WO2018079579A1 - 標的塩基配列を検出する方法、プローブを設計および製造する方法ならびにキット - Google Patents
標的塩基配列を検出する方法、プローブを設計および製造する方法ならびにキット Download PDFInfo
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- WO2018079579A1 WO2018079579A1 PCT/JP2017/038458 JP2017038458W WO2018079579A1 WO 2018079579 A1 WO2018079579 A1 WO 2018079579A1 JP 2017038458 W JP2017038458 W JP 2017038458W WO 2018079579 A1 WO2018079579 A1 WO 2018079579A1
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Definitions
- the present invention relates to a method for detecting a target nucleotide sequence, and more specifically, a method for detecting a target nucleotide sequence containing a single nucleotide mutation such as a single nucleotide polymorphism (SNP), for use in the method.
- the present invention relates to a probe designing method and manufacturing method, and a kit including the probe.
- SNP is a mutation in which one nucleotide in a genome sequence is replaced with a different nucleotide among individuals, and the mutation exists at a frequency exceeding 1% of the whole population of individuals.
- Some SNPs cause individual differences in various phenotypes such as constitution, disease sensitivity, and drug responsiveness. For example, human alcohol resolution is known to depend on SNPs in the ALDH2 gene, and SNPs in the CYP family related to drug metabolism have also been found to contribute to the effects of drugs on individuals. In recent years, specific SNPs have been reported as biomarkers for predicting the postoperative course and therapeutic effect of cancer, and the usefulness of discriminating SNPs is high.
- the technique for discriminating a single base used for discriminating SNPs is not limited to SNP, but can be effectively used for discriminating base sequences including mutations (point mutations) by single base substitution.
- the present invention can be applied to detection of drug-resistant pathogens having a presence frequency of less than 1%. Therefore, it is expected that the demand for a method that can easily identify a single base difference between base sequences will increase.
- a probe detection method is known as a simple method for discriminating SNPs.
- the Tm value of a perfectly matched duplex obtained by hybridizing a detection probe having a base sequence complementary to the base sequence to be detected is different from that of the base sequence to be detected.
- This is a method for discriminating the base sequence based on the difference in Tm value by utilizing the fact that it is about 5 ° C. higher than the Tm value of the single-base mismatch duplex to which the detection probe is hybridized.
- the Tm value fluctuates depending on conditions such as the base composition of the base sequence to be detected and the salt concentration of the reaction sample, and such conditions may become noise and it may be difficult to obtain a stable measurement result. Therefore, a device for reducing such noise has been devised.
- a part of the “detection probe” is hybridized to one of the 3 ′ end side and the 5 ′ end side of the region containing the single nucleotide mutation site of the target nucleic acid, and the other ,
- a partial region of a “partial competitive probe” having a partially matching base sequence other than a single-base mutation site other than a single-base mutation site is hybridized, and the detection probe and a part of the region including the single-base mutation site
- a method in which the remaining regions of the competitive probe compete with each other, that is, both the detection probe and the partially competitive probe can hybridize to the corresponding regions of the target nucleic acid, but both these probes are single nucleotide mutation sites.
- a technique of competing with each other in a region including the ability to discriminate between mutations in the competitive region “region containing a single nucleotide mutation site” can be improved and the stability of hybridization of a single base mismatch can be lowered. The accuracy of discrimination based on the difference can be increased.
- the Tm value is measured by, for example, QP (Quenching / Probe / Primer) method or the like for hybridization or dissociation of a fluorescent dye labeled with a detection probe between a nucleic acid and a detection probe. Methods are used to detect the accompanying quenching or luminescence.
- the fluorescence intensity is measured while changing the temperature of the reaction sample in which the detection probe is added to the nucleic acid sample, and the peak (maximum value) of the curve obtained by first-order differentiation of the temperature-fluorescence intensity curve obtained from the measurement result ( (Corresponding to the temperature at which quenching or light emission occurs) is defined as the Tm value.
- the method of discriminating the SNP based on the difference in the Tm value it is necessary to determine whether the measured Tm value is on the high temperature side or the low temperature side. There is a possibility of bringing.
- the nucleic acid targeted for SNP discrimination is a heterozygote of two SNP alleles
- quenching or luminescence is performed at a temperature between the Tm value on the high temperature side and the Tm value on the low temperature side.
- a peak may be seen.
- the difference between the Tm value of the measurement result and the Tm value on the high temperature side or the low temperature side becomes as small as, for example, about 2 ° C., and is difficult to discriminate.
- the present invention provides a method for detecting a target base sequence, a kit used in the method, and a method for making it possible to easily determine whether or not a target base sequence is present in a nucleic acid sample. It is an object to provide a method for designing and manufacturing the probe used.
- the present inventors have continued intensive research and use a competitive probe that specifically hybridizes to a base sequence of a single base mismatch and inhibits the hybridization of a single base mismatch of a detection probe.
- a detection system capable of detecting only the target base sequence can be realized without causing quenching or light emission at the Tm value on the low temperature side.
- the present inventors first-order differentiated the temperature-fluorescence intensity curve obtained for the reaction sample for hybridization between the detection probe and the nucleic acid, and the peak of the first-order derivative curve is usually accompanied by quenching or luminescence at the Tm temperature.
- the peak of the detection probe can be prevented from appearing by inhibiting hybridization of a single base mismatch of the detection probe, and can clearly be distinguished from the case where the peak due to detection of the target base sequence appears. It came.
- a method for detecting a target base sequence (A) containing a base-mutated nucleotide from a nucleic acid sample comprising the following steps: (1) A detection sample labeled with a fluorescent dye that can be used in a QP (Quenching Probe / Primer) method and a competitive probe are added to a nucleic acid sample to obtain a reaction sample, whereby a target in the reaction sample is obtained.
- QP Quenching Probe / Primer
- a fluorescently labeled detection probe or a competitive probe hybridizes to a target nucleic acid having the base sequence (A); (2) measuring the fluorescence intensity while changing the temperature of the reaction sample; and (3) first derivative of the temperature-fluorescence intensity curve obtained from the measurement result of (2), wherein: (I) The base sequence of the fluorescently labeled detection probe includes a base sequence (A ′) complementary to the target base sequence (A), (Ii) The base sequence of the competitive probe is a base sequence complementary to the non-target base sequence (B) which is the same base sequence as the target base sequence (A) except that the base mutant nucleotide is replaced with an unmutated base nucleotide ( B ') (Iii)
- the base length and base sequence of each of the fluorescently labeled detection probe and the competitive probe and the amount added to the nucleic acid sample are as follows: (A) a control target nucleic acid sample containing a target nucleic acid but substantially free of a non-target nucleic acid having a
- the first derivative curve obtained in (3) comprises determining that the target base sequence (A) is present in the nucleic acid sample, wherein the nucleic acid sample is outside the target nucleic acid,
- the method according to [1] which may also contain a non-target nucleic acid.
- the Tm value of the competitive probe is at least 5 ° C. higher than the Tm value of the fluorescently labeled detection probe for the non-target nucleic acid, and the Tm value of the competitive probe is the Tm value of the fluorescently labeled detection probe for the target nucleic acid.
- the method according to [1] or [2] which is lower.
- the Tm value of the competitive probe is at least 10 ° C. higher than the Tm value of the fluorescently labeled detection probe for the non-target nucleic acid, and the Tm value of the competitive probe is the Tm value of the fluorescently labeled detection probe for the target nucleic acid.
- the Tm value of the competitive probe is at least 10 ° C. higher than the Tm value of the fluorescently labeled detection probe with respect to the non-target nucleic acid, and the Tm value of the competitive probe is the Tm value of the fluorescently labeled detection probe with respect to the target nucleic acid.
- the Tm value of the competitive probe is at least 15 ° C. higher than the Tm value of the fluorescently labeled detection probe for the non-target nucleic acid, and the Tm value of the competitive probe is the Tm value of the fluorescently labeled detection probe for the target nucleic acid.
- the Tm value of the competitive probe is at least 15 ° C. higher than the Tm value of the fluorescently labeled detection probe for the non-target nucleic acid, and the Tm value of the competitive probe is the Tm value of the fluorescently labeled detection probe for the target nucleic acid
- the region on the target nucleic acid that hybridizes with the fluorescently labeled detection probe includes a region that hybridizes with the competitive probe;
- the region on the target nucleic acid that hybridizes with the competitive probe A region that hybridizes with a fluorescently labeled detection probe; or
- a region that hybridizes with a competitive probe on the target nucleic acid matches a region that hybridizes with a fluorescently labeled detection probe, [1] to [ [7] The method according to any one of [7].
- the fluorescent dye usable in the QP (Quenching Probe / Primer) method is at least selected from the group consisting of TAMRA (trademark), BODIPY (trademark) FL, PACIFIC BLUE (trademark) and CR6G (trademark) The method according to any one of [1] to [10], which is one kind of fluorescent dye.
- [12] The method according to any one of [1] to [11], wherein the nucleic acid sample is derived from a living body.
- [13] The method according to any one of [1] to [12], wherein the base-mutated nucleotide is DNA containing a single-base substitution mutation.
- a method for producing a fluorescently labeled detection probe and a competitive probe that can be used in the method according to any one of [1] to [13], the method comprising: (1 ′′) synthesizing oligonucleotides of respective base lengths and sequences designed according to the method described in [14]; and (2 ′′) oligonucleotides of fluorescently labeled detection probes can be used in the QP method. Labeling with a fluorescent dye.
- a specific competitive probe is hybridized to the non-target base sequence, and the single-base mismatch of the fluorescence-labeled detection probe is detected.
- a peak of quenching or luminescence can appear in the measurement result of temperature-fluorescence intensity only when the target base sequence is detected. Therefore, unlike the discrimination method using the difference in Tm value, the presence or absence of the target base sequence is known by the presence or absence of quenching or luminescence peak, and therefore it is affected by conditions such as salt concentration that affect the Tm value. (High robustness), it can be easily determined whether or not the target base sequence is present in the nucleic acid sample.
- FIG. 1 shows detection of a wild type (rpoBsp516Asp) in a nucleic acid sample containing a point mutation at the 516th codon of the rpoB gene (wild type, mutant type, and wild type / mutant hetero) in Example 1. Shows a first derivative curve (thermal dissociation curve) of a temperature-fluorescence intensity curve measured by adding a detection probe and a competitive probe.
- FIG. 2 shows the base sequences of the target nucleic acid and non-target nucleic acid amplified from the rpoB gene, as well as fluorescently labeled detection probes designed for the detection of the target nucleic acid and various competitive probes in Example 2.
- FIG. 1 shows detection of a wild type (rpoBsp516Asp) in a nucleic acid sample containing a point mutation at the 516th codon of the rpoB gene (wild type, mutant type, and wild type / mutant hetero) in Example 1. Shows a first derivative curve
- FIG. 3-1 shows temperature-fluorescence intensity curves obtained for each reaction sample in Example 2.
- FIG. 3-2 shows a curve obtained by first-order differentiation of the temperature-fluorescence intensity curve obtained in Example 2.
- FIG. 4-1 shows the base sequences of the target nucleic acid and non-target nucleic acid amplified from the rpoB gene in Example 3.
- FIG. 4-2 shows the nucleotide sequences of fluorescently labeled detection probes designed for detecting target nucleic acids and various competitive probes in Example 3, and Tm values at which each probe hybridizes with each of target and non-target nucleic acids.
- FIG. 4-3 shows the Tm values of Example 3 for fluorescently labeled detection probes designed for detection of target nucleic acids and various competitive probes that hybridize with target nucleic acids and non-target nucleic acids, respectively.
- FIG. 5 shows a curve obtained by first-order differentiation of the temperature-fluorescence intensity curve obtained in Example 3.
- SNP is used synonymously with SNPs in the technical field.
- SNP is an example of a detection target, and the present invention can be widely applied to techniques for detecting DNA containing a single base substitution mutation.
- scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.
- the detection method according to the present invention is a method for detecting a target base sequence (A) containing a base-mutated nucleotide from a nucleic acid sample, comprising at least the following steps (1) to (3).
- a fluorescently labeled detection probe and a competitive probe are added to a nucleic acid sample to obtain a reaction sample, whereby a fluorescently labeled detection probe or a competitive probe is added to a target nucleic acid having a target base sequence in the reaction sample.
- Hybridize (2) Measuring the fluorescence intensity while changing the temperature of the reaction sample; and (3) First-order differentiation of the temperature-fluorescence intensity curve obtained from the measurement result of (2).
- detecting a target base sequence means determining whether or not the base sequence of a nucleic acid contained in a nucleic acid sample contains the same base sequence as the target base sequence.
- the nucleic acid is not particularly limited as long as it is DNA or RNA, and it may be natural or synthesized. Examples of natural nucleic acids include genomic DNA, mRNA, rRNA, heteronuclear (hn) RNA recovered from living organisms.
- the synthesized nucleic acid is, for example, DNA synthesized by a known chemical synthesis method such as ⁇ -cyanoethyl phosphoramidite method or DNA solid phase synthesis method, or synthesized by a known nucleic acid synthesis method such as PCR. Examples include nucleic acids and cDNA synthesized by reverse transcription.
- a nucleic acid having a base sequence including a target base sequence is referred to as a “target nucleic acid”.
- 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 or the like can be performed by a known method such as a phenol / chloroform method.
- the nucleic acid sample may be derived from a living body.
- the phrase “nucleic acid sample is derived from a living body” is not limited to the meaning that the nucleic acid sample is a sample itself collected from a living body, as long as it starts from a sample collected from a living body.
- nucleic acid samples derived from living organisms By applying the method of the present invention to a nucleic acid sample derived from a living body, it can be used for genetic diagnosis of the living body.
- 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.
- a detection probe or a competitive probe can be hybridized to the single-stranded nucleic acid in step (1) described later.
- Single-stranded extraction of the extracted double-stranded nucleic acid can be performed by a known method such as application of thermal energy.
- “Hybridization” means that single-stranded nucleic acids bind to each other by forming complementary base pairs (for example, the DNA to be detected and the probe to be single-stranded by heat treatment) and forming a complementary base pair. .
- hybridize includes, for example, a case in which a base sequence that is completely complementary to one base sequence hybridizes, and between one base sequence and the other base sequence, for example, 1 -Even if there are some base pairs and parts that cannot form complementary base pairs (mismatched parts), the entire sequence may be hybridized by forming base pairs between complementary parts. .
- the case where a completely complementary base sequence hybridizes to one base sequence is expressed as “perfectly hybridized” or “specifically hybridizes” and includes a mismatch. It may be expressed as “mismatch hybridization” or “non-specific hybridization”.
- examples of the base difference site of “base mutation nucleotide” and “base non-mutation nucleotide” include SNP, insertion mutation, deletion mutation, and mutation site due to repetitive sequence mutation.
- a difference site is a mutation site in the SNP
- the “base-mutated nucleotide” and “base-unmutated nucleotide” are each one nucleotide.
- base-mutated nucleotides two consecutive bases sandwiching the deletion site
- three consecutive bases not including the deletion are referred to as “base-unmutated nucleotides”. It is also possible to apply the present invention.
- base mutation nucleotides a total of three bases of the inserted nucleotide and the adjacent nucleotides on both sides thereof are referred to as “base mutation nucleotides”, and two consecutive bases that do not include insertions are referred to as “bases”.
- base mutation nucleotides two consecutive bases that do not include insertions
- bases two consecutive bases that do not include insertions
- unmutated nucleotide The number of deleted bases or inserted bases may be 2 or more. That is, in the claims attached to the present application, the description that “a base-mutated nucleotide is replaced by a base-unmutated nucleotide” means that “base-mutated nucleotide” and “base-unmutated nucleotide” are several consecutive nucleotides. It includes some cases.
- the relationship between the “base-mutated nucleotide” and the “base-unmutated nucleotide” is, for example, the relative relationship of different bases between the wild type and the mutant type in an allele such as SNP. It can be said that there is. Therefore, typically, a “target base sequence including a base-mutated nucleotide” is a mutant base sequence, and a “non-target base sequence” including a “base unmutated nucleotide” is a wild-type base sequence.
- the "target base sequence including the base-mutated nucleotide” is regarded as the wild-type base sequence, and at the same time, the "non-target base sequence” including the “base unmutated nucleotide” is replaced with the mutant-type base sequence. It may be regarded as a base sequence.
- the latter example will be described in Examples 1 and 2 described later. That is, the term “mutation” and “unmutated” in the term “base-mutated nucleotide” in the present specification does not necessarily mean whether or not the nucleotide is a mutant type, and is a detection target of the present method. It means whether or not the SNP is a different base with reference to the base at the SNP site in the non-target base sequence (a site where mutation due to single base substitution for detection may occur).
- target base sequence refers to a partial sequence of a certain length containing a base-mutated nucleotide in a base sequence of a nucleic acid (target nucleic acid) containing the target base-mutated nucleotide.
- the length of the target base sequence is not particularly limited as long as a probe of the same length having a base sequence complementary to the partial sequence can specifically hybridize under stringent conditions. .
- the length of the target base sequence can be appropriately determined in consideration of the type of the target base sequence, the base sequences of detection probes and competitive probes described later, and the like.
- the “non-target base sequence” refers to the same base sequence as the target base sequence except that the base-mutated nucleotide is replaced with a base-unmutated nucleotide. Since such a non-target base sequence is similar to the target base sequence to be detected even though it is not a detection target, it can hybridize non-specifically with a detection probe having a base sequence complementary to the target base sequence. . Therefore, in the conventional probe method, in addition to the target base sequence, a non-target base sequence may be detected by hybridization with a detection probe. If the detected base sequence is a non-target base sequence, hybridization is performed.
- non-target nucleic acid a nucleic acid having a base sequence including this non-target base sequence is referred to as “non-target nucleic acid”.
- the “detection probe” and the “competitive probe” are those in which one or more selected from the group consisting of nucleotides, nucleotide analogs, and modifications thereof are linked by a phosphodiester bond.
- 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.
- nucleotide analogs include PNA (polyamide nucleotide derivatives), LNA (BNA), ENA (2′-O, 4′-C-Ethylene-bridged nucleic acids), and complexes thereof.
- PNA polyamide nucleotide derivatives
- LNA BNA
- ENA 2′-O, 4′-C-Ethylene-bridged nucleic acids
- complexes thereof 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.
- examples of the modified form include modified deoxyribonucleotide, modified ribonucleotide, modified phosphate-sugar-backbone oligonucleotide, modified PNA, modified LNA (BNA), and modified ENA.
- 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.
- nucleotides and modified nucleotide analogs for example, nucleotides modified with functional groups such as amino groups, carboxyvinyl groups, phosphate groups, methyl groups, nucleotides modified with 2-O-methylation with methyl groups, phosphorothioates, etc. And nucleotides modified with a labeling molecule such as a fluorescent dye described later.
- the detection probe is labeled with a fluorescent dye that can be used in the QP (Quenching Probe / Primer) 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.
- the target base sequence is set so as to have a guanine base inside, and the detection probe is labeled with a fluorescent dye at a site complementary to the guanine base of the target nucleic acid.
- fluorescent dyes examples include TAMRA (trademark) (manufactured by Invitrogen), BODIPY (registered trademark) FL (manufactured by Invitrogen), PACFIC BLUE (trademark) (manufactured by Invitrogen), and CR6G (registered trademark). ) (Manufactured by Invitrogen) and the like.
- TAMRA trademark
- BODIPY registered trademark
- FL manufactured by Invitrogen
- PACFIC BLUE trademark
- CR6G registered trademark
- Each fluorescent dye can be labeled on the detection probe by a commonly used method such as an organic synthesis method.
- the detection probe is also referred to as a “fluorescent label detection probe”.
- the base sequence of the fluorescently labeled detection probe includes a base sequence (A ′) complementary to the target base sequence (A).
- the base sequence of the competitive probe is the base sequence (B ′) complementary to the non-target base sequence (that is, the same base sequence as the target base sequence except that the base-mutated nucleotide is replaced with the base-unmutated nucleotide) (B).
- the “probe base sequence” refers to the full-length base sequence of the region that hybridizes to the nucleic acid of the probe.
- “complementary” in the term “base sequence (A ′) complementary to target base sequence (A)” means that base sequences (A) and (A ′) are completely complementary.
- base sequence (B ′) complementary to the non-target base sequence (B) has a completely complementary relationship between the base sequences (B) and (B ′).
- the base sequence of the fluorescently labeled detection probe includes “a base sequence complementary to the target base sequence” when the base sequence (A ′) of the fluorescently labeled detection probe is complementary to the target base sequence (A).
- the base sequence of the competitive probe includes “a base sequence complementary to the non-target base sequence” means that the base sequence (B ′) of the competitive probe is complementary to the non-target base sequence (B); This includes the case where the base sequence (B ′) of the competitive probe is complementary to the base sequence of the region including the non-target base sequence (B) on the non-target nucleic acid and longer than the non-target base sequence (B).
- step (1) a fluorescent labeled detection probe or a competitive probe is hybridized to a target nucleic acid having a target base sequence in a reaction sample.
- the base length and base sequence of each of the fluorescently labeled detection probe and the competitive probe and the amount added to the nucleic acid sample are determined so as to satisfy predetermined conditions, which will be described later.
- the reaction conditions for hybridization between the target nucleic acid, the fluorescently labeled detection probe, and the competitive probe are not particularly limited, and the normal temperature, The reaction can be performed under conditions such as pH, salt concentration, and buffer solution.
- the Tm value is the temperature at which 50% of the oligonucleotide dissociates from its complementary strand, and is an indicator of the thermal stability of the duplex when the probe is hybridized to a nucleic acid.
- a method for calculating the Tm value a conventional method can be used. For example, in the case of a probe having 17 to 25 bases, it can be estimated by the following formula (Wallace formula).
- Tm 2 ⁇ (number of A in sequence + number of T in sequence) + 4 ⁇ (number of G in sequence + number of C in sequence)
- the Tm value can be calculated by, for example, a conventionally known MELTCALC software (http: /www.meltcalc.com/), or can be determined by a neighbor method (Nearest Neighbor Method). .
- the hybridization reaction is preferably carried out 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.
- the target nucleic acid is preferentially hybridized with a fluorescent label detection probe containing a base sequence complementary to the target base sequence.
- the fluorescence is quenched by hybridization of the fluorescently labeled detection probe to the nucleic acid.
- the target nucleic acid may hybridize to the target nucleic acid at a certain rate.
- the rate at which the competitive probe hybridizes to the target nucleic acid is such that the rate of hybridization of the fluorescently labeled detection probe to the target nucleic acid is sufficiently high so that its detection is not substantially hindered.
- the competitive probe preferentially hybridizes to the non-target nucleic acid in this step. As a result, much of the mismatch hybridization of the fluorescently labeled detection probe to the non-target nucleic acid is inhibited, and quenching or luminescence due to the mismatch hybridization is substantially not detected.
- a target nucleic acid having a target base sequence complementary to the base sequence of the fluorescent label detection probe is present in the nucleic acid sample.
- quenching or luminescence is not detected, it can be seen that there is no target base sequence complementary to the base sequence of the fluorescently labeled detection probe in the nucleic acid sample.
- Quenching or luminescence of the fluorescent dye labeled on the fluorescent label detection probe can be detected by performing steps (2) and (3).
- step (2) by measuring the fluorescence intensity while changing the temperature of the reaction sample, a temperature-fluorescence intensity curve representing the change in fluorescence intensity is obtained.
- Methods for measuring fluorescence intensity while changing the temperature of the reaction sample include, for example, a commercially available real-time PCR apparatus (ABI Prism (registered trademark) 7900HT, ABI Prism 7700 (Applied Biosystems), iCycler iQ (TM) Real-Time PCR Detection System (BIO-RAD), MX3000p, MX3005p (Agilent Technologies), etc.) can be used.
- the melting curve analysis function similar to the fluorescence analysis in real-time PCR can be used.
- step (3) the temperature-fluorescence intensity curve obtained in step (2) is first-order differentiated so that the unevenness of the temperature-fluorescence intensity curve can be observed sharply.
- the first-order differentiation of the temperature-fluorescence intensity curve can be easily performed using the dedicated software for the real-time PCR apparatus described above.
- the rate of change in fluorescence intensity increases with the Tm value, and a peak (maximum value) appears in the result of the first derivative.
- the “peak (maximum value)” represents not only the maximum value on the plus side but also the maximum value on the minus side (see Examples).
- a fluorescently labeled detection probe labeled with a fluorescent dye such as TAMRA (trademark), BODIPY (registered tradename) FL, or the like that quenches when approaching a nucleic acid is used
- fluorescence is detected when the temperature falls below the Tm value. Since the fluorescence of the labeled detection probe is quenched by hybridization, a negative peak appears, which may be referred to as a “quenching peak” in this specification.
- the reaction sample contains a non-target nucleic acid that can hybridize with the fluorescence-labeled detection probe with a one-base mismatch
- it competes for hybridization of the fluorescence-labeled detection probe to the non-target nucleic acid.
- the conditions of the fluorescently labeled detection probe and the competitive probe are set so that the peak due to mismatch hybridization does not appear in the result of the first derivative by inhibiting with the probe.
- the base length and base sequence of the fluorescence-labeled detection probe and the competitive probe and the amount added to the nucleic acid sample are as follows: (A) a control target nucleic acid sample containing a target nucleic acid but substantially free of a non-target nucleic acid having a non-target base sequence (B), and substantially free of a target nucleic acid, Adding a fluorescently labeled detection probe and a competitive probe to each of the control non-target nucleic acid samples containing the non-target nucleic acid to obtain a control target reaction sample and a control non-target reaction sample; (B) measuring the fluorescence intensity while changing the temperature of each control reaction sample; and (c) according to the first derivative of the temperature-fluorescence intensity curve obtained from the measurement results, respectively, the control target reaction sample
- the first derivative curve has a peak (maximum value), but the first derivative curve of the control non-target reaction sample is determined to satisfy the functional condition that it has substantially no peak.
- the non-target nucleic acid or target nucleic acid is “substantially” not included is that the non-target nucleic acid or target nucleic acid is not detected in addition to the case where the sample does not contain the non-target nucleic acid or target nucleic acid at all. It also means that only a small amount is included.
- “The first derivative curve of the control non-target reaction sample substantially does not have a peak” means, for example, that even if the first derivative curve of the control non-target reaction sample has a peak, The intensity (maximum amplitude of the fluorescence intensity) is much weaker (flattened) compared to the peak intensity of the first derivative curve of the control target reaction sample, and the peak The case where it does not have a shape may be included.
- the conditions of the base length and base sequence of the fluorescently labeled detection probe and the competitive probe and the amount added to the nucleic acid sample are experimentally determined according to the above procedures (a) to (c) without imposing an excessive burden on those skilled in the art.
- at least two correlated factors of the Tm value of each probe and the added amount (molar ratio) of each probe may be determined.
- suitable conditions for the base length and base sequence of each of the fluorescently labeled detection probe and the competitive probe that are highly likely to satisfy the above functional conditions and the amount added to the nucleic acid sample are as follows. Has been found by the inventors. A person skilled in the art can also determine the base length and base sequence of each of the fluorescently labeled detection probe and the competitive probe and the amount added to the nucleic acid sample so as to satisfy the above conditions based on such an example.
- the present inventors have determined that the Tm value of the competitive probe is at least 5 than the Tm value of the fluorescently labeled detection probe. °C, preferably at least 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C or 20 °C higher
- the Tm value of the competitive probe is lower than the Tm value of the fluorescently labeled detection probe for the target nucleic acid, preferably at least 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C., 7 ° C., 8 It has been found that a lower temperature of 9 ° C., 9 ° C.
- the difference between the Tm value of the competitive probe for the non-target nucleic acid and the Tm value of the fluorescently labeled detection probe is at least 5 ° C., A peak due to mismatch hybridization with a non-target nucleic acid tends to be suppressed.
- the fluorescent label detection probe is easily hybridized with the target nucleic acid. Tend to be sharper.
- the difference between the Tm value of the competitive probe for the non-target nucleic acid and the Tm value of the fluorescently labeled detection probe, and the difference between the Tm value of the competitive probe for the target nucleic acid and the Tm value of the fluorescently labeled detection probe are related to each other. It fluctuates and cannot be set independently. For this reason, depending on the target sequence, it may not be possible to satisfy the combination of the above preferable Tm value conditions.
- the Tm value of the competitive probe is at least 10 ° C., 11 ° C., 12 ° C., 13 ° C., 14 ° C., 15 ° C., 16 ° C., 17 ° C., 18 than the Tm value of the fluorescently labeled detection probe.
- the Tm value of the competitive probe may not be lower than the Tm value of the fluorescently labeled detection probe for the target nucleic acid.
- the Tm value of the competitive probe When the Tm value of the competitive probe is higher than the Tm value of the fluorescently labeled detection probe with respect to the target nucleic acid, hybridization of the fluorescently labeled detection probe to the target nucleic acid is likely to be hindered by the competitive probe, and as a result, the target nucleic acid is detected.
- the peak of the first derivative curve tends to attenuate.
- the effect of the present invention can be achieved as long as the peak due to mismatch hybridization between the fluorescently labeled detection probe and the non-target nucleic acid is sufficiently suppressed (flattened) and the presence or absence of the peak can be visually determined. Can be said to be preferable.
- the Tm value of the competitive probe is at least 10 ° C, preferably 11 ° C, 12 ° C, 13 ° C, 14 ° C, more preferably 15 ° C, 16 ° C, than the Tm value of the fluorescently labeled detection probe. 17 ° C., 18 ° C., 19 ° C. or 20 ° C. higher than the target nucleic acid, and the Tm value of the competitive probe does not exceed the Tm value of the fluorescently labeled probe + 5 ° C., preferably the Tm value of the fluorescently labeled probe + 4 ° C.
- Tm value of the fluorescently labeled probe + 3 ° C., the Tm value of the fluorescently labeled probe + 2 ° C., the Tm value of the fluorescently labeled probe + 1 ° C. or the Tm value of the fluorescently labeled probe may not be exceeded. (Specifically, see the example in which “RB516A-3-P-8” is used as a competitive probe in Example 3 below).
- the added amount of the competitive probe is at least 1 time (ie, 1: 1) in molar ratio with respect to the added amount of the fluorescently labeled detection probe, Preferably at least 2 to 9 times (molar ratio), more preferably at least 10 times (molar ratio), more preferably at least 20, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times 100 times, 150 times, 200 times, 250 times, 300 times, 400 times, 500 times or 1000 times (molar ratio).
- the added amount is 10 or more in terms of the molar ratio of the competitive probe / fluorescently labeled probe, the mismatch of the fluorescently labeled detection probe Tend to be sufficiently suppressed.
- the Tm value of the competitive probe for the non-target nucleic acid and the Tm value of the fluorescently labeled detection probe The larger the difference, the smaller the amount (molar ratio) of competing probe / fluorescently labeled probe tends to be.
- a person skilled in the art can easily determine the difference in the Tm value of the probe and the addition amount of the probe based on the sequence of the target nucleic acid by routine experiments based on the procedure according to the present invention.
- the region on the target nucleic acid that hybridizes with the fluorescently labeled detection probe includes a region that hybridizes with the competitive probe
- the region on the target nucleic acid that hybridizes with the competitive probe includes a region that hybridizes with the fluorescently labeled detection probe
- the region on the target nucleic acid that hybridizes with the competitive probe may be matched with the region that hybridizes with the fluorescently labeled detection probe.
- the “hybridization” in (I) to (III) includes both perfect match hybridization and mismatch hybridization.
- the narrower one of the region that hybridizes with the fluorescently labeled detection probe and the region that hybridizes with the competitive probe corresponds to the target base sequence.
- both the fluorescently labeled detection probe and the competitive probe have the same base length as the target base sequence and can compete and hybridize with each other in a completely matched region.
- the fluorescent labeled detection probe and the competitive probe examples are given.
- the fluorescent labeled detection probe and the competitive probe other than those satisfying those conditions, have a peak in the first derivative curve of the control target reaction sample according to the above procedures (a) to (c), but the control non-target reaction It is sufficient that the first derivative curve of the sample does not substantially have a peak.
- the presence or absence of the target base sequence can be clearly determined based on the presence or absence of the peak of the first derivative curve of the temperature-fluorescence intensity curve.
- the detection method according to the present invention even if the salt concentration in the reaction solution, which is a factor affecting the Tm value, changes to some extent, the discrimination ability is hardly affected, and a stable and accurate detection result can be obtained. There is an advantage that can be.
- the detection method includes the steps (1) to (3) described above, (4)
- the primary differential curve obtained in (3) may include determining that the target base sequence (A) is present in the nucleic acid sample.
- the nucleic acid sample may contain non-target nucleic acids in addition to the target nucleic acid.
- step (4) whether the first derivative curve of the temperature-fluorescence intensity curve obtained in steps (1) to (3) has a shape with a peak or a shape having substantially no peak.
- the presence or absence of the target base sequence (A) can be easily determined.
- the nucleic acid sample may contain non-target nucleic acid in addition to the target nucleic acid. This means that the presence of the target base sequence contained in the heterozygote of the allele is determined by this method, for example, in genetic diagnosis. This means that it is possible to easily determine the presence or absence.
- the present invention can also provide a method for designing a fluorescently labeled detection probe and a competitive probe that can be used in the above detection method.
- the method (1 ′) According to (a) to (c), the first derivative curve of the control target reaction sample has a peak (maximum value), but the first derivative curve of the control non-target reaction sample has substantially no peak. And determining the base length and base sequence of each of the fluorescently labeled detection probe and the competitive probe.
- the present invention can also provide a method for producing a fluorescently labeled detection probe and a competitive probe that can be used in the above detection method.
- the method (1 ′′) synthesizing oligonucleotides of respective base lengths and base sequences designed according to the above-described probe design method; and (2 ′′) using fluorescently labeled detection probe oligonucleotides in the QP method. Labeling with possible fluorescent dyes.
- Oligonucleotides may be artificially synthesized or biosynthesized, and production methods based on the sequences are already well known in the art, and those skilled in the art can select an appropriate method.
- the present invention can also provide a kit for use in the detection method described above.
- the kit according to the present invention includes the above-described fluorescently labeled detection probe, competitive probe, and instructions for use. Instructions for use include, for example, information on the target base sequence that can be detected, information on steps (1) to (3) or steps (1) to (4) of the detection method described above, and fluorescently labeled detection probes and competitors. Information about the amount of each probe added to the nucleic acid sample can be included.
- the kit according to the present invention may be a kit for use in genetic diagnosis.
- the present invention can also provide a genetic diagnosis method using the above detection method.
- the genetic diagnosis method according to the present invention comprises: (1-1) obtaining a nucleic acid sample containing a DNA region to be diagnosed from a sample containing DNA; (1-2) A detection sample labeled with a fluorescent dye that can be used in the QP (Quenching Probe / Primer) method and a competitive probe are added to a nucleic acid sample to obtain a reaction sample.
- a fluorescently labeled detection probe or a competitive probe hybridizes to a target nucleic acid having the target base sequence (A) of (2) measuring fluorescence intensity while changing the temperature of the reaction sample; and (3) first-order differentiation of the temperature-fluorescence intensity curve obtained from the measurement result of (2), wherein fluorescence
- the labeled detection probe and the competitive probe satisfy the same conditions (i) to (iii) as the above-described target base sequence detection method.
- the primary differential curve obtained in step (4) ((3) of the above detection method has a peak in order to make it easier to determine the presence or absence of a gene having a target base sequence. If it has, it may be determined that the target base sequence (A) is present in the nucleic acid sample).
- the nucleic acid of the DNA region is used as a means for obtaining a nucleic acid sample sufficiently containing the nucleic acid of the DNA region to be diagnosed in step (1-1). May be amplified by the LAMP method. As a result, for example, a simple genetic diagnosis method can be provided in a shorter time than an existing genetic diagnosis method using a PCR method.
- the nucleic acid amplified by the LAMP method in this method is an example of the “nucleic acid sample derived from a living body” as described above.
- a person skilled in the art can appropriately design a fluorescent-labeled detection probe and a competitive probe that can achieve the effects of the present invention with respect to various target base sequences with reference to the following examples. Does not require trial and error.
- Example 1 Detection of rpoB gene mutation by addition of competitive probe Using the method for detecting a target nucleotide sequence according to the present invention, the rpoB gene resistance determining region (RRDR) involved in acquiring resistance to rifampicin in M. tuberculosis ), Single nucleotide mutation detection was performed. In this single nucleotide mutation detection, a mutation in which the encoded amino acid was replaced from Asp to Val by substituting one base from A to T in the 516th codon of the base sequence of the rpoB gene was targeted.
- RRDR rpoB gene resistance determining region
- the base sequence of the region including the mutation site is represented by “target nucleic acid having target base sequence” (A allele) or “non-target nucleic acid having non-target base sequence” (T allele).
- a allele target base sequence
- T allele non-target nucleic acid having non-target base sequence
- a “target base sequence including a base-mutated nucleotide” is a wild-type base sequence
- a “non-target base sequence” including a “base unmutated nucleotide” is a mutant-type base sequence. This corresponds to an example of determining whether or not a wild-type base sequence is included.
- the sense strand of the target base sequence in the A allele has “A” at the mutation site, and the sense strand of the non-target base sequence in the T allele has “
- the target to be actually hybridized with the detection probe / competitive probe is the antisense strand of each base sequence.
- the antisense strand complementary to the sense strand of the A allele corresponds to an example of the “target nucleic acid” in the claims.
- the “antisense strand of the target base sequence” complementary to the “sense strand of the target base sequence” corresponds to an example of the “target base sequence” in the claims.
- the antisense strand of the T allele corresponds to an example of “non-target nucleic acid” in the claims
- the antisense strand of a non-target base sequence corresponds to an example of “non-target base sequence” in the claims.
- nucleotide “A” at the mutation site (“T” in the antisense strand of the A allele) For a non-target base sequence that is the subsequent nucleotide “T” (the same base sequence as the target base sequence except that it is replaced by “A” in the antisense strand of the T allele), the non-target base sequence A competitive probe (RB516T-2-P) containing an oligonucleotide having the same base sequence as the sense strand was designed.
- the detection probe (RB516A-2) is incorporated into the antisense strand of the target base sequence (“target base sequence” in the claims) by adopting the same base sequence as the sense strand of each base sequence.
- the competitive probe (RB516T-2-P) has a complementary base sequence, and has a base sequence complementary to the antisense strand of the non-target base sequence (“non-target base sequence” in the claims).
- Oligonucleotides having these designed base lengths and base sequences were synthesized, and the detection probes were labeled with a fluorescent dye TAMRA (trademark) to prepare fluorescently labeled detection probes and competitive probes.
- TAMRA fluorescent dye
- the competitive probe was not labeled with a fluorescent dye, but was modified with a phosphate group.
- Nucleic acid samples were prepared as follows: For each of the gene sequence in which the rpoB RRDR sequence is a mutant type and the gene sequence in which the single nucleotide mutation site is a wild type, the following regions in the genomic DNA: Mycobacterium tuberculosis H37Rv ID: ref
- XbaI / XhoI restriction enzyme treatment
- the H37Rv sequence is obtained from the database, and a plasmid that is artificially mutated to become a drug resistant sequence (mutant) is prepared (by an artificial gene synthesis service of Eurofin Genomics). used.
- a plasmid of a sensitive sequence (wild type) was synthesized, and for heterozygous, the synthesized wild type and mutant plasmids were mixed and used.
- a LAMP reaction was performed for 90 minutes at 65 ° C. using the following primers on the template. The LAMP reaction was performed using a Loopamp (registered trademark) real-time turbidity measurement apparatus LA200 (Teramecs).
- the composition of the reaction solution was as follows: 20 mM Tricine pH 8.8, 50 mM KCl, 8 mM MgSO 4 , 1.4 mM ATP, 1.4 mM GTP, 1.4 mM CTP, 1.4 mM TTP, 0.5% Tween 20, 16U Bst-Large Fragment.
- a fluorescently labeled detection probe final concentration 0.04 ⁇ M
- a competitive probe final concentration 1.2 ⁇ M
- the probe is added before the LAMP amplification reaction in order to prevent an increase in the risk of carry-over contamination of the amplification product when the container containing the amplification product after the reaction is opened.
- the T allele in the mutant template and the A allele in the wild type template were each specifically amplified.
- a reaction sample corresponding to a product obtained by adding the fluorescently labeled detection probe (final concentration 0.04 ⁇ M) and the competitive probe (final concentration 1.2 ⁇ M) to the amplification product was obtained.
- reaction samples were heated by an apparatus MX3005p (Agilent technologies) at 99 ° C. for 5 minutes, then cooled at a rate of ⁇ 2 ° C./30 seconds, and the fluorescence value was measured every 30 seconds.
- a differential value was calculated using software dedicated to MX3005p (Agilent technologies).
- a first derivative curve (thermal dissociation curve) shown in FIG. 1 was obtained.
- the obtained thermal dissociation curve has a peak.
- T allele non-target nucleic acid
- the target nucleic acid (A allele) and the non-target nucleic acid (T allele) The same experiment was also conducted on the mixture with).
- the obtained thermal dissociation curve (shown by a one-dot chain line in FIG. 1) has a weaker peak than the peak obtained in the case of only the target nucleic acid (A allele). Therefore, since the thermal dissociation curve has a peak, it can be determined that the individual has a wild-type single nucleotide unmutated.
- Example 2 Competitive probe design conditions (1) (Comparison with a partially competitive probe) Regarding the rpoB RRDR gene similar to that in Example 1, the probe design conditions for the detection of single nucleotide mutations were examined.
- a target nucleic acid having a target base sequence as shown in FIG. 2 A allele, SEQ ID NO: 9 (sequence seen in the 3 ′ ⁇ 5 ′ direction)) and a non-target nucleic acid having a non-target base sequence (T allele, SEQ ID NO: 10) (Sequence viewed in the 3 ′ ⁇ 5 ′ direction)) was used for the control target nucleic acid sample and the control non-target nucleic acid sample, respectively.
- the target strands in the base sequence represented by SEQ ID NO: 9 and SEQ ID NO: 10 are the same as the sense strand of the target base sequence and the sense strand of the non-target base sequence.
- a detection probe and a competitive probe each having a base sequence complementary to a non-target base sequence in the base sequence represented by These detection probes and competitive probes have the same base length, and have the same base sequence except that one base complementary to the nucleotide (“T” or “A”) at the mutation site is different.
- T nucleotide
- A nucleotide
- two types of partially competitive probes were designed. Partially competitive probe-1 and partially competitive probe-2 are designed so that a part of their base sequences can hybridize to a common region with the detection probe on the target nucleic acid.
- a nucleic acid sample that contains a target nucleic acid but does not substantially contain a non-target nucleic acid, and a target nucleic acid is substantially
- the detection probe final concentration 0.04 ⁇ M
- the competitive probe final concentration 1.2 ⁇ M
- the fluorescence intensity was measured in the same manner as in Example 1 while changing the temperature.
- the first derivative curve of the temperature-fluorescence intensity curve obtained for the control non-target nucleic acid sample when only the detection probe is used, or when a partially competitive probe is used together with the detection probe, the first derivative curve of the temperature-fluorescence intensity curve obtained for the control non-target nucleic acid sample. However, it had a quenching peak at a lower temperature than the quenching peak of the first derivative curve for the control target nucleic acid sample. This is a quenching peak due to mismatch hybridization of the detection probe with a non-target nucleic acid. For example, depending on conditions such as salt concentration, the temperature at which the quenching peak due to mismatch hybridization appears may shift to a high temperature, and it may be mistaken for the quenching peak due to perfect match hybridization with the target nucleic acid of the detection probe. There is.
- Example 3 Competitive probe design conditions (2) (Tm value) For the same rpoB RRDR gene as in Example 1 and Example 2, in order to examine the Tm value conditions for designing a competitive probe, the binding strength to a fluorescently labeled detection probe, a competitive probe, and each target nucleic acid / non-target nucleic acid was calculated as a Tm value, and the relationship with the presence or absence of a quenching peak in the first derivative curve of the temperature-fluorescence intensity curve described above was examined.
- the target nucleic acid was a T allele (mutant base sequence) and the non-target nucleic acid was an A allele (wild type base sequence).
- a target nucleic acid having a target base sequence as shown in FIG. 4A T allele, SEQ ID NO: 10 (sequence viewed in the 3 ′ ⁇ 5 ′ direction)
- a non-target nucleic acid having a non-target base sequence A The allele, SEQ ID NO: 9 (sequence seen in the 3 ′ ⁇ 5 ′ direction)) was used for the control target nucleic acid sample and the control non-target nucleic acid sample, respectively.
- a detection probe that perfectly matches the T allele that is, completely complementary to the target base sequence in the base sequence represented by SEQ ID NO: 10) is labeled with the fluorescent dye BODIPY (registered trademark) FL.
- a fluorescently labeled probe (as a fluorescently labeled detection probe) was prepared.
- 10 competing probes having different Tm values that perfectly match the A allele (that is, completely complementary to the non-target base sequence in the base sequence represented by SEQ ID NO: 9) were prepared.
- a plurality of competing probes having different Tm values were prepared by changing the base length of the competing probe variously from a fluorescent base probe shorter than the fluorescent labeled probe to a base longer than the fluorescent labeled probe.
- Example 2 Similar to Example 1, using one fluorescently labeled probe that perfectly matches the T allele (final concentration 0.04 ⁇ M) and ten competitive probes that perfectly match the A allele and have different Tm values (final concentration 1.2 ⁇ M) The presence or absence of a quenching peak was measured by this method.
- the Tm values for the target nucleic acid and the non-target nucleic acid of the fluorescently labeled probe and the competitive probe were as shown in the table of FIG. 4-2 and the graph of FIG. 4-3.
- Tm value for perfect match (fluorescent labeled probe for T allele, competitive probe for A allele) and mismatch (fluorescent labeled probe for A allele, competitive probe for T allele) is Meltcalc 99 free (http://www.meltcalc.com) /) And setting conditions: Na eq. [MM] Calculated under the condition of 50.
- the quenching peak when the T allele is used as a template is observed.
- it decays stepwise and increases to 9.9 ° C (when "RB516A-3-P-9" or “RB516A-3-P-10" is used as a competitive probe) using T allele as a template
- the competitive probe hybridizes more stably to the target nucleic acid than the fluorescently labeled detection probe. This is probably because hybridization of the fluorescently labeled detection probe to the target nucleic acid is inhibited, and detectable quenching is reduced.
- the first derivative curve of the control target reaction sample (sample containing the T allele) has an extinction peak
- the first derivative curve of the control non-target reaction sample has a substantial quenching peak.
- the Tm value of the competitive probe is 10.1 ° C. higher than the Tm value of the fluorescently labeled probe with respect to the non-target nucleic acid, and the Tm value of the competitive probe is the fluorescently labeled probe with respect to the target nucleic acid. It has been found that lower than the Tm value of
- the Tm value of the competitive probe exceeds the Tm value of the fluorescently labeled probe with respect to the target nucleic acid, if the difference is 6.1 ° C. or less, the first derivative curve of the control target reaction sample is Since it has an extinction peak and the first derivative curve of the control non-target reaction sample has substantially no quenching peak, it is considered to be sufficiently applicable to the present invention.
- the Tm value of the competitive probe is at least 10 ° C. higher than the Tm value of the fluorescently labeled probe, and for the target nucleic acid, the Tm value of the competitive probe is the Tm value of the fluorescently labeled probe + 6.
- Tm value of the competitive probe for the target nucleic acid exceeds the Tm value of the fluorescently labeled probe
- the Tm value of the competitive probe for the non-target nucleic acid also tends to be higher.
- the competitive probe for the non-target nucleic acid The Tm value of the competitive probe can be 15 ° C. or more higher than the Tm value of the fluorescently labeled probe (specifically, referring to FIG. 4-2, when the competitive probe “RB516A-3-P-8” is used, the Tm value of the competitive probe) Is 16.8 ° C.
- the Tm value of the competitive probe is at least 15 ° C. higher than the Tm value of the fluorescently labeled probe, and for the target nucleic acid, the Tm value of the competitive probe is Tm value of the fluorescently labeled probe + 5 ° C. It can be said that it does not exceed is an example of another preferable Tm value condition.
- Example 1 and 2 an example of detecting a target base sequence having a wild-type base sequence is described, and in Example 3 above, mutation is reversed in contrast to Examples 1 and 2 above.
- Example 3 mutation is reversed in contrast to Examples 1 and 2 above.
- the example of the examination of the design condition of the probe for detecting the base sequence of the type has been explained, whether the target base sequence is the wild type base sequence or the mutant base sequence is not particularly limited Absent.
- the method described in Example 3 may be applied to the examination of probe design conditions for detecting a wild-type base sequence, contrary to Example 3 described above.
- one fluorescently labeled probe that perfectly matches the A allele and a plurality of competing probes with different Tm values that perfectly match the T allele are designed, and the presence or absence of a quenching peak is measured in the same manner as described above. do it.
- Example 4 Example of examination of probe design conditions (Tm value) for detection of single nucleotide mutations in other genes
- the present inventors also detected single nucleotide mutations other than rpoB RRDR according to the detection of the present invention. It showed that the method can be applied.
- the design conditions (Tm value) of the detection probe and the competitive probe were examined for the purpose of discriminating a specific single nucleotide mutation in a human gene.
- a target base sequence having an appropriate length including the target single base mutation site is determined, a detection probe complementary to the target base sequence is prepared, labeled with a fluorescent dye, and fluorescent. A labeled detection probe was used.
- the base length of the competing probe is variously changed from one shorter than the detection probe to one longer than the detection probe, and the control target reaction sample and the control non-target reaction sample as described above.
- An experiment was conducted to obtain a first derivative curve for and a probe design condition for detecting a single nucleotide mutation was examined. From this experiment, as the conditions of the base length and base sequence of each competitive probe, the Tm value of the competitive probe is at least 5 ° C.
- the first derivative curve of the control target reaction sample shows a quenching peak. Yes, but the first derivative curve of the control non-target reaction sample has substantially no quenching peak (or even if it has a quenching peak, it is clearly compared to the quenching peak of the first derivative curve of the control target reaction sample) The result was a weak extinction peak, clearly distinguishable from the control target reaction sample.
- the difference between the Tm value of the competitive probe for the non-target nucleic acid and the Tm value of the detection probe is less than 5 ° C.
- the quenching peak due to mismatch hybridization between the detection probe and the non-target nucleic acid is not suppressed
- the first derivative curve of the control non-target reaction sample also showed a quenching peak similar to the first derivative curve of the control target reaction sample.
- the Tm value of the competitive probe was higher than the Tm value of the detection probe for the target nucleic acid, the result was that the quenching peak itself due to the detection of the target nucleic acid was small.
- the competitive probe is more stably hybridized to the target nucleic acid than the detection probe, so that detection by hybridization of the detection probe to the target nucleic acid is inhibited.
- the Tm value of the competitive probe for the same non-target nucleic acid is the same as that of the non-target nucleic acid except that the Tm value of the competitive probe is at least 15 ° C. higher than the Tm value of the detection probe. It was found that the shape of the first derivative curve of the control non-target reaction sample becomes more flat compared to the case where the value is about 5-10 ° C. higher than the Tm value of the detection probe.
- the specificity (thermal stability of hybridization) of the competitive probe to the non-target nucleic acid is significantly increased relative to the thermal stability of hybridization of the detection probe to the non-target nucleic acid. It is considered that the quenching peak due to mismatch hybridization with low specificity can be more reliably suppressed.
- suitable Tm value conditions are the target nucleic acid sequence itself to be detected, and which region of the sequence the fluorescently labeled detection probe / competitive probe covers. And may vary in a complicated manner depending on the concentration of each probe added, the salt concentration in the sample, and the like.
- those skilled in the art will achieve the effects of the present invention by conducting experiments based on the procedure according to the present invention with reference to the preferred Tm values found from the above-mentioned Examples 3 and 4. It is possible to determine the base length and base sequence of the fluorescently labeled detection probe and the competitive probe of the present invention, and it does not require excessive trial and error.
- the present invention provides a means for easily and reliably discriminating differences between single bases such as SNPs, and strict management of conditions such as salt concentration of reaction samples that affect Tm values unlike conventional methods. By making it unnecessary, it is possible to simplify the SNP typing technology, and there is a wide range of applications such as genetic testing, drug discovery, and medical diagnosis.
Abstract
Description
[1]核酸試料から、塩基変異ヌクレオチドを含む標的塩基配列(A)を検出する方法であって、該方法は、以下のステップ:
(1)核酸試料に、QP(Quenching Probe/Primer)法に使用可能な蛍光色素で標識された検出プローブと、競合プローブとを加えて、反応試料を得ること、これによって、反応試料中の標的塩基配列(A)を有する標的核酸に、蛍光標識検出プローブまたは競合プローブがハイブリダイズする;
(2)反応試料の温度を変化させながら、蛍光強度を測定すること;および
(3)(2)の測定結果から得られた温度-蛍光強度曲線を一次微分すること
を含み、ここで、
(i)蛍光標識検出プローブの塩基配列は、標的塩基配列(A)に相補的な塩基配列(A’)を含み、
(ii)競合プローブの塩基配列は、塩基変異ヌクレオチドが塩基未変異ヌクレオチドに置き換わっている以外は標的塩基配列(A)と同じ塩基配列である非標的塩基配列(B)に相補的な塩基配列(B’)を含み、
(iii)蛍光標識検出プローブおよび競合プローブ夫々の塩基長および塩基配列ならびに核酸試料への添加量は、以下の手順:
(a)標的核酸は含まれているが、非標的塩基配列(B)を有する非標的核酸は実質的に含まれていない対照標的核酸試料、および、標的核酸は実質的に含まれないが、非標的核酸は含まれている対照非標的核酸試料の夫々に、蛍光標識検出プローブと競合プローブとを加えて、対照標的反応試料および対照非標的反応試料を得ること;
(b)各対照反応試料の温度を変化させながら、蛍光強度を測定すること;ならびに
(c)測定結果から得られた温度-蛍光強度曲線を夫々、一次微分すること
に従うと、対照標的反応試料の一次微分曲線がピーク(極大値)を有するが、対照非標的反応試料の一次微分曲線がピークを実質的に有さないように、決定されている、前記方法。
(4)(3)で得られた一次微分曲線がピークを有する場合、核酸試料中に標的塩基配列(A)が存在すると判断すること
を含み、ここで核酸試料が、標的核酸の外に、非標的核酸をも含んでいてもよい、[1]に記載の方法。
[3]非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より少なくとも5℃高く、かつ、標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より低い、[1]または[2]に記載の方法。
[5]非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より少なくとも10℃高く、かつ、標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値+5℃を超えない、[1]または[2]に記載の方法。
[6]非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より少なくとも15℃高く、かつ、標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値+5℃を超えない、[1]または[2]に記載の方法。
[8](I)標的核酸上の、蛍光標識検出プローブとハイブリダイズする領域が、競合プローブとハイブリダイズする領域を含むか;(II)標的核酸上の、競合プローブとハイブリダイズする領域が、蛍光標識検出プローブとハイブリダイズする領域を含むか;または、(III)標的核酸上の、競合プローブとハイブリダイズする領域が、蛍光標識検出プローブとハイブリダイズする領域と一致する、[1]~[7]のいずれかに記載の方法。
[10]核酸試料への競合プローブの添加量が、蛍光標識検出プローブの添加量の少なくとも20倍(モル比)である、[1]~[9]のいずれかに記載の方法。
[11]QP(Quenching Probe/Primer)法に使用可能な蛍光色素が、TAMRA(商標)、BODIPY(登録商標) FL、PACIFIC BLUE(商標)およびCR6G(登録商標)からなる群から選択される少なくとも1種の蛍光色素である、[1]~[10]のいずれかに記載の方法。
[13]塩基変異ヌクレオチドが、一塩基置換の変異を含むDNAである、[1]~[12]のいずれかに記載の方法。
[14][1]~[13]のいずれかに記載の方法に使用可能な蛍光標識検出プローブおよび競合プローブを設計する方法であって、該方法は、
(1’)(a)~(c)に従い、対照標的反応試料の一次微分曲線がピーク(極大値)を有するが、対照非標的反応試料の一次微分曲線がピークを実質的に有さないように、蛍光標識検出プローブおよび競合プローブ夫々の塩基長および塩基配列を決定すること
を含む、前記方法。
(1”)[14]に記載の方法に従って設計された、夫々の塩基長および塩基配列のオリゴヌクレオチドを合成すること;および
(2”)蛍光標識検出プローブのオリゴヌクレオチドを、QP法に使用可能な蛍光色素で標識すること
を含む、前記方法。
[16][1]~[13]のいずれかに記載の方法において用いられるためのキットであって、蛍光標識検出プローブ、競合プローブおよび使用説明書を含む、前記キット。
なお、本明細書において、用語SNPは、当該技術分野におけるSNPsと同義で用いる。また、本明細書において、用語SNPが用いられて説明されている事項については、基本的には、「SNP」を「一塩基置換の変異を含むDNA」に置き換えたとしても、同様の説明が成り立つ。つまり、以下の説明において、SNPは検出の対象の一例であり、本発明は、一塩基置換の変異を含むDNAを検出する技術に広く適用され得る。
本明細書中に別記のない限り、本発明に関して用いられる科学的および技術的用語は、当業者に通常理解されている意味を有するものとする。一般的に、本明細書中に記載された分子生物学、微生物学、遺伝子およびタンパク質および核酸化学に関して用いられる用語およびその技術は、当該技術分野においてよく知られ、通常用いられているものとする。本明細書中に参照される全ての特許、出願および他の出版物は、その全体を参照することにより本明細書に組み込まれる。
本発明に係る検出方法は、以下のステップ(1)~(3)を少なくとも含む、核酸試料から、塩基変異ヌクレオチドを含む標的塩基配列(A)を検出する方法である。
(1)核酸試料に、蛍光標識検出プローブと、競合プローブとを加えて、反応試料を得ること、これによって、反応試料中の標的塩基配列を有する標的核酸に、蛍光標識検出プローブまたは競合プローブがハイブリダイズする;
(2)反応試料の温度を変化させながら、蛍光強度を測定すること;および
(3)(2)の測定結果から得られた温度-蛍光強度曲線を一次微分すること。
核酸は、DNAまたはRNAであれば特に限定されず、天然のものであっても合成されたものであってもよい。天然の核酸としては、例えば、生物から回収されたゲノムDNA、mRNA、rRNA、ヘテロ核(hn)RNA等がある。また、合成された核酸として、例えば、β-シアノエチルホスフォロアミダイト法、DNA固相合成法等の公知の化学的合成法により合成されたDNAや、PCR等の公知の核酸合成法により合成された核酸、逆転写反応により合成されたcDNA等がある。
本明細書において、標的塩基配列を含む塩基配列を有する核酸を、「標的核酸」という。
なお、核酸試料中に含有される核酸が、二本鎖核酸である場合、あらかじめ一本鎖核酸にしておくことが好ましい。一本鎖核酸を用いることにより、後述するステップ(1)において、該一本鎖核酸に、検出プローブまたは競合プローブをハイブリダイズさせることができる。抽出された二本鎖核酸の一本鎖化は、熱エネルギーを加える等の公知の手法により行うことができる。
「ハイブリダイズ」とは、一本鎖核酸同士が(例えば熱処理により一本鎖化した検出対象のDNAとプローブとが)相補的な塩基対を形成して結合し二本鎖となることをいう。本明細書において、「ハイブリダイズ」には、一方の塩基配列に対して完全に相補的な塩基配列がハイブリダイズする場合、および、一方の塩基配列と他方の塩基配列との間で、例えば1~数塩基対、相補的な塩基対を形成できない部分(ミスマッチの部分)があっても、相補性を有する部分同士で塩基対を形成することで配列全体としてはハイブリダイズする場合が、含まれる。本明細書においては、一方の塩基配列に対して完全に相補的な塩基配列がハイブリダイズすることを「完全マッチのハイブリダイズ」「特異的にハイブリダイズする」と表現し、ミスマッチを含む場合を「ミスマッチのハイブリダイズ」「非特異的にハイブリダイズする」と表現することがある。
典型的には、かかる相違部位は、SNPにおける変異部位であり、「塩基変異ヌクレオチド」および「塩基未変異ヌクレオチド」は、夫々1つのヌクレオチドである。しかし、例えば、塩基変異が1塩基の欠失変異である場合、欠失部位を挟んだ連続する2塩基を「塩基変異ヌクレオチド」とし、欠失を含まない連続する3塩基を「塩基未変異ヌクレオチド」として本発明を適用することも可能である。また、例えば、塩基変異が1塩基の挿入変異である場合、挿入されたヌクレオチドとその両隣に連続するヌクレオチドの合計3塩基を「塩基変異ヌクレオチド」とし、挿入を含まない連続する2塩基を「塩基未変異ヌクレオチド」として本発明を適用することも可能である。欠失塩基数または挿入塩基数が2塩基以上であってもよい。すなわち、本願に添付の特許請求の範囲における「塩基変異ヌクレオチドが塩基未変異ヌクレオチドに置き換わっている」という記載は、「塩基変異ヌクレオチド」および「塩基未変異ヌクレオチド」が夫々連続する数個のヌクレオチドであるような場合も包含する。
本明細書中において、この非標的塩基配列を含む塩基配列を有する核酸を、「非標的核酸」という。
ここで、ヌクレオチドアナログとは、非天然のヌクレオチドであり、天然のヌクレオチドであるデオキシリボヌクレオチド(DNA)やリボヌクレオチド(RNA)と同様の機能を有するものをいう。すなわち、ヌクレオチドアナログは、ヌクレオチドと同様にリン酸ジエステル結合により鎖を形成することができ、かつ、ヌクレオチドアナログを用いて形成されたプライマーやプローブは、ヌクレオチドのみを用いて形成されたプライマーやプローブと同様に、PCRやハイブリダイズに用いることができる。このようなヌクレオチドアナログとして、例えば、PNA(ポリアミドヌクレオチド誘導体)、LNA(BNA)、ENA(2’-O,4’-C-Ethylene-bridged nucleic acids)、およびこれらの複合体等がある。ここで、PNAは、DNAやRNAのリン酸と5炭糖からなる主鎖をポリアミド鎖に置換したものである。また、LNA(BNA)は、リボヌクレオシドの2’部位の酸素原子と4’部位の炭素原子がメチレンを介して結合している2つの環状構造を持つ化合物である。
ここで、修飾体としては、たとえば、修飾デオキシリボヌクレオチド、修飾リボヌクレオチド、修飾ホスフェート-糖-骨格オリゴヌクレオチド、修飾PNA、修飾LNA(BNA)、修飾ENA等がある。ここで、ヌクレオチドやヌクレオチドアナログの修飾に用いられる物質は、本発明の効果を損なわない限り、特に限定されるものではなく、ヌクレオチド等の修飾に通常用いられる物質を用いることができる。修飾ヌクレオチドや修飾ヌクレオチドアナログとして、たとえば、アミノ基、カルボキシビニル基、リン酸基、メチル基等の官能基により修飾されたヌクレオチド等、メチル基により2-O-メチル化修飾されたヌクレオチド等、ホスホロチオエート化修飾されたヌクレオチド等、後述する蛍光色素等の標識分子により修飾されたヌクレオチド等がある。
QP法に使用可能な蛍光色素としては、例えば、TAMRA(商標)(インビトロジェン社製)、BODIPY(登録商標) FL(インビトロジェン社製)、PACFIC BLUE(商標)(インビトロジェン社製)、CR6G(登録商標)(インビトロジェン社製)等が挙げられる。各蛍光色素の検出プローブへの標識は、有機合成方法等の通常用いられる方法により行うことができる。
以下、検出プローブを、「蛍光標識検出プローブ」ともいう。
「蛍光標識検出プローブの塩基配列」が「標的塩基配列に相補的な塩基配列を含む」ことは、蛍光標識検出プローブの塩基配列(A’)が標的塩基配列(A)に相補的である場合と、蛍光標識検出プローブの塩基配列(A’)が、標的核酸上の標的塩基配列(A)を含み標的塩基配列(A)よりも長い領域の塩基配列に相補的である場合とを包含する。
「競合プローブの塩基配列」が「非標的塩基配列に相補的な塩基配列を含む」ことは、競合プローブの塩基配列(B’)が非標的塩基配列(B)に相補的である場合と、競合プローブの塩基配列(B’)が、非標的核酸上の非標的塩基配列(B)を含み非標的塩基配列(B)よりも長い領域の塩基配列に相補的である場合とを包含する。
まず、ステップ(1)において、反応試料中の標的塩基配列を有する標的核酸に、蛍光標識検出プローブまたは競合プローブをハイブリダイズさせる。蛍光標識検出プローブおよび競合プローブ夫々の塩基長および塩基配列ならびに核酸試料への添加量は、所定の条件を満たすように決定されているが、その条件については後述する。標的核酸と、蛍光標識検出プローブと、競合プローブとのハイブリダイズの反応条件は、特に限定されるものではなく、蛍光標識検出プローブおよび競合プローブのTm値等を考慮した上で、通常の温度、pH、塩濃度、緩衝液等の条件下で行うことができる。
Tm=2×(配列中のAの数+配列中のTの数)+4×(配列中のGの数+配列中のCの数)
また、より正確性を期すために、Tm値は、例えば、従来公知のMELTCALCソフトウェア(http:/www.meltcalc.com/)等により算出でき、隣接法(Nearest Neighbor Method)によって決定することもできる。
(a)標的核酸は含まれているが、非標的塩基配列(B)を有する非標的核酸は実質的に含まれていない対照標的核酸試料、および、標的核酸は実質的に含まれないが、非標的核酸は含まれている対照非標的核酸試料の夫々に、蛍光標識検出プローブと競合プローブとを加えて、対照標的反応試料および対照非標的反応試料を得ること;
(b)各対照反応試料の温度を変化させながら、蛍光強度を測定すること;ならびに
(c)測定結果から得られた温度-蛍光強度曲線を夫々、一次微分すること
に従うと、対照標的反応試料の一次微分曲線がピーク(極大値)を有するが、対照非標的反応試料の一次微分曲線がピークを実質的に有さなくなる、という機能的な条件を満たすように決定されている。
非標的核酸または標的核酸が「実質的に」含まれないことは、試料中に当該非標的核酸または標的核酸が全く含まれない場合に加えて、当該非標的核酸または標的核酸が検出されない程度に少量しか含まれない場合も意味する。「対照非標的反応試料の一次微分曲線がピークを実質的に有さなくなる」とは、例えば、厳密には、対照非標的反応試料の一次微分曲線がピークを有していても、そのピークの強度(蛍光強度の最大の振れ幅)が、対照標的反応試料の一次微分曲線のピークの強度と比較して大幅に弱くなり(平坦化し)、対照標的反応試料の一次微分曲線のようなピークの形状を有さなくなる場合を含んでもよい。
蛍光標識検出プローブ/競合プローブとのモル比にも依るが、非標的核酸に対する競合プローブのTm値と蛍光標識検出プローブのTm値との差が、少なくとも5℃であると、蛍光標識検出プローブと非標的核酸とのミスマッチのハイブリダイズによるピークが抑制され易い傾向にある。また、標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より低いと、標的核酸に対する蛍光標識検出プローブのハイブリダイズがし易くなり、標的核酸を検出する際、一次微分曲線のピークがより鋭くなる傾向にある。
なお、非標的核酸に対する競合プローブのTm値と蛍光標識検出プローブのTm値との差、および標的核酸に対する競合プローブのTm値と蛍光標識検出プローブのTm値との差は、相互に関連して変動し、独立に設定することはできない。このため、標的とする配列によっては、上記の好適なTm値条件の組み合わせを満たせない場合があり得る。例えば、非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より、少なくとも10℃、11℃、12℃、13℃、14℃、15℃、16℃、17℃、18℃、19℃または20℃高いとき、標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より低くならない場合があり得る。標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より高くなると、標的核酸に対する蛍光標識検出プローブのハイブリダイズが競合プローブに妨げられ易くなり、その結果、標的核酸を検出する際の一次微分曲線のピークが減衰する傾向が生じる。しかし、かかる場合もまた、蛍光標識検出プローブと非標的核酸とのミスマッチのハイブリダイズによるピークが十分に抑制(平坦化)されてピークの有無が視覚的に判断できるのであれば、本発明の効果が達成されるので、好適といえる。例えば、非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より、少なくとも10℃、好ましくは11℃、12℃、13℃、14℃、さらに好ましくは15℃、16℃、17℃、18℃、19℃または20℃高く、かつ、標的核酸に対し、競合プローブのTm値が蛍光標識プローブのTm値+5℃を超えない、好ましくは、蛍光標識プローブのTm値+4℃、蛍光標識プローブのTm値+3℃、蛍光標識プローブのTm値+2℃、蛍光標識プローブのTm値+1℃または蛍光標識プローブのTm値を超えない場合も、好適であり得ることが見出されている(具体的には、以下の実施例3の、競合プローブとして「RB516A-3-P-8」を使用した例を参照)。
検出すべき標的核酸の配列それ自体や、検出・競合プローブが該配列のどの領域をカバーするかにも依るが、一般に、非標的核酸に対する競合プローブのTm値と蛍光標識検出プローブのTm値との差が大きくなるほど、競合プローブ/蛍光標識プローブの添加量(モル比)は小さくなる傾向にある。当業者は、標的核酸の配列を元に、プローブのかかるTm値の差と添加量とを、本発明に係る手順に基づき、ルーチンな実験で容易に決定することができる。
(I)標的核酸上の、蛍光標識検出プローブとハイブリダイズする領域が、競合プローブとハイブリダイズする領域を含むか;
(II)標的核酸上の、競合プローブとハイブリダイズする領域が、蛍光標識検出プローブとハイブリダイズする領域を含むか;または、
(III)標的核酸上の、競合プローブとハイブリダイズする領域が、蛍光標識検出プローブとハイブリダイズする領域と一致することを含めてもよい。
なお、この(I)~(III)でいう「ハイブリダイズ」は、完全マッチのハイブリダイズもミスマッチのハイブリダイズも含む。(I)または(II)の場合には、蛍光標識検出プローブとハイブリダイズする領域および競合プローブとハイブリダイズする領域のうち、いずれか狭いほうの領域が、標的塩基配列に相当する。(III)の場合には、蛍光標識検出プローブと競合プローブとが、ともに標的塩基配列と同じ塩基長を有し、完全に一致した領域で互いに競合してハイブリダイズし得る。
これにより、本発明に係る検出方法では、温度-蛍光強度曲線の一次微分曲線のピークの有無に基づいて、標的塩基配列の有無を明確に判別することができる。さらに、本発明に係る検出方法では、Tm値に影響を与える要因である反応液中の塩濃度がある程度変化しても、判別能に影響が生じにくく、安定して正確な検出結果を得ることができるという利点がある。
(4)(3)で得られた一次微分曲線がピークを有する場合、核酸試料中に標的塩基配列(A)が存在すると判断すること
を含んでもよい。ここで核酸試料が、標的核酸の外に、非標的核酸をも含んでいてもよい。かかるステップ(4)では、ステップ(1)~(3)で得られた温度-蛍光強度曲線の一次微分曲線が、ピークを有する形状であるか、ピークを実質的に有さない形状であるかによって、標的塩基配列(A)の有無を容易に判断することができる。ここで核酸試料が、標的核酸の外に、非標的核酸をも含んでいてもよいということは、この方法により、例えば遺伝子診断等において、対立遺伝子のヘテロ接合体に含まれる標的塩基配列の存在の有無をも容易に判断することができるということである。
本発明は、上記の検出方法に使用可能な蛍光標識検出プローブおよび競合プローブを設計する方法を提供することもできる。該方法は、
(1’)(a)~(c)に従い、対照標的反応試料の一次微分曲線がピーク(極大値)を有するが、対照非標的反応試料の一次微分曲線がピークを実質的に有さないように、蛍光標識検出プローブおよび競合プローブ夫々の塩基長および塩基配列を決定すること
を含む。
このようなプローブの設計方法により、SNPの1塩基の違いを高精度で検出し、温度-蛍光強度曲線の一次微分曲線のピークの有無によって標的塩基配列の有無を容易に判断することが可能となるようなプローブセットを実現することができる。
また、本発明は、上記の検出方法に使用可能な蛍光標識検出プローブおよび競合プローブを製造する方法を提供することもできる。該方法は、
(1”)上記のプローブの設計方法の方法に従って設計された、夫々の塩基長および塩基配列のオリゴヌクレオチドを合成すること;および
(2”)蛍光標識検出プローブのオリゴヌクレオチドを、QP法に使用可能な蛍光色素で標識すること
を含む。オリゴヌクレオチドは、人工的に合成させても、生合成させてもよく、その配列に基づく製造方法は当該技術分野において既に周知であり、当業者は適切な方法を選択することができる。
また、本発明は、上記の検出方法において用いられるためのキットを提供することもできる。本発明に係るキットは、上記の蛍光標識検出プローブ、競合プローブおよび使用説明書を含む。使用説明書は、例えば、検出できる標的塩基配列についての情報、上記の検出方法のステップ(1)~(3)またはステップ(1)~(4)についての情報、および、蛍光標識検出プローブおよび競合プローブ夫々の、核酸試料への添加量についての情報等を含み得る。
本発明に係るキットは、遺伝子診断に用いられるためのキットであってもよい。
さらに、本発明は、上記の検出方法を利用した遺伝子診断の方法を提供することもできる。本発明に係る遺伝子診断の方法は、
(1-1)DNAを含む検体から、診断対象のDNA領域を含む核酸試料を得ること;
(1-2)核酸試料に、QP(Quenching Probe/Primer)法に使用可能な蛍光色素で標識された検出プローブと、競合プローブとを加えて、反応試料を得ること、これによって、反応試料中の標的塩基配列(A)を有する標的核酸に、蛍光標識検出プローブまたは競合プローブがハイブリダイズする;
(2)反応試料の温度を変化させながら、蛍光強度を測定すること;および
(3)(2)の測定結果から得られた温度-蛍光強度曲線を一次微分すること
を含み、ここで、蛍光標識検出プローブおよび競合プローブは、上記の標的塩基配列の検出方法と同じ条件(i)~(iii)を満たすものである。本発明に係る遺伝子診断の方法は、標的塩基配列を有する遺伝子の有無の判断をより容易にするため、上記の検出方法のステップ(4)((3)で得られた一次微分曲線がピークを有する場合、核酸試料中に標的塩基配列(A)が存在すると判断すること)を含んでもよい。
また、本発明に係る遺伝子診断の方法は、さらに簡便な方法とするため、ステップ(1-1)において診断対象のDNA領域の核酸を十分に含む核酸試料を得る手段として、該DNA領域の核酸をLAMP法により増幅させてもよい。これにより、例えばPCR法を用いた既存の遺伝子診断方法と比較して、より短時間で簡便な遺伝子診断方法を提供することができる。なお、本方法におけるLAMP法による増幅後の核酸は、上述のとおり「生体に由来する核酸試料」の一例である。
本発明に係る標的塩基配列を検出する方法を用いて、結核菌のリファンピシン耐性獲得に関わるrpoB遺伝子の耐性決定領域(rifampicin resistance determining region、RRDR)を対象として、一塩基変異検出を実施した。この一塩基変異検出では、rpoB遺伝子の塩基配列の516番目のコドン中の、1塩基がAからTに置換されることによってそのコードするアミノ酸がAspからValに置換される変異を標的とした。この変異部位を含む領域の塩基配列は、「標的塩基配列を有する標的核酸」(Aアレル)または「非標的塩基配列を有する非標的核酸」(Tアレル)で表される。なお、実施例1は、「塩基変異ヌクレオチドを含む標的塩基配列」を野生型の塩基配列とし、「塩基未変異ヌクレオチド」を含む「非標的塩基配列」を変異型の塩基配列として、核酸試料中に野生型の塩基配列が含まれるか否かを判別する例に相当する。
ここで、前提として、実施例1では、Aアレル中の標的塩基配列のセンス鎖が上記変異部位に「A」を有し、Tアレル中の非標的塩基配列のセンス鎖が上記変異部位に「T」を有するが、実際に検出プローブ・競合プローブをハイブリダイズさせる対象は、夫々の塩基配列のアンチセンス鎖である。このため、実施例1の内容を、本願に添付の特許請求の範囲の記載に照らすと、Aアレルのセンス鎖に相補するアンチセンス鎖が、請求項中の「標的核酸」の一例に相当し、「標的塩基配列のセンス鎖」に相補する「標的塩基配列のアンチセンス鎖」が、請求項中の「標的塩基配列」の一例に相当する。同様に、Tアレルのアンチセンス鎖が、請求項中の「非標的核酸」の一例に相当し、非標的塩基配列のアンチセンス鎖が、請求項中の「非標的塩基配列」の一例に相当する。
上記のrpoB RRDRの配列が変異型である遺伝子配列と、その一塩基変異部位の配列が野生型である遺伝子配列との夫々について、ゲノムDNA中の以下の領域:
Mycobacterium tuberculosis H37Rv ID: ref|NC_000962.3|760767-761516
(米国国立生物工学情報センター(NCBI)のGenBank内で検索可能。)
をサブクローニングしたプラスミドを制限酵素処理(XbaI/XhoI)して得られたサンプルを、鋳型として用いた。より具体的には、該データベースからそのH37Rvの配列を取得し、人為的に薬剤耐性配列(変異型)となるよう変異させたプラスミドを作製して(ユーロフィンジェノミクス社の人工遺伝子合成サービスによる)使用した。同様に、感受性配列(野生型)のプラスミドも合成しており、ヘテロについては、合成した野生型と変異型のプラスミドを混合して使用した。
上記の鋳型に対して、以下のプライマーを用い、65℃で90分間LAMP反応を行った。LAMP反応は、Loopamp(登録商標)リアルタイム濁度測定装置 LA200(Teramecs)により行った。
20mM Tricine pH8.8、50mM KCl、8mM MgSO4、1.4mM ATP、1.4mM GTP、1.4mM CTP、1.4mM TTP、0.5% Tween20、16U Bst-Large Fragment。
上記の反応により、変異型の鋳型中のTアレルおよび野生型の鋳型中のAアレルを、夫々特異的に増幅させた。これにより、増幅産物に、蛍光標識検出プローブ(最終濃度0.04μM)と競合プローブ(最終濃度1.2μM)とが添加されたものに相当する、反応試料が得られた。これらの反応試料を、装置MX3005p(Agilent technologies)により、99℃で5分間加熱後、-2℃/30秒の速度で降温させ、30秒毎に蛍光値を測定した。測定によって得られた、温度-蛍光強度曲線に相当するデータについて、MX3005p(Agilent technologies)専用ソフトウェアにより微分値を算出した。図1に示す一次微分曲線(熱解離曲線)が得られた。
実施例1と同様のrpoB RRDRの遺伝子について、一塩基変異の検出のためのプローブの設計条件を検討した。図2に示すとおりの標的塩基配列を有する標的核酸(Aアレル、配列番号9(3’→5’方向に見た配列))および非標的塩基配列を有する非標的核酸(Tアレル、配列番号10(3’→5’方向に見た配列))を、夫々、対照標的核酸試料および対照非標的核酸試料に用いた。これらの標的核酸および非標的核酸に対して、標的塩基配列のセンス鎖および非標的塩基配列のセンス鎖と同一の(すなわち、配列番号9で表される塩基配列中の標的塩基配列および配列番号10で表される塩基配列中の非標的塩基配列に夫々相補的な)塩基配列を有する検出プローブおよび競合プローブを夫々設計した。これらの検出プローブと競合プローブとは、互いに同じ塩基長を有し、互いに変異部位のヌクレオチド(「T」または「A」)に相補的な1塩基が異なる以外は同じ塩基配列を有する。
本発明における競合プローブを使用した場合と比較するため、2通りの部分競合型プローブを設計した。部分競合型プローブ-1および部分競合型プローブ-2は、その塩基配列の一部が、標的核酸上の検出プローブと共通の領域に対してハイブリダイズし得るように設計されている。
同様に、対照標的核酸試料および対照非標的核酸試料に、夫々、検出プローブ(最終濃度0.04μM)および部分競合型プローブ-1または-2(最終濃度1.2μM)を添加した各反応試料についても、温度を変化させながら蛍光強度を測定した。
さらに、対照実験として、対照標的核酸試料および対照非標的核酸試料に、夫々、検出プローブ(最終濃度0.04μM)のみを添加して、温度を変化させながら蛍光強度を測定した。結果を図3-1に示す。
一方、検出プローブと共に上記の競合プローブを用いた場合には、対照非標的核酸試料についての一次微分曲線が、消光ピークを実質的に有さなかった。したがって、この競合プローブは、本発明の検出方法に上記検出プローブと共に使用可能であり、この競合プローブを用いることによって、標的核酸が実質的に含まれていなければ実質的に消光ピークが現れない検出系を実現することができる。
この結果は、特許文献1に記載の発明に対する本発明の効果の顕著性を如実に表した例ともいえる。
実施例1および実施例2と同様のrpoB RRDRの遺伝子について、競合プローブ設計のためのTm値条件を検討するために、蛍光標識検出プローブと競合プローブ、夫々の標的核酸・非標的核酸に対する結合力をTm値として算出し、上述した温度-蛍光強度曲線の一次微分曲線における消光ピークの有無との関係性を調べた。
本実施例では実施例1・実施例2とは異なり、標的核酸をTアレル(変異型の塩基配列)とし、非標的核酸をAアレル(野生型の塩基配列)とした。すなわち、図4-1に示すとおりの標的塩基配列を有する標的核酸(Tアレル、配列番号10(3’→5’方向に見た配列))、および非標的塩基配列を有する非標的核酸(Aアレル、配列番号9(3’→5’方向に見た配列))を、夫々、対照標的核酸試料および対照非標的核酸試料に用いた。本実施例では、Tアレルに完全マッチする(すなわち、配列番号10で表される塩基配列中の標的塩基配列に完全に相補的な)検出プローブを、蛍光色素BODIPY(登録商標) FLで標識することによって、蛍光標識プローブ(蛍光標識検出プローブとして)を作製した。また、Aアレルに完全マッチする(すなわち、配列番号9で表される塩基配列中の非標的塩基配列に完全に相補的な)Tm値の異なる競合プローブ10種を作製した。Tm値の異なる複数の競合プローブは、蛍光標識プローブに対して競合プローブの塩基長を、蛍光標識プローブより数塩基短いものから蛍光標識プローブより数塩基長いものまで様々に変更することで作製した。
一方、Tアレルに対する競合プローブ(Aアレル完全マッチ)のTm値が蛍光標識プローブ(Tアレル完全マッチ)のTm値よりも-1.8℃以上高くなるとTアレルを鋳型としたときの消光ピークが段階的に減衰し、9.9℃高くなったとき(競合プローブとして「RB516A-3-P-9」または「RB516A-3-P-10」を使用したとき)Tアレルを鋳型としたときの消光ピークは消失した。これは、理論上、標的核酸に対する競合プローブのTm値が蛍光標識検出プローブのTm値よりも高いときには、標的核酸に対して競合プローブの方が蛍光標識検出プローブよりも安定的にハイブリダイズするので、標的核酸に対する蛍光標識検出プローブのハイブリダイズが阻害され、検出可能な消光が減少するからであると考えられる。
ここで、標的核酸に対する競合プローブのTm値が蛍光標識プローブのTm値を上回っている場合には、非標的核酸に対する競合プローブのTm値もさらに高くなる傾向があり、例えば非標的核酸に対する競合プローブのTm値が蛍光標識プローブのTm値より15℃以上高くなり得る(具体的に、図4-2を参照すると競合プローブ「RB516A-3-P-8」を用いた場合に競合プローブのTm値が蛍光標識プローブのそれより16.8℃高い)。したがって、例えば、非標的核酸に対し、競合プローブのTm値が蛍光標識プローブのTm値より少なくとも15℃高く、かつ、標的核酸に対し、競合プローブのTm値が蛍光標識プローブのTm値+5℃を超えないことも、別の好適なTm値条件の一例といえる。
本発明者らは、rpoB RRDR以外の一塩基変異の検出についても、本発明の検出方法を適用することができることを示した。本実施例では、実施例1~3とは異なり、ヒトの遺伝子中のある特定の一塩基変異の判別を目的として、検出プローブおよび競合プローブの設計条件(Tm値)を検討した。本実施例では、まず、対象とする一塩基変異部位を含む適当な長さの標的塩基配列を決定し、標的塩基配列に対して相補的な検出プローブを作製して蛍光色素で標識し、蛍光標識検出プローブとした。さらに、同じ検出プローブに対して競合プローブの塩基長を、検出プローブより数塩基短いものから検出プローブより数塩基長いものまで様々に変更し、上記のように対照標的反応試料および対照非標的反応試料について一次微分曲線を求める実験を行い、一塩基変異の検出のためのプローブの設計条件を検討した。この実験からは、競合プローブ夫々の塩基長および塩基配列の条件として、非標的核酸に対し、競合プローブのTm値が、検出プローブのTm値より少なくとも5℃高く、かつ、標的核酸に対し、競合プローブのTm値が、検出プローブのTm値より低く、かつ競合プローブ/蛍光標識検出プローブの添加量(モル比)が30/1である場合に、対照標的反応試料の一次微分曲線が消光ピークを有するが、対照非標的反応試料の一次微分曲線が消光ピークを実質的に有さなくなる(あるいは、消光ピークを有していても、対照標的反応試料の一次微分曲線の消光ピークに比べて明らかに弱い消光ピークであり、対照標的反応試料の場合とは明らかに区別可能である)という結果が得られた。
しかし、非標的核酸に対する競合プローブのTm値と検出プローブのTm値との差が5℃に満たない場合には、検出プローブと非標的核酸とのミスマッチのハイブリダイズによる消光ピークが抑制されず、対照非標的反応試料の一次微分曲線にも、対照標的反応試料の一次微分曲線と似たような消光ピークが見られる結果となった。一方、標的核酸に対し、競合プローブのTm値を、検出プローブのTm値より高くした場合には、標的核酸の検出による消光ピークそのものが小さくなるという結果が得られた。これは、標的核酸に対して競合プローブの方が検出プローブよりも安定的にハイブリダイズするため、標的核酸に対する検出プローブのハイブリダイズによる検出が阻害されるからである。
また、上記条件において、非標的核酸に対し、競合プローブのTm値を、検出プローブのTm値より少なくとも15℃高くした以外は上記条件と同じ場合には、同じ非標的核酸に対する競合プローブのTm値を検出プローブのTm値より5~10℃程度高くした場合と比較して、対照非標的反応試料の一次微分曲線の形状がより平坦に近づくことが見出された。このことから、本発明において、非標的核酸に対する競合プローブの特異性(ハイブリダイゼーションの熱安定性)を、非標的核酸に対する検出プローブのハイブリダイゼーションの熱安定性に対してより大幅に高めることで、特異性が低いミスマッチのハイブリダイズによる消光ピークをより確実に抑制することができると考えられる。
Claims (16)
- 核酸試料から、塩基変異ヌクレオチドを含む標的塩基配列(A)を検出する方法であって、該方法は、以下のステップ:
(1)核酸試料に、QP(Quenching Probe/Primer)法に使用可能な蛍光色素で標識された検出プローブと、競合プローブとを加えて、反応試料を得ること、これによって、反応試料中の標的塩基配列(A)を有する標的核酸に、蛍光標識検出プローブまたは競合プローブがハイブリダイズする;
(2)反応試料の温度を変化させながら、蛍光強度を測定すること;および
(3)(2)の測定結果から得られた温度-蛍光強度曲線を一次微分すること
を含み、
ここで、
(i)蛍光標識検出プローブの塩基配列は、標的塩基配列(A)に相補的な塩基配列(A’)を含み、
(ii)競合プローブの塩基配列は、塩基変異ヌクレオチドが塩基未変異ヌクレオチドに置き換わっている以外は標的塩基配列(A)と同じ塩基配列である非標的塩基配列(B)に相補的な塩基配列(B’)を含み、
(iii)蛍光標識検出プローブおよび競合プローブ夫々の塩基長および塩基配列ならびに核酸試料への添加量は、以下の手順:
(a)標的核酸は含まれているが、非標的塩基配列(B)を有する非標的核酸は実質的に含まれていない対照標的核酸試料、および、標的核酸は実質的に含まれないが、非標的核酸は含まれている対照非標的核酸試料の夫々に、蛍光標識検出プローブと競合プローブとを加えて、対照標的反応試料および対照非標的反応試料を得ること;
(b)各対照反応試料の温度を変化させながら、蛍光強度を測定すること;ならびに
(c)測定結果から得られた温度-蛍光強度曲線を夫々、一次微分すること
に従うと、対照標的反応試料の一次微分曲線がピーク(極大値)を有するが、対照非標的反応試料の一次微分曲線がピークを実質的に有さないように、決定されている、前記方法。 - さらに以下のステップ:
(4)(3)で得られた一次微分曲線がピークを有する場合、核酸試料中に標的塩基配列(A)が存在すると判断すること
を含み、
ここで核酸試料が、標的核酸の外に、非標的核酸をも含んでいてもよい、請求項1に記載の方法。 - 非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より少なくとも5℃高く、かつ、
標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より低い、請求項1または2に記載の方法。 - 非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より少なくとも10℃高く、かつ、
標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より低い、請求項1または2に記載の方法。 - 非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より少なくとも10℃高く、かつ、
標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値+5℃を超えない、請求項1または2に記載の方法。 - 非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より少なくとも15℃高く、かつ、
標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値+5℃を超えない、請求項1または2に記載の方法。 - 非標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より少なくとも15℃高く、かつ、
標的核酸に対し、競合プローブのTm値が、蛍光標識検出プローブのTm値より低い、請求項1または2に記載の方法。 - (I)標的核酸上の、蛍光標識検出プローブとハイブリダイズする領域が、競合プローブとハイブリダイズする領域を含むか;
(II)標的核酸上の、競合プローブとハイブリダイズする領域が、蛍光標識検出プローブとハイブリダイズする領域を含むか;または、
(III)標的核酸上の、競合プローブとハイブリダイズする領域が、蛍光標識検出プローブとハイブリダイズする領域と一致する、請求項1~7のいずれか一項に記載の方法。 - 核酸試料への競合プローブの添加量が、蛍光標識検出プローブの添加量の少なくとも10倍(モル比)である、請求項1~8のいずれか一項に記載の方法。
- 核酸試料への競合プローブの添加量が、蛍光標識検出プローブの添加量の少なくとも20倍(モル比)である、請求項1~9のいずれか一項に記載の方法。
- QP(Quenching Probe/Primer)法に使用可能な蛍光色素が、TAMRA(商標)、BODIPY(登録商標) FL、PACIFIC BLUE(商標)およびCR6G(登録商標)からなる群から選択される少なくとも1種の蛍光色素である、請求項1~10のいずれか一項に記載の方法。
- 核酸試料が、生体に由来する、請求項1~11のいずれか一項に記載の方法。
- 塩基変異ヌクレオチドが、一塩基置換の変異を含むDNAである、請求項1~12のいずれか一項に記載の方法。
- 請求項1~13のいずれか一項に記載の方法に使用可能な蛍光標識検出プローブおよび競合プローブを設計する方法であって、該方法は、
(1’)(a)~(c)に従い、対照標的反応試料の一次微分曲線がピーク(極大値)を有するが、対照非標的反応試料の一次微分曲線がピークを実質的に有さないように、蛍光標識検出プローブおよび競合プローブ夫々の塩基長および塩基配列を決定すること
を含む、前記方法。 - 請求項1~13のいずれか一項に記載の方法に使用可能な蛍光標識検出プローブおよび競合プローブを製造する方法であって、該方法は、
(1”)請求項14に記載の方法に従って設計された、夫々の塩基長および塩基配列のオリゴヌクレオチドを合成すること;および
(2”)蛍光標識検出プローブのオリゴヌクレオチドを、QP法に使用可能な蛍光色素で標識すること
を含む、前記方法。 - 請求項1~13のいずれか一項に記載の方法において用いられるためのキットであって、蛍光標識検出プローブ、競合プローブおよび使用説明書を含む、前記キット。
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- 2017-10-25 WO PCT/JP2017/038458 patent/WO2018079579A1/ja unknown
- 2017-10-25 CN CN201780066441.3A patent/CN109890978A/zh active Pending
- 2017-10-25 KR KR1020197012351A patent/KR102438915B1/ko active IP Right Grant
- 2017-10-25 US US16/344,413 patent/US11390911B2/en active Active
- 2017-10-25 AU AU2017351263A patent/AU2017351263B2/en active Active
- 2017-10-25 EP EP17865651.8A patent/EP3533880A4/en not_active Ceased
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2022
- 2022-06-06 US US17/833,324 patent/US20220298558A1/en active Pending
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Also Published As
Publication number | Publication date |
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EP3533880A1 (en) | 2019-09-04 |
US11390911B2 (en) | 2022-07-19 |
US20200056228A1 (en) | 2020-02-20 |
EP3533880A4 (en) | 2020-06-03 |
JPWO2018079579A1 (ja) | 2019-09-26 |
US20220298557A1 (en) | 2022-09-22 |
CA3041445A1 (en) | 2018-05-03 |
KR102438915B1 (ko) | 2022-08-31 |
AU2017351263A1 (en) | 2019-05-23 |
AU2017351263B2 (en) | 2023-12-21 |
CN109890978A (zh) | 2019-06-14 |
KR20190066031A (ko) | 2019-06-12 |
JP7025342B2 (ja) | 2022-02-24 |
US20220298558A1 (en) | 2022-09-22 |
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