ACAC(FIP-3)、ACACAC(FIP-4)を挿入したものはプライマーの挿入部分がループアウトして鋳型核酸にアニーリングする。従って、目的の産物が生成されている場合、その増幅産物は当該AccIで切断される。
塩基挿入なし(FIP-1)のものは、立ち上がり時間は29分であった。FIPプライマーの3’末端側から4塩基目にAC(FIP-2)、ACAC(FIP-3)、ACACAC(FIP-4)、TGTG(FIP-5)、TCTC(FIP-6)を挿入した試料の立ち上がり時間は、32分、37分、38分、38分、37分であった。さらに、FIPプライマー3’末端側から6塩基目にAC(FIP-7)、ACAC(FIP-8)、ACACAC(FIP-9)、TGTG(FIP-10)、TCTC(FIP-11)を挿入した試料の立ち上がり時間は、30分、38分、47分、32分、35分であった。ネガティブコントロールでは増幅が立ち上がらなかった。このことから、塩基挿入したことにより、50分以内には問題なく増幅は立ち上がり、増幅産物が得られることが明らかになった。また、図13で示したように、制限酵素により目的産物が増幅されていることが確認された。
FIPプライマーの3’末端側から4塩基目にACAC(FIP-3)、TGTG(FIP-5)、TCTC(FIP-6)を挿入したプライマーを用いて増幅を行った。B3、F3、BIP、LBcプライマーについては共通で使用した(表1参照)。鋳型として、ヒトゲノムを添加したものと、当該ヒトゲノムを添加せずに変わりに滅菌水を添加したものの2種類を調製した。その後、得られた増幅産物についてチップ検出を行った。
検出に使用した合成DNAオリゴ核酸プローブを表3に示した。本例では、核酸プローブとして、FIPプライマーの3’末端側から4塩基目にACAC挿入したもの(プローブ2;配列番号17)、TGTG挿入したもの(プローブ3:配列番号18)、TCTC挿入したもの(プローブ4;配列番号19)、および鋳型配列に由来する領域の相補鎖となる部分に一塩基の変異を入れたもの(プローブ5;配列番号20)を準備した。ネガティブコントロールとして、MTHFR遺伝子配列とは無関係な配列を有するネガティブプローブ(プローブ1;配列番号16)を用いた。以上5種のプローブは、金電極に固定化するために3’末端側をチオール修飾した。
上記の核酸プローブを、金電極へ固定化した。核酸プローブの固定化は、チオールと金との強い共有結合性を利用して行った。核酸プローブ溶液を金電極上にスポットし、25℃で1時間静置した。その後、1mMメルカプトヘキサノール溶液に浸し、0.2×SSC溶液で洗浄した。同一プローブは各4電極にスポットした。各核酸プローブの電極の位置を以下に示す。洗浄後、超純水で洗浄、風乾し、電機化学的検出用DNAチップを得た。
電極とそこに固定化する核酸プローブとの対応は次の通りである。
5-8電極 ACAC検出用プローブ(核酸プローブ2)
9-12電極 TGTG検出用プローブ(核酸プローブ3)
13-16電極 TCTC検出用プローブ(核酸プローブ4)
17-20電極 ACAC 1塩基変異導入プローブ(核酸プローブ5)
[ハイブリダイゼーション]
上記のように作製した電気化学的検出用DNAチップを、2×SSCの塩を添加したLAMP産物に浸漬し、55℃で10分間静置させて、ハイブリダイゼーションを行った。その後、電気化学的検出用DNAチップを0.2×SSC溶液に48℃で10分間浸漬して洗浄した。次いで、電気化学的検出用DNAチップを、挿入剤であるヘキスト33258溶液を50μM含むリン酸緩衝液中に1分間浸漬した。その後、ヘキスト33258溶液の酸化電流応答を測定した。
図14Aは、ヒトゲノムを添加して増幅したACAC(FIP-3)挿入プライマーセットの増幅産物と滅菌水を添加して増幅したTGTG(FIP-5)およびTCTC(FIP-6)挿入プライマーセットの増幅産物を混合したものである。図14Bは、ヒトゲノムを添加して増幅したTGTG(FIP-5)挿入プライマーセットの増幅産物と滅菌水を添加して増幅したACAC(FIP-3)およびTCTC(FIP-6)挿入プライマーセットの増幅産物を混合したものである。同様に図14Cは、ヒトゲノムを添加して増幅したTCTC(FIP-6)挿入プライマーセットの増幅産物と滅菌水を添加して増幅したACAC(FIP-3)およびTGTG(FIP-5)挿入プライマーセットの増幅産物を混合したものである。チップ検出の結果、図14Aでは、ACACを検出する核酸プローブ2から高いシグナルが得られた。図14Bでは、TGTGを検出する核酸プローブ3から高いシグナルが得られた。同様に、図14Cでは、TCTCを検出する核酸プローブ4から高いシグナルが得られた。このことから、本発明のタグ配列を挿入したプライマーで、複数検体を同時にDNAチップ検出できることが示された。また、図14Aについて、洗浄条件を厳しくすることで、ACAC検出用プローブと1塩基違いの核酸プローブ5について、核酸プローブ2と比較してシグナルが低くなっており、鋳型配列由来の領域に変異を位置させることにより、変異が検出可能であることが示唆された。
Claims (10)
- 複数検体の解析方法であって、
(a)前記検体毎に異なる配列を有するタグ配列を含む第1のプライマーと、前記第1のプライマーと対で使用される第2のプライマーとを含むプライマーセットを検体毎に準備する工程と、ここで、前記タグ配列は、前記第1のプライマーが、前記検体毎の鋳型核酸中の鋳型配列にハイブリダイズしたときにループアウトするように設計されている;
(b)前記検体毎に独立した反応系で、前記検体毎に対応する前記プライマーセットを用いて、前記検体毎の鋳型核酸を増幅し、前記タグ配列が導入された増幅産物を得る工程と;
(c)前記検体毎に得られた前記増幅産物を1つに混合する工程と;
(d)前記当該タグ配列を含む標的配列を検出する配列を有し、基体上に固定化された核酸プローブと、工程(c)で混合された前記増幅産物とを反応させる工程と;
(e)工程(d)において生じたハイブリダイゼーション量を検出することにより、各検体について前記標的核酸の有無および/または量を検出する工程と;
を具備する解析方法。 - 前記核酸プローブは、前記タグ配列と前記検体毎の鋳型配列に由来する配列とを含む、標的配列を検出する配列を有することを特徴とする請求項1記載の解析方法。
- 請求項1に記載の解析方法であって、 前記プライマーセットが、以下の1~9からなる群より少なくとも1選択され;
前記鋳型配列の5’末端側よりF3領域、F2領域、LF領域、F1領域、3’末端側よりB3c領域、B2c領域、LBc領域、B1c領域を設定したとき、
1. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、および5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもつBIPプライマー;
2. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもつFIPプライマー、および5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー;
3. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、および5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー;
4. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもつBIPプライマー、F3領域と同じ配列をもつF3プライマー、およびB3c領域と相補的な配列をもつB3プライマー;
5. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもつFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー、F3領域と同じ配列をもつF3プライマー、およびB3c領域と相補的な配列をもつB3プライマー;
6. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー、F3領域と同じ配列をもつF3プライマー、およびB3c領域と相補的な配列をもつB3プライマー;
7. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもつBIPプライマー、F3領域と同じ配列をもつF3プライマー、B3c領域と相補的な配列をもつB3プライマー、LF領域と相補的な配列をもつLFcプライマー、およびLBc領域と同じ配列をもつLBcプライマー;
8. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもつFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー、F3領域と同じ配列をもつF3プライマー、B3c領域と相補的な配列をもつB3プライマー、LF領域と相補的な配列をもつLFcプライマー、およびLBc領域と同じ配列をもつLBcプライマー;
9. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー、F3領域と同じ配列をもつF3プライマー、B3c領域と相補的な配列をもつB3プライマー、LF領域と相補的な配列をもつLFcプライマー、およびLBc領域と同じ配列をもつLBcプライマー;
(b)の増幅がLAMP法である方法。 - 検体核酸中の複数の部分核酸配列の解析方法であって、
(a)前記部分核酸配列毎に、異なる配列を有するタグ配列を含む第1のプライマーと、前記第1のプライマーと対で使用される第2のプライマーとを含むプライマーセットを前記核酸部分配列毎に準備する工程と、ここで、前記タグ配列は、前記第1のプライマーが、前記核酸部分配列毎の鋳型配列にハイブリダイズしたときにループアウトするように設計されている;
(b)前記プライマーセットを用いて、前記核酸部分配列毎の鋳型配列をマルチ増幅し、前記タグ配列が導入された増幅産物を得る工程と;
(c)前記タグ配列を含む標的配列を検出するための、基体上に固定化された核酸プローブと、工程(b)で得られた前記増幅産物とを反応させる工程と;
(d)工程(c)において生じたハイブリダイゼーション量を検出することにより、各核酸配列について前記標的核酸の有無および/または量を検出する工程と;
を具備する解析方法。 - 前記核酸プローブは、前記タグ配列と前記核酸部分配列毎の鋳型配列に由来する配列とを含む標的配列を検出する配列を有することを特徴とする請求項4記載の解析方法。
- 請求項4記載の解析方法であって、 前記プライマーセットが、以下の1~9からなる群より少なくとも1選択され;
鋳型配列の5’末端側よりF3領域、F2領域、LF領域、F1領域、3’末端側よりB3c領域、B2c領域、LBc領域、B1c領域を設定したとき、
1. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、および5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもつBIPプライマー;
2. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもつFIPプライマー、および5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー;
3. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、および5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー;
4. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもつBIPプライマー、F3領域と同じ配列をもつF3プライマー、およびB3c領域と相補的な配列をもつB3プライマー;
5. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもつFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー、F3領域と同じ配列をもつF3プライマー、およびB3c領域と相補的な配列をもつB3プライマー;
6. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー、F3領域と同じ配列をもつF3プライマー、およびB3c領域と相補的な配列をもつB3プライマー;
7. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもつBIPプライマー、F3領域と同じ配列をもつF3プライマー、B3c領域と相補的な配列をもつB3プライマー、LF領域と相補的な配列をもつLFcプライマー、およびLBc領域と同じ配列をもつLBcプライマー;
8. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもつFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー、F3領域と同じ配列をもつF3プライマー、B3c領域と相補的な配列をもつB3プライマー、LF領域と相補的な配列をもつLFcプライマー、およびLBc領域と同じ配列をもつLBcプライマー;
9. 5’末端側にF1と相補的な配列をもち3’末端側にF2と同じ配列をもち、且つF2配列内に検体によって異なるタグ配列を挿入したFIPプライマー、5’末端側にB1cと同じ配列をもち3’末端側にB2cと相補的な配列をもち、且つB2c配列内に検体によって異なるタグ配列を挿入したBIPプライマー、F3領域と同じ配列をもつF3プライマー、B3c領域と相補的な配列をもつB3プライマー、LF領域と相補的な配列をもつLFcプライマー、およびLBc領域と同じ配列をもつLBcプライマー;
(b)の増幅がLAMP法である方法。 - 請求項1に記載の複数検体を解析する方法において使用するためのアッセイキットであって、
検体毎に異なる配列を有するタグ配列を含み、前記タグ配列は、前記検体毎の鋳型核酸中の鋳型配列にハイブリダイズしたときにループアウトするように設計された第1のプライマーと、前記第1のプライマーと対で使用される第2のプライマーとを含むプライマーセットと、
基体と、前記基体上に固定化された前記タグ配列を含む標的配列に相補的な核酸プローブとを具備するDNAチップと
を具備するアッセイキット。 - 前記核酸プローブは、前記タグ配列及び前記検体中の鋳型配列に由来する配列を含む標的配列に相補的な核酸プローブであることを特徴とする請求項7記載のアッセイキット。
- 請求項4に記載の複数の核酸部分配列の解析方法において使用するためのアッセイキットであって、
前記核酸部分配列毎に異なる配列を有するタグ配列を含み、前記タグ配列は、前記核酸部分配列毎の鋳型配列にハイブリダイズしたときにループアウトするように設計された第1のプライマーと、前記第1のプライマーと対で使用される第2のプライマーとを含むプライマーセットと、
基体と、前記基体上に固定化された前記タグ配列及び前記核酸部分配列毎の鋳型配列に由来する配列とを含む標的配列に相補的な核酸プローブとを具備するDNAチップと
を具備するアッセイキット。 - 前記核酸プローブは、前記タグ配列及び前記核酸部分配列毎の鋳型配列に由来する配列を含む標的配列に相補的な核酸プローブであることを特徴とする請求項9記載のアッセイキット。
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JP (1) | JP4528885B1 (ja) |
KR (1) | KR101378214B1 (ja) |
CN (1) | CN102459630B (ja) |
WO (1) | WO2011001496A1 (ja) |
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JPWO2017043114A1 (ja) * | 2015-09-07 | 2018-09-27 | 株式会社ファスマック | 等温増幅反応産物の多項目同時検出方法 |
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CN102459630A (zh) | 2012-05-16 |
KR101378214B1 (ko) | 2014-03-27 |
JPWO2011001496A1 (ja) | 2012-12-10 |
KR20120024873A (ko) | 2012-03-14 |
US8673595B2 (en) | 2014-03-18 |
US20120171673A1 (en) | 2012-07-05 |
CN102459630B (zh) | 2014-04-30 |
JP4528885B1 (ja) | 2010-08-25 |
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