WO2019229792A1 - Procédé de détection d'un fragment d'acide nucléique cible - Google Patents

Procédé de détection d'un fragment d'acide nucléique cible Download PDF

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WO2019229792A1
WO2019229792A1 PCT/JP2018/020305 JP2018020305W WO2019229792A1 WO 2019229792 A1 WO2019229792 A1 WO 2019229792A1 JP 2018020305 W JP2018020305 W JP 2018020305W WO 2019229792 A1 WO2019229792 A1 WO 2019229792A1
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nucleic acid
represented
following formula
acid fragment
probe
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PCT/JP2018/020305
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Japanese (ja)
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太郎 上野
久皇 鈴木
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株式会社ニコン
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present invention relates to a method for detecting a target nucleic acid fragment. More specifically, the present invention relates to a method for detecting a target nucleic acid fragment and a kit for detecting a target nucleic acid fragment.
  • ⁇ -TAS Micro-Total Analysis Systems
  • Non-Patent Document 1 describes a method of performing rolling circle amplification on a DNA microarray.
  • Rolling circle amplification is a type of nucleic acid amplification that synthesizes a complementary strand using a single-stranded or double-stranded circular nucleic acid as a template.
  • the method according to one embodiment is a method for detecting a target nucleic acid fragment consisting of a first part ⁇ and a second part ⁇ , comprising (a) the target nucleic acid fragment and a third part ⁇ , A capture probe immobilized on a solid support, a nucleic acid region ⁇ ′ complementary to the first portion ⁇ , a nucleic acid region ⁇ ′ complementary to the third portion ⁇ , a nucleic acid region ⁇ ′, A nucleic acid region ⁇ ′ complementary to the second portion ⁇ is reacted with a single-stranded or circular nucleic acid probe linked in this order, and the end of the first portion ⁇ of the target nucleic acid fragment; The end of the third portion ⁇ of the capture probe is bound, the nucleic acid region ⁇ ′ of the circular nucleic acid probe is the first portion ⁇ , the nucleic acid region ⁇ ′ is the third portion ⁇ , Obtaining a complex in which the nucleic acid region ⁇ ′
  • the partial nucleic acid region in the single-stranded nucleic acid amplified by hybridizing a detection probe labeled with a labeling substance having the same sequence as the nucleic acid region to the partial nucleic acid region of the single-stranded nucleic acid Detecting.
  • a kit for detecting a target nucleic acid fragment detects a target nucleic acid fragment represented by the following formula (3), and is represented by the following formula (1) or (8).
  • ⁇ , ⁇ , ⁇ ′, and ⁇ each represent a single-stranded nucleic acid fragment having an arbitrary base sequence, and ⁇ ′, ⁇ ′, and ⁇ ′ represent ⁇ , ⁇ , respectively, , Represents a single-stranded nucleic acid fragment consisting of a base sequence complementary to ⁇ .
  • (A)-(c) is a schematic diagram explaining the detection method of the target nucleic acid fragment in case a capture probe is a nucleic acid array.
  • (A) to (f) are fluorescence micrographs of typical spots in Experimental Example 1.
  • (A) And (b) is a graph which shows the fluorescence intensity of Cy3 digitized based on the fluorescence micrograph in Experimental example 2.
  • FIG. 1 is a schematic diagram explaining the detection method of the target nucleic acid fragment in case a capture probe is a nucleic acid array.
  • (A) to (f) are fluorescence micrographs of typical spots in Experimental Example 1.
  • (A) And (b) is a graph which shows the fluorescence intensity of Cy3 digitized based on the fluorescence micrograph in Experimental example 2.
  • the present invention provides a method for detecting a target nucleic acid fragment consisting of a first part ⁇ and a second part ⁇ , wherein (a) the target nucleic acid fragment has a third part ⁇ .
  • a single-stranded or circular nucleic acid probe in which a nucleic acid region ⁇ ′ complementary to the second portion ⁇ is linked in this order is reacted with the end of the first portion ⁇ of the target nucleic acid fragment;
  • the end of the third part ⁇ of the capture probe is bound, the nucleic acid region ⁇ ′ of the circular nucleic acid probe is the first part ⁇ , and the nucleic acid region ⁇ ′ is the third part ⁇ .
  • a detection probe labeled with a labeling substance having the same sequence as the region is hybridized to a partial nucleic acid region of the single-stranded nucleic acid, and the partial nucleic acid region in the amplified single-stranded nucleic acid is And a step of detecting.
  • the method of the first embodiment includes a target nucleic acid fragment represented by the following formula (3), a single-stranded nucleic acid probe represented by the following formula (1), and the following formula (2) bound to a solid phase.
  • a step of contacting the third part represented by formula (6) to form a complex represented by the following formula (6), ⁇ and ⁇ in the complex represented by the following formula (6), and ⁇ ′ And ⁇ ′ are bonded to each other to form a complex represented by the following formula (7), and ⁇ - ⁇ - ⁇ in the complex represented by the following formula (7) is used as a primer.
  • a probe having a sequence complementary to a part of the nucleic acid fragment represented by the following formula (9) is hybridized, and the presence of the nucleic acid fragment represented by the following formula (9) is detected. It can be said that it is.
  • ⁇ , ⁇ , ⁇ , and ⁇ represent single-stranded nucleic acid fragments each having an arbitrary base sequence
  • ⁇ ′, ⁇ ′, ⁇ ', ⁇ ' represents a single-stranded nucleic acid fragment having a base sequence complementary to ⁇ , ⁇ , ⁇ , ⁇ , respectively.
  • the method of the first embodiment is a method for detecting a target nucleic acid fragment consisting of a first part ⁇ and a second part ⁇ , comprising the step (a): a sample containing the target nucleic acid fragment; A solid phase carrier on which a capture probe having a portion ⁇ is fixed; a nucleic acid region ⁇ ′ complementary to the first portion ⁇ ; a nucleic acid region ⁇ ′ complementary to the third portion ⁇ ; and a nucleic acid region ⁇ And a nucleic acid probe in which a nucleic acid region ⁇ ′ complementary to the second portion ⁇ is linked in this order, and the nucleic acid region ⁇ ′ is in contact with the third portion ⁇ and the nucleic acid region forming a complex wherein ⁇ ′ is hybridized with the first part ⁇ and the nucleic acid region ⁇ ′ is hybridized with the second part ⁇ , and the first part of the target nucleic acid fragment in the complex the end of ⁇ is bound to the end of
  • a step of synthesizing a single-stranded nucleic acid having a simple base sequence and step (c): a probe having a sequence complementary to a part of the single-stranded nucleic acid synthesized in the step (b) And the step of hybridizing to the single-stranded nucleic acid synthesized in step (b) and detecting the presence of the single-stranded nucleic acid synthesized in step (b).
  • ⁇ , ⁇ , ⁇ , ⁇ ′, ⁇ ′, ⁇ ′, and ⁇ ′ represent the same meaning as in the above formulas (1) to (3) and (6) to (9).
  • the amplification reaction of the single-stranded nucleic acid proceeds only when the target nucleic acid fragment, the capture probe and the nucleic acid probe are hybridized to form a complex of formula (7). Differences in units can be detected. In addition, since the detection probe is hybridized to the detection sequence present in the amplification product and detected, the target nucleic acid fragment can be detected even in a minute amount.
  • a plurality of types of target nucleic acid fragments in a sample can be easily and simultaneously detected in one reaction container.
  • rolling circle amplification which is an isothermal amplification reaction, is used, it is easy to reduce the size of the inspection equipment.
  • each step will be described.
  • ⁇ and ⁇ , and ⁇ ′ and ⁇ ′ in the complex represented by the above formula (6) are bonded to form a complex represented by the above formula (7).
  • the nucleic acid fragment represented by the above formula (1) may be referred to as a “padlock probe”.
  • the nucleic acid fragment represented by the above formula (2) may be referred to as “capture probe”.
  • Target nucleic acid fragment represented by formula (3) is a target nucleic acid fragment to be detected.
  • the target nucleic acid may be DNA or RNA.
  • RNA include micro RNA (miRNA), messenger RNA (mRNA), and non-coding RNA (ncRNA).
  • the target nucleic acid fragment needs to be a single-stranded nucleic acid.
  • a single-stranded nucleic acid may be prepared by heat denaturation, alkali denaturation or the like, and this may be used as a target nucleic acid fragment.
  • the target nucleic acid is preferably contained in a biological sample.
  • biological samples include blood, urine, saliva and the like.
  • the target nucleic acid fragment is bound to the nucleic acid fragment represented by the above formula (2) bound to the solid phase in the step described later.
  • the binding is preferably T4 DNA ligase, E. coli. It is carried out with a ligase having an activity of linking the 5'-phosphorylated end and 3'-OH end of adjacent DNA strands by a phosphodiester bond, represented by E. coli DNA ligase, heat-resistant DNA ligase and the like. For this reason, it is preferable that a phosphate group exists at the 5 'end of the target nucleic acid fragment.
  • a phosphate group is present at the 5 'end of miRNA. For this reason, when the target nucleic acid fragment is miRNA, this step may be carried out as it is.
  • a phosphate at the ⁇ -position of ATP represented by T4 polynucleotide kinase
  • T4 polynucleotide kinase is used as a polynucleotide (double-stranded and single-stranded DNA, RNA) and A kinase having catalytic activity for transfer or exchange of the nucleoside 3′-monophosphate to the 5′-hydroxyl terminus can be reacted to add a phosphate group to the 5 ′ terminus of the target nucleic acid fragment.
  • any phosphate group can be added before binding to the nucleic acid fragment represented by the above formula (2) bound to the solid phase. It may be done in stages.
  • the length of the target nucleic acid fragment may be about 15 to 500 bases, about 30 to 500 bases, or about 30 to 200 bases.
  • the target nucleic acid fragment may be fragmented to a length within the above range by, for example, ultrasonic treatment, enzyme treatment or the like.
  • Nucleic acid fragment represented by formula (2) (capture probe) >> The capture probe is bound to the solid phase at the 5 ′ end.
  • the solid phase include a substrate and particles.
  • the capture probe and the solid phase may be directly bonded or may be bonded via a linker.
  • the linker include polyethylene glycol, polyacrylamide, polyether, polyester, polyurethane, polysaccharides, copolymers thereof, and nucleic acid fragments.
  • the capture probe may have a linker on the 5 ′ end side of ⁇ in the above formula (2). Therefore, it can be said that the capture probe is a nucleic acid fragment containing ⁇ in the above formula (2).
  • a plurality of capture probes may be arranged in an array on the solid phase. That is, the capture probe may be a nucleic acid array.
  • the capture probe represented by the above formula (2) and the target nucleic acid fragment represented by the above formula (3) are bound in a sequence-specific manner. Therefore, when the capture probe is a nucleic acid array, it becomes possible to detect the target nucleic acid fragment by arranging it at individual positions on the nucleic acid array in a sequence-specific manner.
  • the capture probes arranged on the nucleic acid array preferably have different base sequences at each position on the nucleic acid array.
  • the solid phase is preferably a nucleic acid array in which a plurality of types of capture probes are immobilized at different positions.
  • a first capture probe that hybridizes with the first nucleic acid is fixed at the first position of the solid phase
  • a second capture probe that hybridizes with the second nucleic acid is fixed at the second position.
  • the target nucleic acid fragment is combined with the capture probe in a process described later.
  • the binding is preferably performed by ligase as described above. For this reason, it is preferable that a hydroxyl group is present at the 3 'end of the capture probe.
  • the padlock probe has a base sequence complementary to both the target nucleic acid fragment represented by the above formula (3) and the capture probe. Therefore, in this step, when the target nucleic acid fragment represented by the above formula (3), the padlock probe, and the capture probe bound to the solid phase are brought into contact with each other, they hybridize in regions complementary to each other, and A complex represented by formula (6) is formed.
  • ⁇ is a capture probe, and the 5 ′ end is bound to the solid phase. Moreover, it hybridizes with ⁇ ′ of the padlock probe.
  • ⁇ - ⁇ is a target nucleic acid fragment represented by the above formula (3), and hybridizes with ⁇ ′ and ⁇ ′ of the padlock probe, respectively.
  • the complex represented by the above formula (6) is not bound between ⁇ and ⁇ , and between ⁇ ′ and ⁇ ′.
  • the target nucleic acid fragment can be adjacent to the capture probe in a sequence-specific manner. Therefore, when the capture probe is a nucleic acid array, it becomes possible to detect the target nucleic acid fragment by arranging it at individual positions on the nucleic acid array in a sequence-specific manner.
  • a phosphate group is present at the 5 'end of miRNA. For this reason, when the target nucleic acid fragment is miRNA, this step may be carried out as it is.
  • a phosphate group is not present at the 5 ′ end of the target nucleic acid fragment, a phosphate at the ⁇ -position of ATP, represented by T4 polynucleotide kinase, is used as a polynucleotide (double-stranded and single-stranded DNA, RNA) and A phosphate group can be added to the 5 ′ end of the target nucleic acid fragment by reacting a kinase having catalytic activity for transfer or exchange of the nucleoside 3′-monophosphate to the 5′-hydroxyl end.
  • the phosphate group When a phosphate group is added to the 5 'end of the target nucleic acid fragment, the phosphate group may be added at any stage as long as it is before binding to a capture probe bound to a solid phase.
  • the padlock probe is connected in a ring shape in the process described later. Specifically, the padlock probe is coupled between ⁇ ′ and ⁇ ′. This binding is performed by ligase in the same manner as the binding between the target nucleic acid fragment and the capture probe described above. Therefore, it is preferable that a phosphate group is present at the 5 ′ end of ⁇ ′ of the padlock probe and a hydroxyl group is present at the 3 ′ end of ⁇ ′. When a phosphate group does not exist at the 5 ′ end of ⁇ ′ of the padlock probe, a phosphate group can be added in the same manner as the phosphate group added to the target nucleic acid fragment described above.
  • ⁇ ′ is a base sequence for detecting the presence of the target nucleic acid fragment represented by the above formula (3).
  • ⁇ and ⁇ , and ⁇ ′ and ⁇ ′ in the complex represented by the above formula (6) are bonded to form a complex represented by the above formula (7).
  • ⁇ and ⁇ , and ⁇ ′ and ⁇ ′ are preferably bound by ligase.
  • the target nucleic acid fragment represented by the above formula (3) is bound on the solid phase.
  • the target nucleic acid fragment represented by the above formula (3) serves as a primer for rolling circle amplification described later.
  • the padlock probe becomes a circular single-stranded nucleic acid, which becomes a template for rolling circle amplification described later.
  • the complementary strand of the circular single-stranded nucleic acid represented by the above formula (8) is synthesized by rolling circle amplification using ⁇ - ⁇ - ⁇ in the complex represented by the above formula (7) as a primer.
  • a nucleic acid fragment represented by the above formula (9) is obtained.
  • the nucleic acid fragment represented by the above formula (9) may be referred to as the amplified single-stranded nucleic acid.
  • Rolling circle amplification can be performed by adjusting the temperature to a temperature at which the DNA polymerase exhibits activity in the presence of dNTP and DNA polymerase as substrates.
  • Rolling circle amplification is an isothermal amplification reaction system. For this reason, it can implement with a simple apparatus.
  • Examples of the DNA polymerase for performing rolling circle amplification include ⁇ 29 DNA polymerase, Csa DNA polymerase, Bst DNA polymerase, and the like.
  • the reaction temperature for rolling circle amplification is, for example, about 30 ° C. when ⁇ 29 DNA polymerase is used, about 60 ° C. when Csa DNA polymerase is used, and about 65 ° C. when Bst DNA polymerase is used. It is.
  • reaction time for rolling circle amplification may be, for example, 1 to 24 hours, for example, 1 to 12 hours, for example, 1 to 6 hours.
  • nucleic acid fragment represented by the above formula (9) is obtained.
  • the target nucleic acid fragment binds to the 3 ′ end side of the capture probe bound to the solid phase, and further complements the padlock probe on the 3 ′ end side of the target nucleic acid fragment.
  • the padlock probe At the position where the target nucleic acid fragment represented by the above formula (3) is arranged at the position where the capture probe is present and the target nucleic acid fragment represented by the above formula (3) is further arranged, the padlock probe Thus, a nucleic acid fragment complementary to is repeatedly amplified. The amplification product is anchored to the solid phase.
  • the nucleic acid fragment represented by the above formula (9) is contacted with a probe having a sequence complementary to a part of the nucleic acid fragment represented by the following formula (9) and hybridized, and the above formula (9)
  • the partial nucleic acid region in the nucleic acid fragment represented by 9) is detected.
  • the probe having a sequence complementary to a part of the nucleic acid fragment represented by the following formula (9) is a probe having the same sequence as a part of the nucleic acid region of the padlock probe.
  • a probe that specifically hybridizes to at least a part of ⁇ is brought into contact with the nucleic acid fragment represented by the above formula (9) to detect ⁇ in the nucleic acid fragment represented by the above formula (9).
  • the probe is, for example, a detection probe labeled with a labeling substance. Examples of the labeling substance include fluorescent dyes, enzymes, and magnetic substances.
  • ⁇ is a nucleic acid fragment complementary to ⁇ 'of the padlock probe, and the copy number is increased by rolling circle amplification in step (c).
  • the detection target in this step may be a part other than ⁇ .
  • it may be any of ⁇ , ⁇ , and ⁇ , or may be a portion that extends over these plural portions.
  • the binding sequence is ⁇ in the nucleic acid fragment. Therefore, a detection probe having a sequence complementary to at least a part of ⁇ can be used as the detection probe. In other words, a detection probe having the same base sequence as at least a part of ⁇ ′ can be used.
  • a detection probe having the same sequence as at least a part of ⁇ ′, a single-stranded nucleic acid represented by the above formula (1) or the above formula coexists. Since the detection probe having the same sequence as at least a part of ⁇ ′ has a smaller molecular weight than the nucleic acid fragment represented by the above formula (1) or the circular single-stranded nucleic acid represented by the above formula (8). , It binds preferentially to the nucleic acid fragment represented by formula (2).
  • nucleic acid fragment represented by the above formula (1) or the circular single-stranded nucleic acid represented by the above formula (8) to bind to the capture probe represented by the above formula (2), In some cases, the amount of nucleic acid fragment represented by Formula (9), which is a rolling cycle amplification product, is reduced. Therefore, when performing real-time detection described later, a detection region ⁇ ′ is provided in the single-stranded nucleic acid represented by the above formula (1) or the circular single-stranded nucleic acid represented by the above formula (8), and ⁇ is used as a detection probe. 'It is preferable to prepare a probe.
  • the detection of the probe hybridized with the nucleic acid fragment represented by the above formula (9) can be performed by, for example, a fluorescence microscope, a spectrophotometer, a GMR sensor or the like according to the label attached to the probe.
  • the signal can be detected using a detection system that does not require B / F separation, that is, separation of bound and free substances.
  • detection systems that do not require B / F separation include signal detection using a total reflection illumination fluorescence microscope, signal detection using surface plasmon resonance, signal detection using fluorescence resonance energy transfer, and giant magnetoresistance effect Signal detection and the like.
  • Step (b) and step (c) may be performed simultaneously. That is, the synthesis of the nucleic acid fragment represented by the above formula (9) by rolling circle amplification may be performed in the presence of a probe that specifically hybridizes to at least a part of ⁇ in the above formula (9). Thereby, for example, the step (b) and the step (c) can be simultaneously performed, and ⁇ in the nucleic acid fragment represented by the above formula (9) can be detected in real time.
  • the temperature control mechanism can be simplified by using an isothermal amplification reaction system.
  • the reaction reagent can have a single liquid configuration, the test chip structure can be simplified.
  • the detection time can be shortened by evaluating the initial signal speed.
  • quantification can be performed based on the amount of change in signal, the influence of variation in lung fluorescence intensity is excluded, and more accurate quantification is possible.
  • reaction time until reaching a certain fluorescence intensity can be quantified as an index, so measurement should be performed using an inexpensive detector having only a single-digit dynamic range. Is possible. That is, the dynamic range requirement required for the detection system is relaxed.
  • ⁇ 1 , ⁇ 2 , ⁇ 3 , ⁇ 1 , ⁇ 2 , ⁇ 3 , ⁇ , ⁇ 1 , ⁇ 2 , and ⁇ 3 are each a single strand having an arbitrary base sequence.
  • Represents a nucleic acid fragment, ⁇ 1 ′, ⁇ 2 ′, ⁇ 3 ′, ⁇ 1 ′, ⁇ 2 ′, ⁇ 3 ′, ⁇ ′, ⁇ 1 ′, ⁇ 2 ′, and ⁇ 3 ′ represent ⁇ 1 and ⁇ 2 , ⁇ 3 , ⁇ 1 , ⁇ 2 , ⁇ 3 , ⁇ , ⁇ 1 , ⁇ 2 , ⁇ 3 and a single-stranded nucleic acid fragment consisting of a complementary base sequence.
  • S represents a solid phase and P represents a probe.
  • the capture probes represented by ⁇ 1 , ⁇ 2 , and ⁇ 3 are each bound to a solid phase to form a nucleic acid array.
  • three types of target nucleic acid fragments represented by ⁇ 1 - ⁇ 1 , ⁇ 2 - ⁇ 2 , and ⁇ 3 - ⁇ 3 can be detected simultaneously.
  • step (a) described above is performed.
  • FIG. 1A a complex of a padlock probe, a capture probe, and a target nucleic acid fragment is formed.
  • step (b) described above is performed.
  • step (b) described above is performed.
  • the complementary strand of the padlock probe is repeatedly bound to the solid phase-capture probe-target nucleic acid fragment.
  • step (c) described above is performed.
  • the three types of padlock probes used all have the same base sequence of ⁇ ′. Therefore, all target nucleic acid fragments on the nucleic acid array can be simultaneously detected using one type of probe P that specifically hybridizes to at least a part of ⁇ .
  • the probe P is labeled with a fluorescent dye.
  • the method of 2nd Embodiment is a method of detecting the target nucleic acid fragment represented by following formula (3), Comprising: The target nucleic acid fragment represented by following formula (3) and the following formula (4) are represented.
  • the probe specifically hybridizes to at least a part of ⁇ to the nucleic acid fragment represented by the following formula (9) To detect ⁇ in the nucleic acid fragment represented by the following formula (9).
  • ⁇ , ⁇ , ⁇ , and ⁇ each represent a single-stranded nucleic acid fragment having an arbitrary base sequence
  • ⁇ ′, ⁇ ′, ⁇ ', ⁇ ' represents a single-stranded nucleic acid fragment having a base sequence complementary to ⁇ , ⁇ , ⁇ , ⁇ , respectively.
  • the method of the second embodiment is mainly different from the method of the first embodiment in the step (a2). More specifically, the method of the second embodiment is a circular nucleic acid in which the 5 ′ end of ⁇ ′ and the 3 ′ end of ⁇ ′ of the padlock probe are previously bound, and the circular nucleic acid binds to the solid phase.
  • the method is mainly different from the method of the first embodiment in that it is pre-hybridized with the captured probe, and the other points are the same as the method of the first embodiment.
  • the complex represented by the above formula (4) since the complex represented by the above formula (4) is formed in advance, the target nucleic acid fragment represented by the above formula (3) can be efficiently contacted. A complex represented by the above formula (10) can be obtained.
  • the target nucleic acid fragment represented by the above formula (3), the nucleic acid fragment represented by the above formula (1), and the above formula (2) bound to a solid phase are represented.
  • the nucleic acid fragment is contacted to form a complex represented by the above formula (6).
  • the target nucleic acid fragment represented by the above formula (3), the nucleic acid fragment represented by the above formula (1), and the nucleic acid fragment represented by the above formula (2) bound to a solid phase are simultaneously used.
  • hybridization of the target nucleic acid fragment represented by the above formula (3) having a high collision frequency with the nucleic acid fragment represented by the above formula (1) occurs preferentially, and in the above formula (6), In some cases, the probability that a complex is formed is reduced, and the detection sensitivity is lowered.
  • nucleic acid fragment represented by the above formula (1) and the nucleic acid fragment represented by the above formula (2) bonded to a solid phase are brought into contact with each other in advance and represented by the above formula (4). Detection sensitivity can be improved by reliably forming a complex.
  • the unreacted nucleic acid fragment represented by the formula (1) is removed before contacting the complex represented by the formula (4) with the target nucleic acid fragment represented by the formula (3).
  • the complex represented by the above formula (6) can be formed more efficiently.
  • the method of 3rd Embodiment is a method of detecting the target nucleic acid fragment represented by following formula (3), Comprising: The target nucleic acid fragment represented by following formula (3) and the following formula (5) are represented.
  • a probe having a sequence complementary to a part of the fragment is hybridized to detect the presence of the nucleic acid fragment represented by the following formula (9).
  • step (c) in the method of the third embodiment the probe specifically hybridizes to at least a part of ⁇ to the nucleic acid fragment represented by the following formula (9) To detect ⁇ in the nucleic acid fragment represented by the following formula (9).
  • ⁇ , ⁇ , ⁇ , and ⁇ represent single-stranded nucleic acid fragments each having an arbitrary base sequence
  • ⁇ ′, ⁇ ′, ⁇ ', ⁇ ' represents a single-stranded nucleic acid fragment having a base sequence complementary to ⁇ , ⁇ , ⁇ , ⁇ , respectively.
  • the method of the third embodiment is mainly different from the method of the first embodiment in the step (a3). More specifically, the method of the third embodiment is mainly different from the method of the first embodiment in that the padlock probe is pre-hybridized with the capture probe bound to the solid phase, and the other points are the same. Is the same as the method of the first embodiment.
  • the method of the third embodiment is different from the method of the second embodiment mainly in that the 5 ′ end of ⁇ ′ and the 3 ′ end of ⁇ ′ of the padlock probe are not bonded in advance. Is the same as the method of the first embodiment.
  • the complex represented by the above formula (5) since the complex represented by the above formula (5) is formed in advance, the target nucleic acid fragment represented by the above formula (3) can be efficiently contacted. A complex represented by the above formula (6) can be obtained. Moreover, the composite represented by the said Formula (5) can be manufactured more simply than the composite represented by the said Formula (4) in 2nd Embodiment.
  • the target nucleic acid fragment represented by the above formula (3), the nucleic acid fragment represented by the above formula (1), and the above formula (2) bound to a solid phase are represented.
  • the nucleic acid fragment is contacted to form a complex represented by the above formula (6).
  • the target nucleic acid fragment represented by the above formula (3), the nucleic acid fragment represented by the above formula (1), and the nucleic acid fragment represented by the above formula (2) bound to a solid phase are simultaneously used.
  • hybridization of the target nucleic acid fragment represented by the above formula (3) having a high collision frequency with the nucleic acid fragment represented by the above formula (1) occurs preferentially, and in the above formula (6), In some cases, the probability that a complex is formed is reduced, and the detection sensitivity is lowered.
  • nucleic acid fragment represented by the above formula (1) and the nucleic acid fragment represented by the above formula (2) bonded to a solid phase are brought into contact with each other and represented by the above formula (5). Detection sensitivity can be improved by reliably forming a complex.
  • the unreacted nucleic acid fragment represented by the formula (1) is removed before contacting the complex represented by the formula (5) with the target nucleic acid fragment represented by the formula (3).
  • the complex represented by the above formula (6) can be formed more efficiently.
  • the present invention includes a nucleic acid fragment represented by the following formula (1) and a nucleic acid fragment represented by the following formula (2) bound to a solid phase, the following formula (3):
  • the kit for detecting the target nucleic acid fragment represented by these is provided.
  • ⁇ , ⁇ , ⁇ ′, and ⁇ each represent a single-stranded nucleic acid fragment having an arbitrary base sequence, and ⁇ ′, ⁇ ′, and ⁇ ′ represent ⁇ , ⁇ , respectively, , Represents a single-stranded nucleic acid fragment consisting of a base sequence complementary to ⁇ .
  • the kit of the present embodiment can be suitably used for performing the method of the first embodiment described above.
  • the nucleic acid fragment (padlock probe) represented by the formula (1) the nucleic acid fragment (capture probe) represented by the formula (2), and the target nucleic acid represented by the formula (3)
  • the solid phase to which the fragment and the capture probe are bound is the same as described above.
  • the padlock probe and the capture probe may form a complex represented by the following formula (4) or (5).
  • the kit in which the padlock probe and the capture probe form a complex represented by the above formula (4) can be suitably used for carrying out the method of the second embodiment described above.
  • kits in which a padlock probe and a capture probe form a complex represented by the above formula (5) can be suitably used for carrying out the method of the third embodiment described above.
  • the kit of this embodiment may further include a probe that specifically hybridizes to at least part of the base sequence ⁇ complementary to ⁇ ′ of the padlock probe.
  • the probe that specifically hybridizes to at least a part of the nucleic acid fragment ⁇ is the same as described above.
  • the capture probe bound to the solid phase is preferably a nucleic acid array.
  • the capture probe is a nucleic acid array, multiple types of target nucleic acid fragments in a sample can be easily and simultaneously detected in one reaction container.
  • the kit of this embodiment may further contain a nucleic acid ligase, and may further contain a nucleic acid polymerase.
  • the nucleic acid ligase and nucleic acid polymerase are the same as described above.
  • a nucleic acid array was prepared by binding a capture probe to a substrate.
  • the capture probe used was a DNA fragment.
  • As the substrate an epoxy group-modified glass substrate was used.
  • eight reaction chambers were formed on one substrate, and two kinds of capture probes were bonded to each of the five reaction chambers.
  • the capture probes used are shown in Table 1 below.
  • reaction solution for rolling circle amplification contained sodium chloride having a final concentration of 0 mM, 50 mM, or 150 mM, and further contained a target nucleic acid fragment (final concentration 25 nM), a padlock probe, T4 DNA ligase, ⁇ 29 DNA polymerase, and a Cy3 labeled probe (SEQ ID NO: 1).
  • the target nucleic acid fragments and padlock probes used are shown in Table 1 below.
  • the Cy3-labeled probe was capable of hybridizing to the complementary strand of any padlock probe used.
  • the target nucleic acid fragment “Target DNA” (SEQ ID NO: 4) was able to hybridize to both the padlock probes “PD1503” (SEQ ID NO: 3) and “PD1505” (SEQ ID NO: 8).
  • FIG. 2 (c) is a fluorescence micrograph of the reaction chamber 3.
  • (D) is a fluorescence micrograph of the reaction chamber 5
  • FIG. 2 (e) is a fluorescence micrograph of the reaction chamber 6
  • FIG. 2 (f) is a fluorescence micrograph of the reaction chamber 7.
  • the rolling circle amplification reaction solution contained sodium chloride having a final concentration of about 150 mM, the detection efficiency of the target nucleic acid fragment tended to increase.
  • Example 2 (Detection of target nucleic acid fragment 2) The same reaction as in Experimental Example 1 was performed except that the concentration of sodium chloride in the reaction solution was fixed at a final concentration of 150 mM and the final concentration of the target nucleic acid was changed to 6.25 nM, 12.5 nM, and 25 nM. Cy3 fluorescence was observed at each spot of the nucleic acid array.
  • the capture probes, target nucleic acid fragments, and padlock probes used are shown in Table 2 below.
  • FIG. 3A shows the result when “c1503” is used as the capture probe
  • FIG. 3B shows the result when “c1505” is used as the capture probe.

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Abstract

L'invention concerne un procédé de détection d'un fragment d'acide nucléique cible comprenant une première partie α et une deuxième partie δ, le procédé comprenant : (a) une étape de mise en réaction d'un fragment d'acide nucléique cible, d'une sonde de capture qui a une troisième partie β et est immobilisée sur un support solide, et d'une sonde d'acide nucléique monocaténaire ou circulaire dans laquelle une région α' complémentaire de α, une région β' complémentaire de β, une région γ', et une région δ' complémentaire de δ sont liées dans l'ordre indiqué sous une forme monocaténaire ou circulaire, de liaison l'extrémité d'α et l'extrémité de β, et d'obtention d'un complexe dans lequel la région α' de la sonde d'acide nucléique circulaire est hybridée avec α, la région β' est hybridée avec β, et la région δ' est hybridée avec δ ; (b) une étape de synthèse d'un acide nucléique simple brin ayant une séquence de base complémentaire de la sonde d'acide nucléique circulaire par amplification par cercle roulant dans laquelle la sonde d'acide nucléique circulaire est utilisée en tant que modèle ; et (c) une étape d'hybridation d'une sonde de détection qui a la même séquence qu'une partie de la région d'acide nucléique de la sonde d'acide nucléique monocaténaire ou circulaire à l'acide monocaténaire, et la détection d'une séquence complémentaire dans l'acide nucléique monocaténaire amplifié.
PCT/JP2018/020305 2018-05-28 2018-05-28 Procédé de détection d'un fragment d'acide nucléique cible WO2019229792A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020177141A1 (en) * 1999-04-20 2002-11-28 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020177141A1 (en) * 1999-04-20 2002-11-28 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HATCH, A. ET AL.: "Rolling circle amplification of DNA immobilized on solid surfaces and its application to multiplex mutation detection", GENET. ANAL., vol. 15, no. 2, 1999, pages 35 - 40, XP000879740, DOI: 10.1016/S1050-3862(98)00014-X *
SCHOPF, E. ET AL.: "Sensitive and selective viral DNA detection assay via microbead-based rolling circle amplification", BIOORG. MED. CHEM. LETT., vol. 18, no. 22, 2008, pages 5871 - 5874, XP025627158, DOI: 10.1016/j.bmcl.2008.07.064 *

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