WO2015182601A1 - Procédé de détection d'un acide nucléique cible - Google Patents

Procédé de détection d'un acide nucléique cible Download PDF

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WO2015182601A1
WO2015182601A1 PCT/JP2015/065082 JP2015065082W WO2015182601A1 WO 2015182601 A1 WO2015182601 A1 WO 2015182601A1 JP 2015065082 W JP2015065082 W JP 2015065082W WO 2015182601 A1 WO2015182601 A1 WO 2015182601A1
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nucleic acid
target nucleic
group
capture probe
base
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PCT/JP2015/065082
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Japanese (ja)
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泰亮 平野
慎二郎 澤田
日笠　雅史
健造 藤本
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東レ株式会社
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Priority to JP2015528483A priority Critical patent/JPWO2015182601A1/ja
Publication of WO2015182601A1 publication Critical patent/WO2015182601A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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

Definitions

  • the present invention relates to a method for detecting a target nucleic acid using hybridization between a capture probe and a target nucleic acid.
  • nucleic acids various nucleic acids / nucleic acid complementarity such as Northern blotting or Southern blotting can be used to examine the relationship between various genes and their biological function expression.
  • proteins the function and expression of proteins can be examined using protein-protein reactions, as typified by Western blotting.
  • a capture probe made of a nucleic acid is used for the purpose of detecting a target nucleic acid to be examined.
  • a capture probe made of a nucleic acid is used for simultaneous detection of a plurality of types of target nucleic acids using a DNA chip or a DNA microarray in which a large number of capture probes are immobilized on a support.
  • the target nucleic acid sequence and abundance are examined by bringing the capture probe immobilized on the support into contact with the target nucleic acid and examining the complementarity due to the presence or absence of hybridization between the capture probe and the target nucleic acid.
  • a method is generally used in which a label is introduced into the target nucleic acid and the signal of the label is detected after contact with the capture probe.
  • Patent Document 1 a method using a capture probe into which a photoreactive group is introduced is known (Patent Document 1).
  • a capture probe introduced with a photoreactive group and a target nucleic acid are hybridized, and the hybridized capture probe and the target nucleic acid are irradiated with light to form a covalent bond (photocrosslinking) between the two, After removing nucleic acids other than the target nucleic acid not forming a covalent bond by washing, the target nucleic acid is efficiently detected by irradiating the hybridized capture probe and target nucleic acid with light again to cleave the covalent bond. ing.
  • the capture probe cross-hybridizes (non-specific adsorption) not only with the target nucleic acid but also with nucleic acids other than the target nucleic acid, and a covalent bond is introduced between the strands in this state.
  • An object of the present invention is to suppress cross-hybridization between a capture probe introduced with a photoreactive group and a nucleic acid other than the target nucleic acid, and to selectively perform hybridization with the target nucleic acid.
  • the present invention can suppress the above problem, that is, cross-hybridization between the capture probe and a nucleic acid other than the target nucleic acid by inserting a spacer in addition to the capture probe into which the photoreactive group has been introduced. , Which enables selective hybridization with a target nucleic acid of interest.
  • a target nucleic acid can be detected with high accuracy and high sensitivity.
  • the nucleic acid other than the target nucleic acid is, for example, due to interstrand repulsion due to a mismatch base, and the distance between the photoreactive group and the nucleic acid other than the target nucleic acid is larger than the distance capable of photocrosslinking. It is presumed that the photocrosslinking reaction after cross-hybridization is suppressed.
  • a method for detecting a target nucleic acid comprising a step of hybridizing a target nucleic acid contained in a specimen and a capture probe, At least one nucleobase in the nucleic acid molecule of the capture probe is substituted with a photoreactive group, and one or more spacers are inserted in the capture probe, After the step of hybridizing the target nucleic acid and the capture probe, the complex formed by the hybridization of the target nucleic acid and the capture probe is irradiated with light so that the photoreactive group and the target nucleic acid Forming a covalent bond with the nucleobase of Detection method.
  • each spacer is a divalent organic group inserted into the main chain of the nucleic acid, and one spacer has a space for one nucleotide.
  • each spacer is an alkylene group, a poly (oxyalkylene) group, a base-deficient sugar chain group, or a non-natural base-introduced nucleoside group.
  • Each spacer is represented by formula I, formula II or formula III:
  • n is an integer of 3 to 12.
  • x is an integer of 1 to 10
  • y is an integer of 1 to 5
  • z is an integer of 1 to 10.
  • R represents a hydrogen atom, a tert-butyldimethylsiloxy group, a formula IV or a formula V:
  • the capture probe is immobilized on a solid support, the target nucleic acid is labeled, the solid support is washed after the light irradiation, and is present on the solid support after washing.
  • the present invention it is possible to selectively perform hybridization with a target nucleic acid by suppressing cross-hybridization between the capture probe and a nucleic acid other than the target nucleic acid.
  • the target nucleic acid can be detected with high accuracy and high sensitivity.
  • the method of the present invention is particularly effective for detection of trace samples that require high sensitivity, such as detection of gene mutations and polymorphisms that are susceptible to cross-hybridization with nucleic acids other than the target nucleic acid, and miRNA detection. It is.
  • target nucleic acid used in the detection method of the present invention examples include, but are not limited to, genes such as pathogenic bacteria and viruses, causative genes of genetic diseases, and the like, and parts thereof.
  • Samples containing these target nucleic acids include body fluids such as blood, serum, plasma, urine, feces, spinal fluid, saliva, wipes, various tissue fluids, various tissues, paraffin-embedded specimens (FFPE) and sections thereof.
  • body fluids such as blood, serum, plasma, urine, feces, spinal fluid, saliva, wipes, various tissue fluids, various tissues, paraffin-embedded specimens (FFPE) and sections thereof.
  • FFPE paraffin-embedded specimens
  • Various foods and drinks, and dilutions thereof may be mentioned, but are not limited thereto.
  • the target nucleic acid may be a sample nucleic acid extracted from blood or cells by a conventional method, and DNA or RNA extracted from the sample can be used.
  • the DNA may be single-stranded or double-stranded, and DNA such as chromosomal DNA, viral DNA, bacteria, mold, etc., cDNA obtained by reverse transcription of RNA, fragments that are part of them, etc. can be used. It is not limited.
  • RNA small RNA such as messenger RNA, ribosomal RNA, and micro RNA (miRNA) and fragments that are a part thereof can be used, but are not limited thereto.
  • chemically synthesized DNA or RNA can be used as the target nucleic acid.
  • the sample nucleic acid may contain a nucleic acid component (non-target nucleic acid) other than the target nucleic acid to be measured.
  • non-target nucleic acids may be removed in consideration of the difference in properties from the target nucleic acid, or may be used as a test substance without being removed.
  • the target nucleic acid may be amplified by a nucleic acid amplification method such as PCR. In this case, the measurement sensitivity can be greatly improved.
  • a nucleic acid amplification product is used as a target nucleic acid
  • the amplified nucleic acid can be labeled by performing amplification in the presence of a nucleoside triphosphate labeled with a fluorescent substance or the like.
  • the method of the present invention can be used for detection by distinguishing the presence or absence of a target nucleic acid in a specimen, virus genotype, bacterial species and strain, mold species and strain, and the like.
  • the method of the present invention can be preferably used for detecting the presence or absence of a mutation in a target nucleic acid in a sample.
  • the target nucleic acid mutation means that the target nucleic acid has a long chain length, for example, deletion, duplication, fusion of a gene sequence in genomic DNA, deletion, duplication, fusion of a transcript, or a short chain length of a target nucleic acid, for example, Examples include detection of miRNA family sequences.
  • the method of the present invention can be particularly preferably used for detection of gene polymorphism in a specimen.
  • detection of gene polymorphism include detection of SNP (single nucleotide polymorphism) of genomic DNA and RNA that is a transcript thereof.
  • the target nucleic acid can be applied as it is, or a fragmented product of the target nucleic acid can be applied.
  • the “target nucleic acid” includes a target nucleic acid fragment containing a region in the target nucleic acid that hybridizes with the capture probe. Is also used to include.
  • the length of the target nucleic acid is not particularly limited as long as the capture probe is hybridized. However, when the target nucleic acid is long (for example, 1500 bases or more, particularly 4000 bases or more), an appropriate length is obtained by fragmentation. It is preferable to apply a fragmented product obtained by fragmentation.
  • the fragmented product does not need to select a specific nucleic acid fragment from the resulting nucleic acid fragments, and the fragmented product can be directly used in the method of the present invention, thereby improving the detection sensitivity.
  • Methods for cleaving the target nucleic acid for fragmentation include methods of cleaving with ultrasonic waves, methods of cleaving with enzymes, methods of cleaving with restriction enzymes, methods using a nebulizer, methods of cleaving with acids and alkalis, etc. Can be used.
  • the method of cutting with ultrasonic waves it is possible to cut to a desired length by controlling the output intensity and irradiation time of the ultrasonic waves irradiated to the target nucleic acid.
  • the length of the target nucleic acid is not particularly limited as long as it can hybridize with the capture probe, but is usually 10 bases or more, preferably 10 bases to 1500 bases, more preferably 15 bases to 1000 bases.
  • the base is more preferably about 18 to 1000 bases.
  • the target nucleic acid is 1500 bases or more, particularly 4000 bases or more, it is preferable to use a target nucleic acid that has been cleaved to a length in this range by the fragmentation treatment described above.
  • a label can be bound to the target nucleic acid.
  • a label that can be used a known substance used for labeling such as a protein-binding substance, a fluorescent dye, a phosphorescent dye, and a radioisotope can be used.
  • An example of a protein binding substance is biotin. Biotin can bind to avidin or streptavidin. Avidin or streptavidin bound with a fluorescent dye, or bound with an enzyme such as alkaline phosphatase or horseradish peroxidase can be used. When alkaline phosphatase or horseradish peroxidase is used, each substrate is added and the substrate reacts with the enzyme, resulting in a luminescence reaction. The luminescence reaction is detected using a plate reader or a CCD camera.
  • a fluorescent dye that is easy to measure and easily detects a signal may be used.
  • cyanine cyanine 2
  • aminomethylcoumarin fluorescein
  • indocarbocyanine cyanine 3
  • cyanine 3.5 aminomethylcoumarin
  • tetramethylrhodamine rhodamine red
  • Texas red indocarbocyanine
  • cyanine 5 cyanine 5.5
  • known fluorescent dyes such as cyanine 7, oyster, BODIPY dye, phycoerythrin, and the like.
  • the detection of the fluorescent dye can be performed with a fluorescence microscope, a fluorescence scanner, a fluorescence spectrophotometer, or the like.
  • a semiconductor fine particle having a light emitting property may be used as a marker.
  • semiconductor fine particles include cadmium selenium (CdSe), cadmium tellurium (CdTe), indium gallium phosphide (InGaP), chalcopyrite fine particles, silicon (Si), and the like.
  • the present invention can be applied to quantification of a target nucleic acid by measuring the signal intensity of the labeled body when the target nucleic acid to which the labeled body is bound is used.
  • the “detection method” of the present invention includes the case where the quantification is accompanied.
  • Measured signal is compared with noise.
  • the signal value (S) of the target nucleic acid hybridized with the capture probe is compared with the signal value (noise value (N)) of a nucleic acid other than the target nucleic acid attached to the capture probe.
  • the ratio of the values is the S / N ratio, and in the present invention, the detection accuracy is represented by the S / N ratio. The larger the S / N ratio value, the higher the detection accuracy, and the smaller the S / N ratio value, the lower the detection accuracy.
  • nucleic acid derivatives such as DNA, RNA, PNA (peptide nucleic acid), and LNA (Locked Nucleic Acid) can be used as the capture probe.
  • derivative refers to a modified nucleotide (for example, halogen, alkyl such as methyl, alkoxy such as methoxy, nucleotide and base rearrangement including thio, carboxymethyl, etc., double bond saturation, deamination Or a chemically modified derivative such as a derivative containing a nucleotide subjected to substitution of oxygen molecule with sulfur molecule.
  • a single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a single-stranded nucleic acid having a base sequence complementary to the base sequence or a part thereof, it is used as a capture probe in the present invention.
  • the capture probe used in the present invention may be a commercially available probe or a probe obtained from a living cell. Particularly preferred as a capture probe is a nucleic acid.
  • nucleic acids called oligonucleic acids having a length of up to 200 bases can be easily artificially synthesized with a synthesizer.
  • the capture probe only needs to contain a sequence complementary to the target nucleic acid sequence, and any region in the target nucleic acid may be selected.
  • a plurality of types of capture probes that hybridize with different regions of the target nucleic acid can also be used.
  • a sequence complementary to either the sense strand or the antisense strand can be selected as a capture probe.
  • the size of the region that hybridizes with the entire region or a partial region of the target nucleic acid in the capture probe is not particularly limited, but is usually about 10 to 200 bases, preferably about 18 to 200 bases.
  • the total length of the capture probe (when the capture probe also includes a region that does not hybridize with the target nucleic acid, the total length including the region) is not particularly limited, but is usually 10 to 200 bases, preferably About 18 to 200 bases.
  • the specificity is selected from the nucleic acid sequences that can be contained in the sample nucleic acid, for example, by distinguishing and detecting the type of virus infecting the patient. It is preferable to select a high sequence region.
  • At least one nucleobase in the nucleic acid molecule is substituted with a photoreactive group, and one or more spacers are inserted in the capture probe. More preferably, 1 to 3 photoreactive groups are substituted in the capture probe.
  • the photoreactive group is an organic group (photoreactive site) whose reactivity in the organic synthesis reaction is activated by irradiation with light of a specific wavelength.
  • the probe after the nucleobase in the probe has been substituted with a photoreactive group can hybridize with the target nucleic acid in the same manner as the nucleobase before substitution to form a complex.
  • a 3-cyanovinylcarbazole group and derivatives thereof As such a photoreactive group, a 3-cyanovinylcarbazole group and derivatives thereof (WO2009 / 066447 (EP2216338, US 2010-274000 A), Yoshinaga Yoshimura et al., Organic Letters 10: 3227-3230 (2008)), p-carbamoylvinylphenol group (Takehiro Ami et al., Organic & Biomolecular Chemistry 5: 2583-2586 (2007)), 4,5 ', 8-trimethylpsoralen group (Akio Kobori et al., Chemistry Letters 38: 272- 273 (2009)) and N 3 -methyl-5-cyanovinyluracil group (Kenzo Fujimoto et al., Chemical Communications: 3177-3179 (2005), etc.), which are incorporated herein by reference.
  • 3-cyanovinylcarbazole group and derivatives thereof are preferable, and 3-cyanovinylcarbazole
  • 3-cyanovinylcarbazole group and derivatives thereof are groups represented by the following formula (I) as described in WO2009 / 066447.
  • R a is a cyano group, an amide group, a carboxyl group, a C 2 -C 7 alkoxycarbonyl group, or hydrogen;
  • R 1 and R 2 are each independently a cyano group, an amide group, , A carboxyl group, a C 2 -C 7 alkoxycarbonyl group, or hydrogen.
  • R a is a cyano group and R 1 and R 2 are hydrogen, it is a 3-cyanovinylcarbazole group.
  • a 3-cyanovinylcarbazole group or a derivative thereof is used as the photoreactive group, it is preferable to design the purine base adjacent to the 5 ′ side of the base in the capture probe.
  • the p-carbamoyl vinylphenol group is a group represented by the following formula (II).
  • the 4,5 ′, 8-trimethylpsoralen group is a group represented by the following formula (III).
  • N 3 -methyl-5-cyanovinyluracil group is a group represented by the following formula (IV).
  • photoreactive groups replace the bases in the nucleotides constituting the nucleic acid, and free bonds in the photoreactive groups represented by the formulas (I) to (IV) are converted into sugars in the nucleotides. Combine directly with the part.
  • a 3-cyanovinylcarbazole group represented by the formula (I) (wherein R a is a cyano group and R 1 and R 2 are hydrogen) binds to deoxyribose as follows.
  • Other photoreactive groups also bind to the sugar.
  • 3-cyanovinylcarbazole group in the general formula (V) represents 3-iodocarbazole (3.52 mmol) and acrylonitrile (7.04 mmol) in dioxane with triphenylphosphine ( 0.53 ⁇ mol), palladium acetate (0.18 ⁇ mol) and triethylamine (4.23 mmol) in the presence of heating at 75 ° C. for 11.5 hours.
  • the conjugate of p-carbamoylvinylphenol and deoxyribose can be obtained in the same manner as described in Takehiro Ami et al., Organic & Biomolecular Chemistry 5: 2583-2586 (2007), similarly p-iodophenol, Hoffer's chlorosugar and It is manufactured by reacting and bonding and reacting with methyl methacrylate.
  • a conjugate of 4,5 ′, 8-trimethylpsoralen group and deoxyribose is obtained in the same manner as described in Akio Kobori et al., Chemistry Letters 38: 272-273 (2009). Manufactured by reacting 5 ', 8-trimethylpsoralen with Hoffer's chlorosugar.
  • the conjugate of N 3 -methyl-5-cyanovinyluracil group and deoxyribose can be obtained similarly to 2-iodinated N 3 -Manufactured by reacting methyluridine with acrylonitrile.
  • deoxyribose is another sugar (for example, ribose or the like)
  • a conjugate of a photoreactive group and a sugar can be obtained in the same manner.
  • a conjugate of a photoreactive group and a sugar is obtained, a nucleic acid containing this can be easily produced by a phosphoramidite method that is a conventional method.
  • a conjugate of 3-cyanovinylcarbazole and deoxyribose represented by the above formula (V) is, in the example of WO2009 / 066447, the conjugate (0.29 mmol) and 4,4-dimethoxytrityl chloride in pyridine.
  • a nucleic acid having a desired base sequence can be synthesized using a commercially available oligonucleotide synthesizer.
  • the nucleic acid itself having a photoreactive group is a well-known one that is used as a reagent that inhibits the expression of a desired gene or the like, and synthesis of a nucleic acid containing a photoreactive group and having a desired base sequence is By requesting the synthesis of a nucleic acid having a desired base sequence and having a desired photoreactive group introduced at a desired position, to be provided as a commercial service. It is also possible to obtain the nucleic acid.
  • the capture probe used in the present invention has one or more spacers.
  • a capture probe in which the nucleobase in the probe is substituted with a photoreactive group and a spacer is inserted can hybridize with the target nucleic acid to form a duplex, and is shared in the duplex by light irradiation. By forming a bond, the target nucleic acid can be captured with high efficiency.
  • the insertion of the spacer increases the structural freedom of the entire probe molecule, for example, increases the repulsion between bases due to mismatched bases, and the distance between the photoreactive group in the capture probe and the nucleic acid other than the target nucleic acid is photocrosslinked. Since it becomes larger than the possible distance, it is presumed that the photocrosslinking reaction after cross-hybridization between the capture probe and the nucleic acid other than the target nucleic acid is suppressed.
  • a divalent organic group to be inserted into the nucleic acid main chain can be preferably used, and it is preferable that one spacer has a space for one nucleotide. .
  • “one spacer has a space for one nucleotide” will be described.
  • T-01 (5′-TCTGAAGTAGATATGGCAGCACATAATGAC-3 ′, SEQ ID NO: 1) is used as the target nucleic acid
  • C-01 (5′-GTCATTATGT (d) CTGCCAQATCT ( d) CTTCAGA-3 ') is used.
  • (d) is a spacer (specifically, a trade name dSpacer which is a sugar chain group lacking a base (specific structure will be described later), and Q is a nucleotide base whose photoreactive group is 3-
  • the target nucleic acid T-01 and the capture probe C-01 are hybridized as follows (the spacer (d) is simply indicated as “d”). .
  • the 23rd d (spacer) from the 5 ′ end of C-01 faces the 8th C from the 5 ′ end of T-01.
  • the 7th G of T-01 adjacent to this is base-paired with the 24th C of C-01, and the 9th A of the other adjacent T-01 is the 22nd of C-01. Base paired with T.
  • the spacer d occupies a space corresponding to one nucleotide corresponding to the eighth C from the 5 ′ end of T-01.
  • the other spacer d in C-01 (11th from the 5 ′ end) occupies a space corresponding to one nucleotide corresponding to the 20th C of T-01.
  • “One spacer has a space for one nucleotide” means such a state.
  • examples of the structure of the spacer include an alkylene group, a poly (oxyalkylene) group (for example, a poly (oxyethylene) group), a base-deficient sugar chain group, and a non-natural base-introduced nucleoside group.
  • some of these spacers are commercially available as nucleic acid spacers, and such commercially available products can be preferably used.
  • the spacer can be inserted into the main chain of the nucleic acid by forming a phosphate structure in the capture probe.
  • R in formula VI is a spacer.
  • the base-deficient sugar chain group refers to a sugar chain group in which a nucleobase part of a natural nucleoside is deleted to form a hydrogen atom or substituted with another organic group.
  • Another organic group in this case includes, for example, a tert-butyldimethylsiloxy group.
  • An unnatural base-introduced nucleoside group refers to a nucleic acid group into which an unnatural base that does not exist in nature is introduced in place of the nucleobase part of a natural nucleoside.
  • Examples of the non-natural base in this case include organic molecules typified by universal bases, and any base can be used as long as it does not interfere with the formation of a double strand when hybridizing with a complementary strand. Good.
  • the spacer is an alkylene group
  • the structure of the spacer is preferably of formula I:
  • n is an integer of 3 to 12 in formula I), and more preferably n is 3 or 12.
  • the spacer is a polyoxyalkylene group
  • the structure of the spacer is preferably the formula II:
  • x is an integer of 1 to 10
  • y is an integer of 1 to 5
  • z is an integer of 1 to 10.
  • x is 2, y is 2 or 5 and z is 2.
  • the structure of the spacer is preferably the formula III:
  • R is a hydrogen atom or a tert-butyldimethylsiloxy group), and more preferably, R is a hydrogen atom.
  • the non-natural base is preferably a hydrophobic base that cannot form a hydrogen bond and has high planarity, and more preferably, R in Formula III is Formula IV or Formula V:
  • one or more spacers are inserted into the capture probe, and a plurality of spacers can be used depending on the length of the capture probe.
  • the length of the capture probe is 30 bases or less, it is preferable to insert one or two.
  • the length of the capture probe is 31 bases or more, it is preferably inserted at a rate of 1 per 6 to 30 bases, more preferably at a rate of 1 per 10 to 20 bases. preferable.
  • the same type of spacers may be used, or different types of spacers may be used in combination.
  • the spacer may be inserted in any part in the capture probe, but it is preferable to insert it in a part that is not opposed to the mismatched base in the nucleic acid other than the target nucleic acid.
  • the number of bases between the spacers is 5 to 30
  • the number is 5 to 20
  • the number is 5 to 12
  • the number of bases between spacers is 5 to 12
  • Spacer insertion requires 0 to 30 bases from the base substituted with the photoreactive group in the capture probe (the number of bases between the spacer and the base substituted with the photoreactive group is 0 to 30). However, it is more preferably performed at a position where about 2 to 22 bases are separated (the number of bases between the spacer and the base substituted with the photoreactive group is 2 to 22).
  • a capture probe in which a nucleobase is substituted with a photoreactive group and a spacer is inserted is, for example, a light having a photoreactive group as a base using a known oligonucleotide synthesizer or peptide synthesizer. It can be produced by a phosphoramidite method using a reactive base derivative, an amidite containing a partial structure of a spacer and an amidite of a natural base as raw materials. By combining these raw materials in a desired order, a capture probe in which a nucleic acid substituted with a photoreactive group or a spacer is introduced at a desired position can be freely designed and manufactured.
  • an amidite disclosed in JP2012-121899A can be used as a raw material.
  • a p-carbamoylvinylphenol group is used as a photoreactive group
  • an amidite shown in the literature [Yoshinaga Yoshimura et al., Bioorganic & Medicinal Chemistry Letters 15: 1293-1301 (2005)] is used. It can be used as a raw material.
  • a 4,5 ′, 8-trimethylpsoralen group is used as a photoreactive group
  • an amidite shown in the literature [Akio Kobori et al., Chemistry Letters 38: 272-273 (2009)].
  • an N 3 -methyl-5-cyanovinyluracil group is used as the photoreactive group
  • the amidite shown in the literature [Kenzo Fujimoto et al., Chemical Communications: 3177-3179 (2005)]. Can be used as a raw material.
  • spacer when an alkylene group is used as the spacer, for example, a product name: Spacer® Phosphoramidite® C3 (manufactured by Glenn Research) or a product name: Spacer® Phosphoramidite® CE C12 (manufactured by Glenn Research) can be used as a raw material.
  • a poly (oxyethylene) group when used as the spacer, for example, a product name: Spacer® Phosphoramidite® 9 (manufactured by Glenn Research) or a product name: Spacer® Phosphoramidite® 18 (manufactured by Glenn Research) can be used as a raw material.
  • a product name: dSpacerdCE Phosphoramidite (manufactured by Glen Research) or a product name: Abasic II Phosphoramidite (manufactured by Glen Research) can be used as a raw material.
  • a non-natural base-introduced nucleoside group for example, product name: 3-Nitropyrrole-CECPhosphoramidite (manufactured by GlenGResearch) or product name: 5-nitroindole-CE Phosphoramidite (manufactured by Glen Research as a raw material) be able to.
  • the capture probe of the present invention may be immobilized on a solid support.
  • a solid support a slide glass, a membrane, beads, or the like can be used.
  • the material of the solid support is not particularly limited, and examples thereof include inorganic materials such as glass, ceramic and silicon, and polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate and silicone rubber.
  • a method for immobilizing a capture probe on a support As a method for immobilizing a capture probe on a support, a method of synthesizing oligonucleic acid on the upper surface of the support and a method of dropping and immobilizing a pre-synthesized oligonucleic acid on the upper surface of the support are known. Both are applicable.
  • the former method includes the method of Ronald et al. (US Pat. No. 5,705,610), the method of Michel et al. (US Pat. No. 6,142,266), and the method of Francesco et al. (US Pat. No. 7,037,659).
  • the support is preferably made of a material resistant to the organic solvent.
  • a glass support having a concavo-convex structure produced using the method described in JP-T-10-503841 can be used.
  • the support is preferably made of a light-transmitting material in order to control DNA synthesis by irradiating light from the back surface of the support. Examples of the latter method include the method of Iwata et al. (Japanese Patent No. 3922454) and the method using a glass capillary.
  • the glass capillary As an example of the glass capillary, a commercially available product such as a self-made glass capillary or a micropipette (manufactured by Micro Support Co., Ltd .; MP-005) can be used, but it is not limited to these methods.
  • a commercially available product such as a self-made glass capillary or a micropipette (manufactured by Micro Support Co., Ltd .; MP-005) can be used, but it is not limited to these methods.
  • the step of hybridizing the target nucleic acid and the capture probe can be performed in the same manner as in the past.
  • the reaction temperature and time are appropriately selected according to the chain length of the nucleic acid to be hybridized. In the case of nucleic acid hybridization, it is usually about 30 to 70 ° C. and about 1 minute to several tens of hours.
  • the complex formed by hybridizing the target nucleic acid and the capture probe contained in the specimen is irradiated with light to form a photoreactive group.
  • a covalent bond is formed between the nucleobase and the nucleobase in the target nucleic acid.
  • the light irradiation can be performed with light containing a wavelength at which it is activated, depending on the photoreactive group used. For example, when a 3-cyanovinylcarbazole group is used as the photoreactive group, light having a wavelength of 340 to 380 nm can be used.
  • a transilluminator As an apparatus for irradiating light, a transilluminator, a black light, a UV-LED, a UV laser, or the like that can irradiate light including the above-described wavelength can be used.
  • the time of light irradiation is a time during which a covalent bond is formed, and is usually about 3 minutes to 7 minutes when the above-described normal light irradiation apparatus is used.
  • the detection of the target nucleic acid can be applied to the quantification of the target nucleic acid by measuring the signal intensity of the label when the target nucleic acid bound to the label is used.
  • the capture probe is immobilized on a solid support, the target nucleic acid is labeled, the solid support is washed after the hybridization step and the light irradiation step described above, and the solid support is washed after washing. The signal from the bound label is detected or measured.
  • the measured signal can be compared with noise.
  • the signal value (S) of the target nucleic acid hybridized with the capture probe is compared with the signal value (noise value (N)) of a nucleic acid other than the target nucleic acid attached to the capture probe.
  • the ratio of the values is the S / N ratio, and in the present invention, the detection accuracy is represented by the S / N ratio. The larger the S / N ratio value, the higher the detection accuracy, and the smaller the S / N ratio value, the lower the detection accuracy.
  • Table 1 shows the nucleic acids used in the following Examples and Comparative Examples.
  • “Q” in the base sequence represents a group having a 3-cyanovinylcarbazole group introduced as a photoreactive group.
  • “(d)” indicates a base-deficient sugar chain group “dSpacer” (trade name) inserted as a spacer
  • “(SC3)” indicates an alkylene group “SpacerC3” as a spacer.
  • (TC3) indicates an alkylene group “SpacerC3” as a spacer.
  • (NP) ”(trade name) is a spacer in which a non-natural base-introduced nucleoside group in which the nucleobase portion is a non-natural base 3-nitropyrrole is inserted.
  • (SC12) (trade name) indicates a spacer inserted with “SpacerC12” which is an alkylene group, and “(ab)” indicates “Abasic” which is a base-deficient sugar chain group as a spacer. ”(Product name) is inserted, and“ (S9) ”is a spacer that is a poly (oxyalkylene) group,“ Spacer9 ”(product ) Shows what was inserted. The spacers used are shown below.
  • T-01 As the target nucleic acid, a synthetic DNA “T-01” having the base sequence represented by SEQ ID NO: 1 was used.
  • T-01 is a synthetic DNA labeled with Cy3 (registered trademark) at the 5 'end and was synthesized by Operon.
  • nucleic acids other than the target nucleic acid examples include synthetic DNAs “M-01”, “M-02”, “M-03”, and “M-04” having the base sequences shown in SEQ ID NOs: 2, 3, 4, and 5.
  • M-01 and M-02 have a base sequence that differs from T-01 by only one base
  • M-03 has a base sequence that differs from T-01 by two bases
  • M-04 has a base sequence different from T-01. It has three different base sequences.
  • M-01 and M-03 are synthetic DNAs labeled with FITC at the 5 'end and were synthesized by Operon.
  • M-02 and M-04 are synthetic DNAs labeled with Cy5 (registered trademark) at the 5 'end and synthesized by Operon.
  • dSpacer is inserted as a spacer in place of the 11th base “G” and the 23rd base “A” of the base sequence shown in SEQ ID NO: 6, and the 18th base “
  • SpacerC3 is inserted as a spacer in place of the 11th base “G” and the 23rd base “A” of the base sequence shown in SEQ ID NO: 6, and the 18th base “T” Instead of this, it is a synthetic DNA in which a 3-cyanovinylcarbazole group is introduced as a photoreactive group and the 5 ′ end is labeled with biotin, and was synthesized by Tsukuba Oligo Service.
  • C-03 represents “NP” (nucleic acid base moiety is 3-nitropyrrole) as a spacer instead of the 11th base “G” and the 23rd base “A” of the base sequence shown in SEQ ID NO: 6.
  • NP nucleic acid base moiety is 3-nitropyrrole
  • a non-natural base-introducing nucleoside group a 3-cyanovinylcarbazole group as a photoreactive group in place of the 18th base “T”, and biotin-labeled 5 ′ end. Synthesized by Tsukuba Oligo Service.
  • Spacer C12 is inserted as a spacer in place of the 11th base “G” and the 23rd base “A” of the base sequence shown in SEQ ID NO: 6, and the 18th base “T” Instead of this, it is a synthetic DNA in which a 3-cyanovinylcarbazole group is introduced as a photoreactive group and the 5 ′ end is labeled with biotin, and was synthesized by Tsukuba Oligo Service.
  • Spacer 9 is inserted as a spacer in place of the 11th base “G” and the 23rd base “A” of the base sequence shown in SEQ ID NO: 6, and the 18th base “T” Instead of this, it is a synthetic DNA in which a 3-cyanovinylcarbazole group is introduced as a photoreactive group and the 5 ′ end is labeled with biotin, and was synthesized by Tsukuba Oligo Service.
  • dSpacer is inserted as a spacer instead of the 15th base “C” and the 21st base “C” of the base sequence shown in SEQ ID NO: 6, and the 18th base “T” Instead of this, it is a synthetic DNA in which a 3-cyanovinylcarbazole group is introduced as a photoreactive group and the 5 'end is labeled with biotin, and was synthesized by Tsukuba Oligo Service.
  • dSpacer is inserted as a spacer in place of the 13th base “T” and the 23rd base “A” of the base sequence shown in SEQ ID NO: 6, and the 18th base “T” Instead of this, it is a synthetic DNA in which a 3-cyanovinylcarbazole group is introduced as a photoreactive group and the 5 'end is labeled with biotin, and was synthesized by Tsukuba Oligo Service.
  • dSpacer is inserted as a spacer in place of the 11th base “G” and the 25th base “T” of the base sequence shown in SEQ ID NO: 6, and the 18th base “T” Instead of this, it is a synthetic DNA in which a 3-cyanovinylcarbazole group is introduced as a photoreactive group and the 5 'end is labeled with biotin, and was synthesized by Tsukuba Oligo Service.
  • dSpacer is inserted as a spacer in place of the 9th base “G” and the 27th base “C” of the base sequence shown in SEQ ID NO: 6, and the 18th base “T” Instead of this, it is a synthetic DNA in which a 3-cyanovinylcarbazole group is introduced as a photoreactive group and the 5 'end is labeled with biotin, and was synthesized by Tsukuba Oligo Service.
  • C-11 has dSpacer inserted as a spacer instead of the 11th base “G” of the base sequence shown in SEQ ID NO: 6, and a photoreactive group instead of the 18th base “T”.
  • a synthetic DNA in which a 3-cyanovinylcarbazole group was introduced and biotin-labeled at the 5 ′ end was synthesized by Tsukuba Oligo Service.
  • dSpacer is inserted as a spacer in place of the 13th base “T” of the base sequence shown in SEQ ID NO: 6, and a photoreactive group is substituted in place of the 18th base “T”.
  • a synthetic DNA in which a 3-cyanovinylcarbazole group was introduced and biotin-labeled at the 5 ′ end was synthesized by Tsukuba Oligo Service.
  • dSpacer is inserted as a spacer in place of the ninth base “G” of the base sequence shown in SEQ ID NO: 6, and a photoreactive group is substituted in place of the 18th base “T”.
  • a synthetic DNA in which a 3-cyanovinylcarbazole group was introduced and biotin-labeled at the 5 ′ end was synthesized by Tsukuba Oligo Service.
  • C-14 is between the 10th base “T” and the 11th base “G” of the base sequence shown in SEQ ID NO: 6, and the 23rd base “A” and the 24th base “C”.
  • a spacer C3 is inserted as a spacer, a 3-cyanovinylcarbazole group is introduced as a photoreactive group in place of the 18th base “T”, and the 5 ′ end is a biotin-labeled synthetic DNA, It was synthesized at Tsukuba Oligo Service.
  • C-15 is a synthesis in which a 3-cyanovinylcarbazole group is introduced as a photoreactive group in place of the 18th base “T” of the base sequence shown in SEQ ID NO: 6, and the 5 ′ end is labeled with biotin. It was DNA and was synthesized by Tsukuba Oligo Service.
  • C-16 is a synthetic DNA having the base sequence represented by SEQ ID NO: 6 and labeled with biotin at the 5 ′ end, and was synthesized by Operon.
  • Example 1 In Example 1, the capture probe C-01 immobilized on the support was brought into contact with the target nucleic acid T-01 and nucleic acids M-01 and M-02 other than the target nucleic acid, and C-01 and the target nucleic acid T-01 were contacted. After the step of hybridizing with T-01, the complex formed by hybridizing T-01 and C-01 is irradiated with light to covalently bond between the photoreactive group and the nucleobase in the target nucleic acid. And T-01 was detected.
  • Hybridization T-01 (100 ⁇ M), M-01 (100 ⁇ M), and M-02 (100 ⁇ M) were mixed at 1 ⁇ L each and diluted with 10 mM phosphate buffer (containing 100 mM sodium chloride and 0.1% Tween 20). The total liquid volume was 200 ⁇ L. Put “Omnifit” (registered trademark of Isis Co., Ltd.) glass column (inner diameter: 3 mm, stainless fritz column stopper of 2 ⁇ m at both ends) in an incubator (set temperature: 50 ° C.) After the mixed solution (200 ⁇ L) was added, 365 nm light was irradiated with black light while stirring for 5 minutes. After filtration of the solution, the beads were washed with 50% DMSO aqueous solution.
  • Detection T-01 (100 ⁇ M), M-01 (100 ⁇ M), and M-02 (100 ⁇ M) are each mixed at 1 ⁇ L, and diluted with 10 mM phosphate buffer (100 mM sodium chloride, containing 0.1% Tween 20).
  • 10 mM phosphate buffer 100 mM sodium chloride, containing 0.1% Tween 20.
  • the fluorescence of a solution (solution A) obtained by adding 50% DMSO aqueous solution (100 ⁇ L) to a solution with a total solution volume of 200 ⁇ L and the filtrate (solution B) obtained in the hybridization step and the washing step were measured.
  • Example 2 Using C-02 as the capture probe and using M-01 and M-02 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-01 and M-02 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 3 Using C-04 as a capture probe and M-01 and M-02 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-01 and M-02 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 4 Using C-05 as the capture probe and M-01 and M-02 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-01 and M-02 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 5 Using C-06 as the capture probe and M-01 and M-02 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-01 and M-02 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 6 Using C-07 as the capture probe and using M-01 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-01 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 7 Using C-08 as a capture probe and M-01 and M-02 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-01 and M-02 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 8 Using C-09 as a capture probe and M-01 and M-02 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-01 and M-02 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 9 Using C-10 as a capture probe and using M-01 and M-02 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-01 and M-02 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 10 Using C-01 as the capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 11 Using C-02 as a capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 12 Using C-03 as a capture probe and using M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 13 Using C-04 as a capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 14 Using C-05 as the capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 15 Using C-06 as the capture probe and M-03 and M-04 as the nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 16 Using C-08 as a capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 17 Using C-09 as a capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 18 Using C-10 as a capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 19 Using C-11 as the capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 20 Using C-12 as a capture probe and using M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 21 Using C-13 as the capture probe and using M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Example 22 Using C-14 as a capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Comparative Example 1 Using C-15 as a capture probe and M-01 and M-02 as nucleic acids other than the target, the same operation as in Example 1 was performed, and the capture rate of T-01 and the attachment of M-01 and M-02 were performed. The rate and S / N ratio were determined. The results are shown in Table 2.
  • Comparative Example 2 Using C-15 as a capture probe and M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Example 1 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • Comparative Example 3 In Comparative Example 3, the capture probe C-16 immobilized on the support was brought into contact with the target nucleic acid T-01 and nucleic acids M-01 and M-02 other than the target nucleic acid, and C-16 and the target nucleic acid T-01 were contacted. And a step of washing to remove nucleic acids (M-01 and M-02) other than the target nucleic acid attached to the support after C-16 or T-01 was detected. .
  • Hybridization T-01 (100 ⁇ M), M-01 (100 ⁇ M), and M-02 (100 ⁇ M) were mixed at 1 ⁇ L each and diluted with 10 mM phosphate buffer (containing 100 mM sodium chloride and 0.1% Tween 20). The total liquid volume was 200 ⁇ L.
  • 10 mM phosphate buffer containing 100 mM sodium chloride and 0.1% Tween 20.
  • the total liquid volume was 200 ⁇ L.
  • an incubator set temperature: 37 ° C.
  • “Omnifit” registered trademark of Isis Co., Ltd.
  • glass column inner diameter: 3 mm, 2 ⁇ m stainless steel frits column stoppers at both ends
  • beads with C-16 immobilized thereon were placed, After the above mixed solution (200 ⁇ L) was added, the mixture was stirred for 5 minutes to hybridize C-20 and T-01. After filtration of the solution, the beads were washed with 50% DMSO aqueous solution.
  • Detection T-01 (100 ⁇ M), M-01 (100 ⁇ M), and M-02 (100 ⁇ M) are each mixed at 1 ⁇ L, and diluted with 10 mM phosphate buffer (100 mM sodium chloride, containing 0.1% Tween 20). Then, the fluorescence of a solution (solution C) obtained by adding a 50% DMSO aqueous solution (100 ⁇ L) to a solution with a total solution volume of 200 ⁇ L and the filtrate (solution D) obtained in the hybridization step and the washing step were measured.
  • Comparative Example 4 Using C-16 as the capture probe and using M-03 and M-04 as nucleic acids other than the target nucleic acid, the same operation as in Comparative Example 3 was performed, and the capture rate of T-01, M-03 and M-04 The adhesion rate and S / N ratio were determined. The results are shown in Table 2.
  • the complex formed by hybridization of the target nucleic acid and the capture probe introduced with the photoreactive group is irradiated with light to covalently bond between the capture probe and the target nucleic acid.
  • target nucleic acid (T-01) was captured, but nucleic acids other than the target (M-01, M-02, M-03, M-04) were hardly attached.
  • M-01 and M-02 have a base sequence that differs by only one base sequence of T-01
  • M-03 has a base sequence that differs from the base sequence of T-01 at two locations.
  • the target nucleic acid (T-01) is accurate even when nucleic acids other than these targets are mixed. We were able to detect well.
  • the spacer is any one of an alkylene group, a poly (oxyethylene) group, a base-deficient sugar chain group, and a non-natural base-introduced nucleoside group
  • the target nucleic acid is comparable. Was detected with high accuracy.
  • the results of Examples 1 and 6 to 9 even when the location where the spacer was inserted was changed, it was possible to detect with the same high accuracy.
  • the target nucleic acid and the capture probe are hybridized after the step of hybridizing the target nucleic acid and the capture probe by inserting a spacer in addition to the capture probe into which the photoreactive group has been introduced.
  • a spacer in addition to the capture probe into which the photoreactive group has been introduced.
  • SEQ ID NO: 1 Synthetic DNA (Cy3 label at the 5 ′ end)
  • SEQ ID NO: 2 Synthetic DNA (FITC labeled at the 5 ′ end)
  • SEQ ID NO: 3 Synthetic DNA (Cy5 labeled at the 5 ′ end)
  • SEQ ID NO: 4 Synthetic DNA (FITC labeled at the 5 ′ end)
  • SEQ ID NO: 5 Synthetic DNA (Cy5 labeled at the 5 ′ end)
  • Sequence number 6 Synthetic DNA (5 'terminal biotinylation)

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Abstract

L'invention concerne un procédé de détection d'un acide nucléique cible au moyen duquel il est possible de détecter un acide nucléique cible avec une haute précision et une haute sensibilité. Le procédé de détection d'un acide nucléique cible comprend une étape consistant à hybrider l'acide nucléique cible contenu dans un échantillon et une sonde de capture. Au moins une base d'acide nucléique dans la molécule d'acide nucléique de la sonde de capture est substituée par un groupe photoréactif et au moins un élément espaceur est inséré dans la sonde de capture. Après l'étape pour l'hybridation de l'acide nucléique cible et de la sonde de capture, le complexe formé par hybridation de l'acide nucléique cible et de la sonde de capture est exposé à de la lumière et une liaison covalente est formée entre le groupe photoréactif et une base d'acide nucléique dans l'acide nucléique cible.
PCT/JP2015/065082 2014-05-27 2015-05-26 Procédé de détection d'un acide nucléique cible WO2015182601A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008527979A (ja) * 2005-01-12 2008-07-31 アプレラ コーポレイション 核酸の選択的増幅のための組成物、方法およびキット
JP2013523128A (ja) * 2010-03-26 2013-06-17 インテグレイテッド ディーエヌエイ テクノロジーズ インコーポレイテッド 核酸のハイブリダイゼーションを強化する方法
WO2014034753A1 (fr) * 2012-08-31 2014-03-06 東レ株式会社 Procédé de détection d'un acide nucléique cible
JP2014187934A (ja) * 2013-03-27 2014-10-06 Toray Ind Inc 標的核酸の検出方法

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JP2008527979A (ja) * 2005-01-12 2008-07-31 アプレラ コーポレイション 核酸の選択的増幅のための組成物、方法およびキット
JP2013523128A (ja) * 2010-03-26 2013-06-17 インテグレイテッド ディーエヌエイ テクノロジーズ インコーポレイテッド 核酸のハイブリダイゼーションを強化する方法
WO2014034753A1 (fr) * 2012-08-31 2014-03-06 東レ株式会社 Procédé de détection d'un acide nucléique cible
JP2014187934A (ja) * 2013-03-27 2014-10-06 Toray Ind Inc 標的核酸の検出方法

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