JP5597939B2 - Method for detecting a target nucleotide sequence using a partially competitive probe - Google Patents

Description

 The present invention relates to a method for detecting a target base sequence having a base mutation site. Specifically, the present invention relates to a method for detecting a target base sequence with high accuracy and high sensitivity using a partially competitive probe in detection of the target base sequence.

  One of the basic means important in genetic engineering is the detection of a nucleic acid having a specific base sequence present in a sample mixture. By detecting the presence of a nucleic acid or gene having a specific base sequence, the expression of the gene of interest can be elucidated to enable new drug discovery, or cells or individuals into which the target gene has been introduced using genetic recombination Can be selected, or genetic defects or changes in humans that cause illness can be detected by genetic diagnosis, enabling diagnosis and prediction before the onset of the illness or at an early stage.

Thus, detection of a nucleic acid having a specific base sequence is a basic means widely used in genetic engineering. For example, the following methods are known.
For a nucleic acid sample having a target base sequence for detection, a nucleic acid probe having a base sequence complementary to the target base sequence is prepared. A target nucleic acid extracted from a cell or the like and subjected to electrophoresis is plotted, and the target nucleic acid is hybridized with the nucleic acid probe. The nucleic acid probe is labeled with a radioisotope or the like in advance so that it can be detected, and the nucleic acid probe hybridized with the nucleic acid is detected by autoradiography to confirm the presence of the nucleic acid having the target base sequence. This method is called the Southern blotting method and is still used with various modifications (see, for example, Non-Patent Document 1 (494-500 tribute)).

  In particular, in recent genetic diagnosis techniques, a DNA chip (DNA microarray) is used for detecting a nucleic acid having a specific base sequence. A large number of sections exist on the substrate of the DNA chip, and DNA probes having various base sequences are immobilized in each section and arranged in an array. A nucleic acid sample derived from a subject previously labeled with a fluorescent dye or the like is dropped onto a DNA chip and hybridized. In the nucleic acid sample, a nucleic acid having a base sequence complementary to the base sequence of the DNA probe is immobilized on the DNA chip by hybridization. A genetic diagnosis can be performed by detecting the immobilized nucleic acid with a fluorescent dye. In this method, various modifications such as nucleic acid adjustment and normalization by competitive hybridization are performed (see, for example, Non-Patent Document 1 (533-535).

"Molecular biology of cells", 4th edition, translated by Keiko Nakamura and Kenichi Matsubara, published by Newton Press, 2004

Thus, in the detection of a nucleic acid having a specific base sequence, hybridization with a nucleic acid probe having a complementary base sequence has long been used as a basic principle that enables specific recognition. However, detection of a nucleic acid having a specific base sequence by hybridization may also hybridize to a nucleic acid having a non-complementary base sequence, and detection accuracy is not always sufficient.
The detection by hybridization utilizes the difference in the thermal stability of the aggregate formed by hybridization, and the hybridization by complementary base pairing also forms non-complementary base pairing. The difference is also only a difference in thermal stability. Therefore, appropriate detection conditions vary depending on the target base sequence, and even under appropriate conditions, conditions for changing thermal stability act equally on both. Therefore, as long as only the difference in thermal stability of hybridization is used as the principle of distinction between the two, there is a problem that a certain noise must be accepted by compromising on the balance of detection specificity and sensitivity. There is a point.

  Furthermore, in recent years, detection of nucleic acids having a base sequence with a single base substitution has been required for new drug discovery and genetic testing. In particular, the technology for detecting base mutations in nucleic acids has great expectations in the field of medical diagnosis. For this purpose, it is necessary to achieve both a specificity that can distinguish and detect sequences that differ by only one base, and a practical sensitivity (S / N ratio).

The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for detecting a target base sequence having a base mutation site with high accuracy and high sensitivity using a partially competitive probe. To do.
Another object of the present invention is to provide a method that allows simple and efficient detection in the detection of a target base sequence.

  The present inventors performed detection using a detection probe having a base sequence complementary to the target base sequence in the presence of a partially competitive probe having a non-complementary base sequence at the target base sequence and the base mutation site. As a result, the detection noise of the target base sequence was reduced, and it was found that detection with higher accuracy and sensitivity was possible, and the present invention was completed.

That is, the present invention provides a method for detecting a target base sequence having the following characteristics.
[1] A method for detecting a target base sequence having a base mutation site,
A partial sequence containing a base mutation site in the target base sequence is a base mutation-containing sequence,
(A) adding a detection probe and a partially competitive probe to a nucleic acid sample;
Hybridizing the detection probe and the partially competitive probe to a target nucleic acid having a target base sequence in the nucleic acid sample;
(B) after the step (a), detecting a detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid,
The base sequence of the detection probe is complementary to a partial region containing a base mutation-containing sequence of the target base sequence,
The base sequence of the partial competitive probe is complementary to a partial region different from the partial region including the base mutation-containing sequence of the target base sequence, and non-complementary at the base mutation site. And
Furthermore, either of following (1) or (2) is satisfied, The detection method of a target base sequence characterized by the above-mentioned.
(1) The detection probe can hybridize with the target nucleic acid on the base mutation-containing sequence and the 5 ′ end side thereof, but cannot hybridize on the 3 ′ end side of the base mutation-containing sequence. Yes, the partially competitive probe is capable of hybridizing with the target nucleic acid at the base mutation-containing sequence and its 3 ′ end, but not at the 5 ′ end of the base mutation-containing sequence. There is.
(2) The detection probe is capable of hybridizing with the target nucleic acid on the base mutation-containing sequence and the 3 ′ end side thereof, but cannot hybridize on the 5 ′ end side of the base mutation-containing sequence. Yes, the partially competitive probe can hybridize with the target nucleic acid on the base mutation-containing sequence and the 5 ′ end side thereof, but cannot hybridize on the 3 ′ end side of the base mutation-containing sequence. There is.
[2] The method for detecting a target base sequence according to the above [1], wherein the base mutation site may be in any position in the base mutation-containing sequence.
[3] The above-mentioned [1] or [2], wherein in the step (b), the detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid is detected using a labeling substance. For detecting a target nucleotide sequence of a bacterium.
[4] The method for detecting a target base sequence according to [3], wherein the labeling substance is a fluorescent dye.
[5] A detection probe hybridized with a portion comprising a base mutation-containing sequence in the target nucleic acid in an assembly in which the target nucleic acid, the detection probe and the partial competition probe were hybridized in the step (b) The method for detecting a target base sequence according to any one of [1] to [4] above, wherein
[6] In the step (b), using the QP (Quenching Probe / Primer) method or the FRET method, detecting a detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid The method for detecting a target base sequence according to the above [4] or [5].

Since the method for detecting a target base sequence of the present invention is a method of partially competing using a partially competitive probe, non-complementary hybridization between the target nucleic acid and the probe is reduced, and the accuracy and performance are increased. The target base sequence can be detected with high sensitivity. Therefore, the target base sequence detection method of the present invention is useful for detecting base mutations such as single nucleotide polymorphisms, and can be used in the field of medical diagnosis and the like.
Furthermore, since non-complementary hybridization can be suppressed without adding a partially competitive probe by simply performing a washing operation, detection can be simplified and efficient.

It is a schematic diagram of aggregate formation in a state where a detection type probe and a partial competition type probe are added to a nucleic acid sample having a single nucleotide mutation, and a target nucleic acid and both probes are present. A target nucleic acid (Target 1) having a target base sequence (first type sequence) or a nucleic acid (Target 2) having a second type sequence, a detection probe (Probe 1), and a partially competitive probe in a target nucleic acid having a single base mutation It is a schematic diagram of the aggregate | assembly which consists of (Probe2). In the target nucleic acid having an insertion mutation, a target nucleic acid (Target 1) having a target base sequence (first type sequence) or a nucleic acid (Target 2) having a second type sequence, a detection probe (Probe 1), a partially competitive probe ( It is a schematic diagram of the aggregate | assembly which consists of Probe2). A target nucleic acid (Target 1) having a target base sequence (first type sequence) or a nucleic acid (Target 2) having a second type sequence, a detection probe (Probe 1), and a partially competitive probe in a target nucleic acid having a deletion mutation It is a schematic diagram of the aggregate | assembly which consists of (Probe2). Target nucleic acid (Target 1) having target base sequence (first type sequence) or nucleic acid (Target 2) having second type sequence, detection type probe (Probe 1), and partial competition type probe It is a schematic diagram of the aggregate | assembly which consists of (Probe2). A target nucleic acid (Target 1) having a target base sequence (first type sequence) or a nucleic acid (Target 2) having a second type sequence, a detection probe (Probe 1), and a partially competitive probe in a target nucleic acid having a single base mutation It is a schematic diagram at the time of detecting the aggregate | assembly which consists of (Probe2) using FRET method. In Example 1, it is the figure which showed the detection result with a temperature change at the time of using a detection type probe (mutant type). In Example 2, it is the figure which showed the detection result with a temperature change at the time of using a detection type probe (wild type).

  The target base sequence detection method of the present invention is a method for detecting a target base sequence having a base mutation site.

In the present invention, the base mutation means that there is a different site in one or more base sequences in the same organism species or the same individual. In the present invention, the base site mutation is performed on the different site in the target base sequence. It is called a part. Examples of the base mutation include single nucleotide polymorphism (SNP), insertion mutation, deletion mutation, and repetitive sequence mutation.
Further, in the present invention, the base mutation detection site refers to a site that forms a base pair with the base mutation site in the target base sequence in the base sequence of the detection probe or the partially competitive probe.

  In the present invention, detecting the target base sequence means detecting whether or not the base sequence of the nucleic acid contained in the nucleic acid sample is the same base sequence as the target base sequence.

The nucleic acid in the nucleic acid sample is not particularly limited as long as it is DNA or RNA, and may be natural or synthesized. Examples of natural nucleic acids include genomic DNA, mRNA, rRNA, hnRNA and the like recovered from living organisms. Moreover, as a synthesized nucleic acid, DNA synthesized by a known chemical synthesis method such as β-cyanoethyl phosphoramidite method, DNA solid phase synthesis method, a nucleic acid synthesized by a known nucleic acid synthesis method such as PCR, Examples include cDNA synthesized by reverse transcription reaction.
In the present specification, a nucleic acid having a target base sequence is referred to as a “target nucleic acid”.

The nucleic acid sample is not particularly limited as long as it is a sample containing nucleic acid, and is preferably a sample obtained by extracting nucleic acid from animals, plants, microorganisms, cultured cells and the like. Extraction of nucleic acids from animals and the like can be performed by a known method such as a phenol / chloroform method.
In addition, when the nucleic acid contained in a nucleic acid sample is a double stranded nucleic acid, it is preferable to make it a single stranded nucleic acid beforehand. By using a single-stranded nucleic acid, in the step (a) described below, the single-stranded nucleic acid can be hybridized with a detection probe and a partially competitive probe. Single-stranded extraction of the extracted double-stranded nucleic acid can be performed by a known method such as application of thermal energy.

The target base sequence is not particularly limited as long as it contains a base mutation site and the base sequence is known to the extent that it can be detected by a hybridization method.
The base mutation site contained in the target base sequence may be a polymorphic site of an inherited mutation such as a gene polymorphism, or may be a mutant site of an acquired mutation such as a somatic mutation.
Further, the target base sequence may have only one base mutation site or may have two or more base mutation sites.

In the present invention, a partial sequence containing a base mutation site in a target base sequence is referred to as a “base mutation-containing sequence”. When the target base sequence has two or more base mutation sites, the base mutation-containing sequence includes all base mutation sites of the target base sequence.
The length of the base mutation-containing sequence is not particularly limited, and can be appropriately determined in consideration of the type of the target base sequence, the base sequence of the detection type probe and the partial competition type probe, and the like.
In the present invention, the base mutation site may be located at any position in the base mutation-containing sequence. For example, it may be the 5 ′ end of the base mutation-containing sequence, the 3 ′ end of the base mutation-containing sequence, or the intermediate site of the base mutation-containing sequence.

In the present invention, the detection type probe and the partial competition type probe are ones in which one or more selected from the group consisting of nucleotides, nucleotide analogs, and modifications thereof are linked by a phosphodiester bond.
Here, the nucleotide analog is a non-natural nucleotide and has the same function as deoxyribonucleotide (DNA) or ribonucleotide (RNA), which are natural nucleotides. That is, nucleotide analogs can form chains by phosphodiester bonds in the same way as nucleotides, and primers and probes formed using nucleotide analogs are primers and probes formed using only nucleotides. Similarly, it can be used for PCR and hybridization. Examples of such nucleotide analogs include PNA (polyamide nucleotide derivatives), LNA (BNA), ENA (2′-O, 4′-C-Ethylene-bridged nucleic acids), and complexes thereof. Here, PNA is obtained by substituting a main chain composed of phosphoric acid and pentose of DNA or RNA with a polyamide chain. LNA (BNA) is a compound having two cyclic structures in which the oxygen atom at the 2 ′ site of the ribonucleoside and the carbon atom at the 4 ′ site are bonded via methylene.
Here, examples of modified products include modified deoxyribonucleotides, modified ribonucleotides, modified phosphate-sugar-backbone oligonucleotides, modified PNAs, modified LNA (BNA), and modified ENA. Here, the substance used for modification of nucleotides and nucleotide analogs is not particularly limited as long as the effects of the present invention are not impaired, and substances usually used for modification of nucleotides and the like can be used. As modified nucleotides and modified nucleotide analogs, for example, nucleotides modified with functional groups such as amino groups, carboxyvinyl groups, phosphate groups, methyl groups, etc., nucleotides modified with 2-O-methylation with methyl groups, phosphorothioates, etc. And nucleotides modified with a labeling molecule such as a fluorescent substance, which will be described later.

The detection probe is characterized in that the base sequence of the detection probe is complementary to a partial region containing a base mutation-containing sequence of the target base sequence.
The base sequence of the detection probe is divided into the following two partial sequences.
(I) A base sequence that is complementary to a base mutation-containing sequence in the target base sequence (hereinafter sometimes referred to as “(I) sequence”).
(II) A base sequence that is complementary to a partial region other than the base mutation-containing sequence in the target base sequence (hereinafter sometimes referred to as “(II) sequence”).

The partial competition type probe includes a partial region in which the base sequence of the partial competition type probe includes the base mutation-containing sequence of the target base sequence, the detection type probe in the target base sequence is different from the complementary part, and the base mutation It is complementary other than the site and non-complementary at the base mutation site.
The base sequence of the partially competitive probe is divided into the following two partial sequences.
(I ′) a base mutation-containing sequence in the target base sequence and a sequence that is complementary at other than the base mutation site and non-complementary at the base mutation site (hereinafter referred to as “(I ′) sequence”) is there.).
(II ′) A sequence that is complementary to a base sequence other than the base mutation-containing sequence in the target base sequence (hereinafter sometimes referred to as “(II ′) sequence”).

Furthermore, the detection probe and the partially competitive probe satisfy either one of the following (1) or (2).
(1) The detection probe is capable of hybridizing with a target nucleic acid on the base mutation-containing sequence and the 5 ′ end thereof, and the partial competition probe comprises a target nucleic acid, the base mutation-containing sequence and It must be able to hybridize at its 3 'end. At this time, the part consisting of (I) sequence and the part consisting of (I ′) sequence hybridize with the target nucleic acid in the base mutation-containing region, respectively, and the part consisting of (II) sequence consists of the target nucleic acid and the base mutation-containing sequence. In the portion consisting of the 5 ′ end, and the portion consisting of the sequence (II ′) hybridizes to the target nucleic acid in the portion consisting of the 3 ′ end of the base mutation-containing sequence.
(2) The detection probe is capable of hybridizing with a target nucleic acid at the base mutation-containing sequence and the 3 ′ end thereof, and the partial competition probe has a target nucleic acid, the base mutation-containing sequence and It must be able to hybridize at its 5 'end. At this time, the part consisting of (I) sequence and the part consisting of (I ′) sequence hybridize with the target nucleic acid in the base mutation-containing region, respectively, and the part consisting of (II) sequence consists of the target nucleic acid and the base mutation-containing sequence. In the portion consisting of the 3 ′ end, and the portion consisting of the sequence (II ′) hybridizes to the target nucleic acid in the portion consisting of the 5 ′ end of the base mutation-containing sequence.

FIG. 1 is a schematic diagram of aggregate formation in a state in which a detection probe and a partially competitive probe are added to a nucleic acid sample, and a target nucleic acid and both probes are present.
The target nucleic acid hybridizes with the part consisting of the (II) sequence of the detection probe and the part consisting of the (II ′) sequence of the partially competitive probe in a region other than the base mutation-containing sequence, Form. The target nucleic acid can hybridize not only with the portion consisting of the (I) sequence of the detection probe but also with the portion consisting of the (I ′) sequence of the partially competitive probe in the base mutation-containing sequence. Therefore, in the nucleic acid sample, the aggregate (B) in which the target nucleic acid and the detection probe are hybridized in the base mutation-containing sequence and the target nucleic acid and the partially competitive probe in the base mutation-containing sequence are hybridized. 1 and the dynamic equilibrium state shown in FIG.
In the present invention, the detection-type probe has a base sequence that is completely complementary to the target nucleic acid at the base mutation detection site in (I) sequence, and the partially competitive probe has the base mutation in (I ′) sequence. It does not have a base sequence that is completely complementary to the target nucleic acid at the detection site. Therefore, in the base mutation-containing sequence of the target nucleic acid, the association between the detection probe and the target nucleic acid has higher thermal stability than the association between the partially competitive probe and the target nucleic acid. Therefore, the aggregate (B) is formed more preferentially than the aggregate (C) from the state (A) where the three members exist alone. Similarly, the aggregate (C) once formed is preferentially transferred to the aggregate (B).
In the method for detecting a target base sequence of the present invention, a detection probe and a partially competitive probe are competitively hybridized to a target nucleic acid using a detection probe and a partially competitive probe. It is presumed that the detection accuracy of the target base sequence is improved by forming a dynamic equilibrium state as shown and preferentially forming an association in which the detection probe hybridizes to the base mutation site such as the association (B). Is done.

  In the present invention, since the thermal stability of the formed aggregate is relatively high, the detection probe is easy to hybridize to the target nucleic acid in the base mutation-containing sequence, and is stable and difficult to dissociate. , "Hybridize preferentially". The thermal stability of the aggregate is represented by a Tm value or the like and can be measured by a conventional method.

The length of the detection type probe and the length of the partial competition type probe can be hybridized between the target nucleic acid and the partial competition type probe under the reaction conditions for hybridizing the target nucleic acid and the detection type probe. As long as the three-party aggregate can be formed, there is no particular limitation.
The length of the portion consisting of the (II ′) sequence of the partially competitive probe is suitable for the target nucleic acid only in the portion consisting of the (II ′) sequence under the reaction conditions for hybridizing the target nucleic acid and the detection probe. It is preferable to be adjusted so that it can be hybridized.

  (I ′) The base sequence of the base mutation detection site in the sequence may be other than a base sequence that is completely complementary to the target base sequence. For example, when the base mutation site consists of two or more bases, at least 1 It is sufficient if the base is non-complementary.

  Since the method for detecting a target base sequence of the present invention has high accuracy in detecting a target base sequence having a base mutation as described above, it can be suitably used particularly for gene polymorphism typing. Here, when the target base sequence is one genotype of gene mutation (hereinafter sometimes referred to as “first type sequence”), the base sequence of the base mutation detection site in the sequence (I ′) is: It is preferably complementary to a base mutation site of a genotype different from the target base sequence (hereinafter sometimes referred to as “second type sequence”). (I ′) Since the base sequence of the base mutation detection site in the sequence is complementary to other genotypes, the genotype of the nucleic acid sample is the same as that of the detection type probe, or It is possible to determine whether they are the same, and the detection accuracy is further improved.

That is, the “second type sequence” is a sequence having a base sequence that differs from the target base sequence only at the base mutation site. For example, when the base mutation to be detected is a gene polymorphism having two types, a wild type and a mutant type, either the wild type or the mutant type is selected as the target base sequence (first type). Sequence) and the other base sequence is the second type sequence. On the other hand, in the case of a gene mutation having two types of normal type and mutant type as seen in cancer, etc., either the normal type or the mutant type base sequence is the target base sequence (first type sequence). ) And the other base sequence is the second type sequence.
In addition, when a base mutation consists of three or more types of genotypes, the sequences of two different genotypes may be the target base sequence (first type sequence) or second type sequence, respectively.

In the method for detecting a target base sequence of the present invention, (a) a detection type probe and a partial competition type probe are added to a nucleic acid sample, and the detection type probe is added to the target nucleic acid having the target base sequence in the nucleic acid sample. And a step of hybridizing the partially competitive probe, and (b) a step of detecting a detection probe hybridized with a portion comprising a base mutation-containing sequence in the target nucleic acid after the step (a), Have
Hereinafter, each step will be described.

  First, in the step (a), a detection type probe and a partial competition type probe are added to a nucleic acid sample, and the detection type probe and the partial competition type are added to a target nucleic acid having a target base sequence in the nucleic acid sample. Hybridize with the probe.

The reaction conditions for the hybridization of the target nucleic acid, the detection probe, and the partially competitive probe are not particularly limited, and the normal reaction conditions such as the Tm values of the detection probe and the partially competitive probe are considered. The reaction can be performed under conditions such as temperature, pH, salt concentration, and buffer solution.
The hybridization reaction is preferably performed in a reaction solution containing a salt having a buffering action. The pH of the reaction solution is preferably in the range of pH 6.5 to 8.5, more preferably in the range of pH 6.7 to 7.7, and the salt concentration of the reaction solution is 5 to 250 mM. The range is preferable, and the range of 10 to 100 mM is more preferable. Examples of the salt having a buffering action include cacodylate, phosphate, and tris salt. The reaction solution preferably contains an alkali metal and / or alkaline earth metal salt, and more preferably contains sodium chloride and / or magnesium chloride.

  Next, in the step (b), after the step (a), a detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid is detected.

The method for detecting a detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid (hereinafter sometimes referred to as “aggregate detection method”) is not particularly limited. This can be done using methods commonly used for detection.
The aggregate detection method of the present invention preferably uses a labeling substance. The labeling substance is not particularly limited as long as it does not interfere with the hybridization between the target nucleic acid, the detection probe and the partially competitive probe, and a labeling substance usually used for labeling nucleic acids can be used. it can. Examples of such a labeling substance include a dye.
The labeling substance of the present invention is preferably a dye, and more preferably a fluorescent dye.
The labeling of each labeling substance to the probe can be performed by using a method usually used for labeling an oligonucleotide, such as an organic synthesis method.

  Examples of suitable methods for detecting an aggregate using a fluorescent dye include detection using a QP (Quenching Probe / Primer) method and detection using a fluorescence resonance energy transfer (FRET) method. .

The QP method is a detection method that utilizes the fact that fluorescence is quenched when a guanine base is spatially close to a certain fluorescent dye.
By labeling the detection probe of the present invention with a fluorescent dye that quenches upon proximity to a guanine base, the presence or absence of proximity between the detection probe and the target nucleic acid or partially competitive probe can be detected. The guanine base may be a target nucleic acid or a partially competitive probe, and is preferably a target nucleic acid. At this time, it is preferable that the detection probe is designed to be quenched when the aggregate (B) in FIG. 1 is formed.

  As a fluorescent dye that quenches by the proximity to the guanine base, it can be performed using a fluorescent dye that is usually used in the QP method. Invitrogen), CR6G (trade name, manufactured by Invitrogen), TAMRA (trade name, manufactured by Invitrogen) and the like.

Preferable examples of the aggregate detection method using the QP method include the following.
In the target base sequence, a base mutation-containing sequence is set so as to have a guanine base inside, and the detection probe is a cytosine base labeled with a fluorescent dye at a site complementary to the guanine base of the target nucleic acid. Design in advance to have When hybridization is performed using a detection probe, a partially competitive probe, and a target nucleic acid, and the target nucleic acid and the detection probe are hybridized, the fluorescent dye of the labeled detection probe and the target nucleic acid Guanine bases come close to each other and quenching of the fluorescent dye occurs. On the other hand, when the target nucleic acid and the partially competitive probe are hybridized, the fluorescent dye and the guanine base are not close to each other, and the fluorescent dye is not quenched.
Thus, when detecting a detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid due to the proximity of the target nucleic acid and the detection probe, the hybridization between the target nucleic acid and the partially competitive probe is , Not necessarily required for detection.

The FRET method is a detection method that uses two different types of fluorescent dyes as a donor and an acceptor and quenches or emits fluorescence by fluorescence resonance energy transfer that occurs when the donor and the acceptor are spatially close to each other.
By labeling the detection probe of the present invention with either the donor or the acceptor and labeling the target nucleic acid or the partially competitive probe with the other, the detection probe and the target nucleic acid or the partially competitive probe The presence or absence can be detected. At this time, it is preferable that both probes are designed so that fluorescence resonance energy transfer occurs when the aggregate (B) of FIG. 1 is formed.

  As the fluorescent dye that can function as a donor or an acceptor, a fluorescent dye that is usually used in the FRET method can be used as appropriate. For example, as donors, Alexa Fluor (registered trademark) 488 (trade name, manufactured by Invitrogen), ATTO 488 (trade name, manufactured by ATTO-TEC GmbH), FAM (trade name, Carboxyfluoresceine), and Hylite Flour (registered trademark). 488 (trade name, manufactured by ASI) and the like, and acceptors include Alexa Fluor (registered trademark) 594 (trade name, manufactured by Invitrogen), ROX (trade name, Carboxy-X-rhodamine), BOBO-3 (product) Name, manufactured by Invitrogen), HEX (trade name, manufactured by Invitrogen), TAMRA (trade name, manufactured by Invitrogen), Hilite Floor (registered trademark) 555 (trade name, manufactured by ASI) and the like.

For example, in a target nucleic acid having a single base mutation, the base mutation-containing sequence of the target base sequence (first type sequence) is 5′-gtac-3 ′, and the base mutation-containing sequence of the second type sequence is 5′-gtgc. When it is −3 ′, the base mutation-containing sequence of the detection probe is 5′-gtac-3 ′. The base mutation-containing sequence of the partially competitive probe is non-complementary to the base sequence of the base mutation site of the target base sequence (first type sequence), and the length of the base mutation detection site is the length of the site of the detection probe. 5′-gcac-3 ′, 5′-gaac-3 ′, and 5′-ggac-3 ′, which are identical to each other, are preferably complementary to the base sequence of the base mutation site of the second type sequence. -3 'is more preferable.
For example, in a target nucleic acid having an insertion mutation, the mutant base mutation-containing sequence that is the target base sequence (first type sequence) is 5′-gtac-3 ′, and the second type sequence is a normal base mutation. When the sequence is 5′-gtc-3 ′, the base mutation-containing sequence of the detection probe is 5′-gtac-3 ′. The base mutation-containing sequence of the partially competitive probe is preferably 5′-gac-3 ′ that is complementary to the base sequence of the base mutation site of the second type sequence.
For example, in a target nucleic acid having a deletion mutation, a mutant base mutation-containing sequence that is a target base sequence (first type sequence) is 5′-gtc-3 ′, and a normal base mutation that is a second type sequence When the containing sequence is 5′-gtgc-3 ′, the base mutation-containing sequence of the detection probe is 5′-gac-3 ′. The base mutation-containing sequence of the partially competitive probe is preferably 5′-gcac-3 ′ that is complementary to the base sequence of the base mutation site of the second type sequence.
In addition, for example, in a target nucleic acid having a repetitive sequence mutation, the mutant base mutation-containing sequence that is the target base sequence (first type sequence) is 5′-atatat-3 ′, and the normal type that is the second type sequence. When the base mutation-containing sequence is 5′-atat-3 ′, the base mutation-containing sequence of the detection type probe is 5′-atat-3 ′. As the base mutation-containing sequence of the partially competitive probe, 5′-atat-3 ′ that is complementary to the base sequence of the base mutation site of the second type sequence is preferable.

FIG. 2 shows a target nucleic acid having a single base mutation (Target 1) having a target base sequence (first type sequence) or a nucleic acid having a second type sequence (Target 2), a detection probe (Probe 1), It is a schematic diagram of the aggregate | assembly which consists of a partial competition type | mold probe (Probe2).
In this figure, the detection probe (Probe 1) has a fluorescent substance as a labeling substance at one end thereof, and a detection method using the fluorescent substance is adopted. However, the detection method of the present invention is not limited to this method. It is not something that can be done. In FIG. 2, the site surrounded by ○ or □ is a base mutation site or a base mutation detection site. In the target nucleic acid (Target 1), the region where the base is described is a portion comprising a base mutation-containing sequence.

Here, the base sequence of the detection probe (Probe 1) is complementary to the target base sequence (first type sequence), and the base sequence of the partially competitive probe (Probe 2) is complementary to the second type sequence. . The target base sequence nucleic acid detection probe set comprising the detection probe (Probe 1) and the partial competition type probe (Probe 2) has a target base sequence (first type sequence) according to the complementarity of the base sequence prepared in advance. Align closely on a target nucleic acid (Target 1) having or a nucleic acid (Target 2) having a second type sequence.
As shown in the upper diagram of FIG. 2, when the target nucleic acid has a target base sequence (first type sequence), bases complementary to the base mutation-containing sequence in the target nucleic acid in the probe sets arranged in close proximity to each other A detection probe (Probe 1) having a sequence hybridizes preferentially. A detection probe (Probe 1) hybridized with a target nucleic acid (Target 1) having a target base sequence (first type sequence) is detected by a fluorescent substance possessed by the detection probe.
In addition, as shown in the lower diagram of FIG. 2, when the nucleic acid has the second type sequence, the partially competitive type having a base sequence complementary to the base mutation-containing sequence in the nucleic acid among the probe sets arranged in close proximity. The probe (Probe 2) hybridizes preferentially. Thereby, it is possible to prevent the detection probe (Probe 1) from being mishybridized with the nucleic acid (Target 2) having the second type sequence and being erroneously detected.
Here, “mishybridization” means that when two nucleic acids are hybridized to form an aggregate, the aggregate contains a non-complementary base pair.

  When quenching or luminescence of a fluorescent dye or the like labeled on the detection probe (Probe 1) is detected, a target base sequence (the first base sequence complementary to the base sequence of the detection probe (Probe 1) is included in the target nucleic acid. It can be seen that there is a target nucleic acid (Target 1) having a (type 1 sequence). If quenching or luminescence is not detected, the target nucleic acid may not have a target base sequence (first type sequence) complementary to the base sequence of the detection probe (Probe 1). Recognize.

  In the present invention, as shown in FIG. 2, the detection-type probe and the partial competition-type probe are allowed to compete with each other in the base mutation-containing sequence in the target nucleic acid, thereby reducing detection noise due to mishybridization and increasing detection efficiency. High accuracy and high sensitivity are possible. And this invention can suppress mishybridization effectively only by adding a partial competition type probe to a reaction solution, and is simple and efficient.

FIGS. 3 to 5 show a target nucleic acid having a target base sequence (Target 1) or a nucleic acid having a second type sequence (Target 2) and a detection probe in a target nucleic acid having an insertion mutation, a deletion mutation, and a repetitive sequence mutation, respectively. It is a schematic diagram of the aggregate | assembly which consists of (Probe1) and a partial competition type | mold probe (Probe2). The explanation in the figure is the same as in FIG.
Further, as shown in FIG. 3, in the case of a target base sequence having an insertion mutation, the base mutation site of the target base sequence is a base inserted into the base mutation site of the second type sequence. As shown, in the case of a target base sequence having a deletion mutation, the base mutation site of the target base sequence is one in which the base is deleted from the base mutation site of the second type sequence.

A preferred example of the method for detecting an aggregate using the FRET method is shown in FIG.
FIG. 6 shows a target nucleic acid having a single base mutation (Target 1) having a target base sequence (Target 1) or a nucleic acid having a second type sequence (Target 2), a detection probe (Probe 1), a partially competitive probe (Probe 2). ) Is a schematic diagram in the case of detecting an aggregate consisting of In FIG. 6, a site surrounded by ○ or □ is a base mutation site or a base mutation detection site. In the target nucleic acid (Target 1), the region where the base is described is a portion comprising a base mutation-containing sequence.
The detection type probe (Probe 1) and the partial competition type probe (Probe 2) are designed in advance so as to be close to each other when the portion consisting of a base mutation-containing sequence in the target nucleic acid and the detection type probe (Probe 1) are hybridized. . When hybridization is performed using these probes and nucleic acids, as shown in the upper diagram of FIG. 6, when the target nucleic acid (Target 1) and the detection probe (Probe 1) are hybridized, these fluorescent dyes In proximity, quenching of the donor fluorescent dye and emission of the acceptor fluorescent dye occur. On the other hand, as shown in the lower diagram of FIG. 6, when a nucleic acid having a second type sequence (Target 2) and a partially competitive probe (Probe 2) are hybridized, these fluorescent dyes are not close to each other, and the fluorescence of the donor Dye quenching and acceptor fluorescent dye emission do not occur.
Thus, when detecting a detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid due to the proximity of the detection probe and the partial competition type probe, the target nucleic acid, the detection type probe, and the partial competition type are detected. The detection type probe hybridized with the portion consisting of the base mutation-containing sequence in the target nucleic acid in the aggregate hybridized with the probe is detected. Therefore, in this case, the target nucleic acid needs to hybridize not only with the detection type probe but also with the partial competition type probe to form an aggregate.

In the present invention, when a detection probe hybridized with a portion consisting of a base mutation-containing sequence in a target nucleic acid is detected using a fluorescent dye such as the QP method, a dye for correcting fluorescence intensity is added to the reaction solution And preferably used.
The correction dye is not particularly limited as long as the detected wavelength does not cross the wavelength of the fluorescent dye used for the detection of the aggregate, and a commonly used fluorescent dye should be used. Can do. For example, Alexa Fluor (registered trademark) 488 (trade name, manufactured by Invitrogen), ATTO 488 (trade name, manufactured by ATTO-TEC GmbH), Alexa Fluor (registered trademark) 594 (trade name, manufactured by Invitrogen), HEX ( Trade name, manufactured by Invitrogen), TAMRA (trade name, manufactured by Invitrogen), and the like.

  When determining whether or not the target base sequence has been detected by using the above-described aggregate detection method, that is, whether or not the target nucleic acid is contained in the nucleic acid sample, the amount of the detected probe is measured. Based on this, it is possible to detect the target base sequence in the nucleic acid sample semi-quantitatively as well as the presence or absence of the target nucleic acid.

In the present invention, it is preferable to use a calibration curve in order to improve the detection accuracy when determining the target base sequence using the result of the method for detecting an aggregate. A calibration curve can be prepared by using a nucleic acid whose base sequence and concentration at the base mutation site are known instead of the nucleic acid sample. Also, the target base sequence can be detected by setting an appropriate threshold value instead of the calibration curve.
In addition, when detecting an aggregate using a fluorescent label such as the QP method, a change in fluorescence intensity due to a temperature change is measured and compared with a nucleic acid (control) with a known base sequence. Can be detected.

  As described above, the present invention can detect a target base sequence semi-quantitatively by using an appropriate threshold or a calibration curve. Therefore, by using a nucleic acid collected from an organism such as a human, the gene of the organism can be used. Can be discriminated whether it is a homozygote or a heterozygote of the genotype of the target base sequence.

  In addition, when the present invention is applied to a DNA chip, it has the advantage that the degree of freedom of selection of hybridization conditions such as temperature is large and management is easy compared to a detection method by normal hybridization. In addition, the DNA chip can be used for a wider range of applications. For example, by arranging a large number of specimens on a substrate and hybridizing a detection probe and a partially competitive probe to the board, target base sequences in a large number of specimens can be easily detected with a single operation. can do. In addition, it is possible to detect a large number of target base sequences in a sample by placing a mixture of a large number of detection probes and partially competitive probes on a substrate and hybridizing the sample to the substrate. it can.

  In addition, a probe used in the above-described method for detecting a target base sequence of the present invention can be made into a kit. For example, a detection probe and a partial competition probe used for detecting a specific base mutation can be used as one kit. The kit may contain other detection reagents. By using such a kit, base mutations can be identified quickly and easily.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

[Example 1: Detection of mutant type]
The oligonucleotides used in this example were those purchased from Japan Bioservice or Operon Biotechnology.
Table 1 shows target oligonucleotides Target1 (wT49; wild type) and Target2 (mT49; mutant) having a single nucleotide mutation, Probe3 (wL27) as a partial competitive probe, and Probe4 (a detection probe). mR27) was used to detect the mutant form of the target gene sequence.
The detection probe Probe4 is a probe complementary to Target2 (mutant), and the cytosine base at the 5 ′ end of the probe was labeled with BODIPY FL (trade name, manufactured by Invitrogen). The partially competitive probe Probe3 is a probe complementary to Target1 (wild type).
First, a mutant homozygous nucleic acid sample (mT) consisting only of Target2, a mutant / wild-type heterozygous nucleic acid sample (wT + mT) obtained by mixing Target2 and Target1 at a ratio of 1: 1, and only Target1 A wild type homozygous nucleic acid sample (wT) was prepared. Next, the detection type probe and the partially competitive type probe (final concentration of 1 pmol / μL each) are added to any one of the above nucleic acid samples (the homozygote has a final concentration of 2 pmol / μL, and the heterozygote has a final concentration of 1 pmol / μL each). 4 kinds of deoxynucleotide triphosphates (final concentration of 250 μM each), fluorescent dye for correcting fluorescence intensity Alexa Fluor (registered trademark) 594 (trade name, manufactured by Invitrogen, final concentration 1 pmol / μL), and reaction buffer 10 × PCR buffer (100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl ₂ ) and 2.5 mM dNTP Mixture were added, and the volume of the reaction solution was adjusted to 40 μL and mixed with pure water. Moreover, what added the pure water instead of the sample was prepared as negative control.
Next, the sample was loaded into a detection container for real-time PCR, and detection was performed using the QP method. As described above, when the QP method is hybridized, the fluorescence is quenched. Specifically, the sample was heated at 35 to 95 ° C., and the fluorescence intensity against the temperature change was measured using F1 (for BODIPY detection, 520 nm) and F2 (for correction fluorescent dye detection, 640 nm) filters. . FIG. 7 shows the result of correcting the fluorescence intensity (F1) of the detection probe with the fluorescence intensity (F2) of the correcting fluorescent dye.

[Example 2: Detection of wild type]
In the same manner as in Example 1 except that Probe 1 (mL27) shown in Table 1 was used as a partially competitive probe and Probe 2 (wR27) shown in Table 1 was used as a detection probe, wild type detection of the target gene sequence was performed. went. The detection probe Probe2 is a probe complementary to Target1 (wild type), and the cytosine base at the 5 ′ end of the probe was labeled with BODIPY FL (trade name, manufactured by Invitrogen). The partially competitive probe Probe1 is a probe complementary to Target2 (mutant type).
FIG. 8 shows the result of correcting the fluorescence intensity (F1) of the detection probe with the fluorescence intensity (F2) of the correcting fluorescent dye.

From the results of FIGS. 7 and 8, when the base type of the detection probe and the nucleic acid sample match, the detection probe and the nucleic acid of the nucleic acid sample are preferentially bound to each other, so that the fluorescence intensity decreases. In addition, the dissociation temperature between the detection probe and the sample increased, but when they did not match, the fluorescence intensity increased and the dissociation temperature decreased. In addition, in the case where only half of the base variants of the detection probe and the nucleic acid of the nucleic acid sample match, the intermediate fluorescence intensity and dissociation temperature between the case where they match and the case where they do not match are shown.
From the above results, it is shown that the target base sequence of a nucleic acid having a plurality of types of base mutations can be detected using the target base sequence detection method of the present invention, which is useful for genotyping. It was.
In addition, the above results were found to be highly accurate and sensitive as compared with the conventional method for detecting a target base sequence by a hybridization method.

The target base sequence detection method of the present invention can be suitably used in fields such as drug discovery, genetic testing, and medical diagnosis.

Claims (6)

A method for detecting a target base sequence having a base mutation site,
A partial sequence containing a base mutation site in the target base sequence is a base mutation-containing sequence,
(A) adding a detection probe and a partially competitive probe to a nucleic acid sample;
Hybridizing the detection probe and the partially competitive probe to a target nucleic acid having a target base sequence in the nucleic acid sample;
(B) after the step (a), detecting a detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid,
The base sequence of the detection probe is complementary to a partial region containing a base mutation-containing sequence of the target base sequence,
The base sequence of the partial competitive probe is complementary to a partial region different from the partial region including the base mutation-containing sequence of the target base sequence, and non-complementary at the base mutation site. And
Furthermore, either of following (1) or (2) is satisfied, The detection method of a target base sequence characterized by the above-mentioned.
(1) The detection probe can hybridize with the target nucleic acid on the base mutation-containing sequence and the 5 ′ end side thereof, but cannot hybridize on the 3 ′ end side of the base mutation-containing sequence. Yes, the partially competitive probe is capable of hybridizing with the target nucleic acid at the base mutation-containing sequence and its 3 ′ end, but not at the 5 ′ end of the base mutation-containing sequence. There is.
(2) The detection probe is capable of hybridizing with the target nucleic acid on the base mutation-containing sequence and the 3 ′ end side thereof, but cannot hybridize on the 5 ′ end side of the base mutation-containing sequence. Yes, the partially competitive probe can hybridize with the target nucleic acid on the base mutation-containing sequence and the 5 ′ end side thereof, but cannot hybridize on the 3 ′ end side of the base mutation-containing sequence. There is.   The method for detecting a target base sequence according to claim 1, wherein the base mutation site may be located at any position in the base mutation-containing sequence.   3. The target base sequence according to claim 1, wherein in the step (b), a detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid is detected using a labeling substance. Detection method.   The method for detecting a target base sequence according to claim 3, wherein the labeling substance is a fluorescent dye.   In the step (b), a detection probe hybridized with a portion consisting of a base mutation-containing sequence in the target nucleic acid in an assembly in which the target nucleic acid, the detection probe, and the partially competitive probe are hybridized is detected. The method for detecting a target base sequence according to any one of claims 1 to 4, wherein:   In the step (b), a detection probe hybridized with a portion consisting of a base mutation-containing sequence in a target nucleic acid is detected using a QP (Quenching Probe / Primer) method or a FRET method. The method for detecting a target base sequence according to claim 4 or 5.

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