JP6846636B2 - Oligonucleotide for nucleic acid detection - Google Patents

Description

The present disclosure relates to a nucleic acid detection oligonucleotide for detecting a specific target nucleic acid obtained by the PCR (Polymerase Chain Reaction) method.

In various fields such as clinical practice and pathology, the PCR method is widely used for the purpose of gene expression analysis, functional analysis, diagnosis, and the like. In general, PCR generally involves (1) dissociating a double-stranded nucleic acid into a single-stranded nucleic acid by heat treatment, and (2) annealing a prima to the single-stranded nucleic acid (template nucleic acid) (binding the prima to the template nucleic acid). By repeating this cycle, the three steps of (3) forming an extension product of the prima using a nucleic acid polymerase (a nucleic acid complementary to a single strand is synthesized) are set as one cycle. , The target gene sequence can be amplified exponentially.

Among them, real-time PCR (Real-time PCR) is a method for monitoring and analyzing the amount of nucleic acid amplification over time (in real time), such as mRNA (messenger RNA) expression analysis, SNP (Single Nucleotide Polymer) analysis, and infectious disease diagnosis. Widely used for various purposes. In real-time PCR, the initial amount of the target gene is calculated from the number of cycles and the amount of PCR final product (final nucleic acid amplification amount) by comparison with a known standard. The PCR end product is optically quantified. For example, when a nucleic acid complementary to a single strand is synthesized, the fluorescence probe bound to the target nucleic acid is decomposed and the emitted fluorescence is measured.

Further, as one of the conventional PCR methods, Patent Document 1 is an intercalator fluorescent dye which has a sequence complementary to a specific base sequence in a target nucleic acid and emits fluorescence when bound to the target nucleic acid. A method using a labeled nucleic acid probe (intercalator fluorescent probe) is disclosed. According to the method, since the probe emits measurable fluorescence by forming a complementary bond with the target nucleic acid, the reaction solution is rapidly heated and lowered with respect to the single-stranded RNA containing a specific base sequence in the sample. It is possible to measure fluorescence at a substantially constant temperature without repeating the operation of doing.

Japanese Unexamined Patent Publication No. 2000-014400

However, although the prior art disclosed in Patent Document 1 has the above-mentioned advantages, as shown in FIG. 5, the lower the initial concentration of the template nucleic acid, the slower the rise of the amplification curve of the PCR product, and further, the exponential function. No increase is seen. Therefore, if the initial concentration of the template nucleic acid is low (trace amount), there is a problem that the fluorescence emitted by the PCR final product cannot be detected.

Therefore, an object of the present disclosure is to provide an oligonucleotide for nucleic acid detection capable of detecting a trace amount of a target nucleic acid with high sensitivity.

In order to achieve the above object, the nucleic acid detection oligonucleotide according to one embodiment of the present disclosure is a nucleic acid detection oligonucleotide modified with a fluorescent substance and a dimming substance that binds to a template nucleic acid, and is a nucleic acid detection oligo. The nucleotides include at least a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide, and the first oligonucleotide binds to the most 3'terminal side of the nucleic acid detection oligonucleotides that bind to the template nucleic acid, 5 The'terminal at least one base has a sequence complementary to the template nucleic acid, and the second oligonucleotide binds to the most 5'terminal side of the nucleic acid detection oligonucleotides that bind to the template nucleic acid, and the 3'end. At least one of the bases has a complementary sequence to the template nucleic acid, and the third oligonucleotide binds to the region between the regions where the first and second oligonucleotides each bind to the template nucleic acid. At least one base other than the 5'end and the 3'end has a sequence complementary to the template nucleic acid, and binds to the template nucleic acid adjacently among the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide. The oligonucleotide to be used is at least one base of the 5'end of the oligonucleotide on the 5'end side of the template nucleic acid when viewed from one side and the 3'end of the oligonucleotide on the 3'end side of the template nucleic acid when viewed from the other side. Has a complementary sequence.

According to the present disclosure, it is possible to provide an oligonucleotide for nucleic acid detection capable of detecting a trace amount of a target nucleic acid with high sensitivity.

Schematic diagram showing a state in which the nucleic acid detection oligonucleotide according to the embodiment is bound to the template nucleic acid. Schematic diagram showing the binding site of the oligonucleotide for nucleic acid detection according to the embodiment. The figure which shows the process which the oligonucleotide for nucleic acid detection which concerns on embodiment binds to a template nucleic acid. The figure explaining how the oligonucleotide for nucleic acid detection which concerns on embodiment fluoresces. PCR amplification curve in the prior art

Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. It should be noted that all of the embodiments described below show a preferred specific example of the present disclosure. Therefore, the numerical values, shapes, materials, components, the arrangement positions and connection forms of the components, the steps and the order of the steps, etc., which are shown in the following embodiments, are examples and are intended to limit the present disclosure. Absent. Therefore, among the components in the following embodiments, the components not described in the independent claims indicating the highest level concept of the present disclosure will be described as arbitrary components.

It should be noted that each figure is a schematic view and is not necessarily exactly illustrated. Further, in each figure, the same reference numerals are given to substantially the same configurations, and duplicate description will be omitted or simplified.

(Embodiment)
Hereinafter, the oligonucleotide for detecting nucleic acid according to the present embodiment will be described with reference to the drawings.

In the present disclosure, the "upstream side" means the 3'end side in the region of the base sequence, and the "downstream side" means the 5'end side in the region of the base sequence.

Further, the “nucleic acid” in the present disclosure means DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).

[A. Oligonucleotide for nucleic acid detection]
Hereinafter, the oligonucleotide for nucleic acid detection according to the present embodiment will be described with reference to FIGS. 1 to 4.

FIG. 1 is a schematic view showing a state in which the nucleic acid detection oligonucleotide 5 according to the present embodiment is bound to the template nucleic acid 100a. FIG. 2 is a schematic diagram showing binding sites 21, 22, 23a to 23d, and 30a to 30e of the nucleic acid detection oligonucleotide 5 according to the present embodiment. FIG. 3 is a diagram illustrating how the nucleic acid detection oligonucleotide 5 according to the present embodiment binds to the template nucleic acid 100a. FIG. 4 is a diagram illustrating how the nucleic acid detection oligonucleotide 5 according to the present embodiment fluoresces.

Hereinafter, the oligonucleotide 5 for nucleic acid detection may be simply referred to as an oligonucleotide 5.

[A-1. Basic structure of oligonucleotide for nucleic acid detection]
As shown in FIGS. 1 to 3, the nucleic acid detection oligonucleotide 5 according to the present embodiment is an oligonucleotide in which both ends thereof are modified with a fluorescent substance 10 and a quenching substance 11. In the nucleic acid detection oligonucleotide 5, since the fluorescent substance 10 and the quenching substance 11 are in close proximity to each other, the function of the fluorescent substance 10 to emit a fluorescent signal is hindered by the quenching substance 11. However, as shown in FIG. 4, in the process of DNA elongation reaction, the nucleic acid detection oligonucleotide 5 bound to the template nucleic acid 100a is decomposed by, for example, the 5'→ 3'exonuclease activity of the Taq DNA polymerase. Since the fluorescent substance 10 and the extinguishing substance 11 modified to the nucleic acid detection oligonucleotide 5 are spatially separated from each other, the fluorescent substance 10 emits a fluorescent signal. The intensity of this fluorescent signal is proportional to the number of molecules of the amplified target nucleic acid.

[A-1-1. Fluorescent and quenching substances]
The fluorescent substance 10 that modifies the nucleic acid detection oligonucleotide 5 according to the present embodiment may not be decomposed or its fluorescence is not attenuated during the nucleic acid amplification step. The fluorescent substance 10 is not particularly limited, but for example, fluoroscein or a derivative thereof (for example, FAM (carboxyfluorescein), JOE (6-carboxy-4', 5'-dichloro2', 7'-dimethoxyfluorescein), FITC (Fluorescein isothiocyanate), TET (Tetrachlorofluorescein), HEX (5'-hexachloro-fluorescein-CE phosphoromidite)), BODICY® series, Rhodamine or derivatives thereof (eg, 5-carboxyrhodamine 6G (eg, 5-carboxyrhodamine 6G) CR6G), tetramethylrhodamine (TAMRA)), Cy® dyes (eg, Cy3, Cy5) and the like may be used. The method for binding the fluorescent dye 10 to the nucleic acid detection oligonucleotide 5 can be carried out according to a usual method. By utilizing the quenching of the fluorescent substance 10, it is possible to use no dye inserted into other double-stranded nucleic acid structures such as an intercalator, and without using two types of probes that cause a FRET (Fluorescence resonance energy transfer) phenomenon. The target nucleic acid sequence can be detected simply and specifically using a probe labeled with one type of fluorescent substance. The modification position of the fluorescent substance 10 in the base sequence of the nucleic acid detection oligonucleotide 5 is not particularly limited, but may be modified to the terminal (within 5 bases from the terminal base), and the terminal base is labeled. It may have been.

Examples of the quenching substance 11 include TAMRA (tetramethyl-rhodamine), DABCYL (4- (4-dimethylaminophenylazo) benzoic acid), BHQ1 (BHQ: Black Hole Quencher (registered trademark)), BHQ2, BHQ3 and the like. However, it is not limited to these.

The fluorescent substance 10 and the quenching substance 11 may modify the fluorescent substance 10 to the 5'end of the nucleic acid detection oligonucleotide 5 and the quenching substance 11 to the 3'end, and the quenching substance 11 may be modified to the nucleic acid detection oligonucleotide 5. The fluorescent substance 10 may be modified at the 5'end and at the 3'end.

[A-1-2. Nucleotide sequence of oligonucleotide for nucleic acid detection]
The nucleic acid detection oligonucleotide 5 according to the present embodiment has a base sequence complementary to the specific base sequence in the template nucleic acid 100a. Therefore, the nucleic acid detection oligonucleotide 5 can specifically bind to the region having a specific base sequence of the template nucleic acid 100a.

The nucleic acid detection oligonucleotide 5 has a sequence in which at least one base is complementary to the template nucleic acid 100a. The nucleic acid detection oligonucleotide 5 may be 7 to 25 consecutive oligonucleotides contained in the complementary strand template nucleic acid 100a, preferably 9 to 25, more preferably 12 to 25, and even more preferably. It may have 15 to 25 nucleotides complementary to it. When the nucleic acid detection oligonucleotide 5 has many base sequences complementary to the template nucleic acid 100a, it becomes easy to bind to the target site (binding sites 21, 22, 23a to 23d in FIG. 2) of the template nucleic acid 100a, and the template This is because the binding force with the nucleic acid 100a is strengthened.

The base sequence of the nucleic acid detection oligonucleotide 5 can be appropriately designed according to the base sequence of the target nucleic acid, as will be described later in the description of the Tm value.

[A-2. Components of oligonucleotides for nucleic acid detection]
The nucleic acid detection oligonucleotide 5 according to the present embodiment contains at least the first oligonucleotide 1, the second oligonucleotide 2, and the third oligonucleotide 3. These oligonucleotides are commonly referred to as "probes".

As shown in FIGS. 1 to 3, the first oligonucleotide 1 binds to the most upstream side of the template nucleic acid 100a among the nucleic acid detection oligonucleotides 5. The first oligonucleotide 1 has a sequence in which at least one base at the 5'end is complementary to the template nucleic acid 100a. The second oligonucleotide 2 binds to the most downstream side of the template nucleic acid 100a among the nucleic acid detection oligonucleotides 5. The second oligonucleotide 2 has a sequence in which at least one base at the 3'end is complementary to the template nucleic acid 100a. The third oligonucleotide 3 binds to the region between the regions where the first oligonucleotide 1 and the second oligonucleotide 2 each bind to the template nucleic acid 100a. The third oligonucleotide 3 has a sequence in which at least one base other than the 5'end and the 3'end is complementary to the template nucleic acid 100a.

Further, the third oligonucleotide 3 may contain a plurality of types of oligonucleotides (third oligonucleotides 3a, 3b, 3c, 3d). By including a plurality of types of third oligonucleotides 3a, 3b, 3c, and 3d (hereinafter referred to as 3a to 3d) having different base sequences, which are designed based on the base sequence of the target nucleic acid, a large amount of the template nucleic acid 100a is used. The fluorescent substance 10 can be applied.

The Tm value of the plurality of types of third oligonucleotides 3a to 3d may be larger than the Tm value of the first oligonucleotide 1 and smaller than the Tm value of the second oligonucleotide 2. The reason will be described later, and the description thereof will be omitted here.

[A-2-1. Relationship between the first, second and third oligonucleotides]
Hereinafter, the relationship between the first, second, and third oligonucleotides (for example, the binding position to the template nucleic acid, etc.) will be described with reference to FIGS. 2 and 3.

As shown in FIG. 2, the nucleic acid detection oligonucleotides 5 according to the present embodiment are the first oligonucleotide 1, the third oligonucleotides 3a to 3d, in order from the 3'terminal side (upstream side) of the template nucleic acid 100a. The second oligonucleotide 2 binds to the template nucleic acid 100a. Of these nucleic acid detection oligonucleotides 5, adjacent oligonucleotides 5 bind to each other at binding sites 30a to 30e. In addition, the individual oligonucleotides 1, 2, 3a to 3d bind to the template nucleic acid 100a at the binding sites 21, 22, 23a to 23d, respectively.

Further, in the present embodiment, the oligonucleotides of the first oligonucleotide 1, the second oligonucleotide 2, and the third oligonucleotides 3a to 3d that are adjacent to each other and bind to the template nucleic acid 100a are the template nucleic acids when viewed from one side. At least one base of the 5'end of the oligonucleotide on the 5'end side (downstream side) of 100a and the 3'end of the oligonucleotide on the 3'end side (upstream side) of the template nucleic acid 100a when viewed from the other side. It has a complementary sequence.

For example, as shown in FIG. 3B, the first oligonucleotide 1 and the third oligonucleotide 3a that are adjacent to each other and bind to the template nucleic acid 100a are on the upstream side of the template nucleic acid 100a when viewed from the third oligonucleotide 3a. It is located downstream of at least one base of the 3'end of the first oligonucleotide 1 (the end of FIG. 3B, modified with the extinguishing substance 11) and the template nucleic acid 100a as viewed from the first oligonucleotide 1. It has a complementary sequence with at least one base of the 5'end of the third oligonucleotide 3a (the end of FIG. 3B, which is modified with the fluorescent substance 10). As described above, the adjacent nucleic acid detection oligonucleotides 5 (here, the first oligonucleotide 1 and the third oligonucleotide 3a) have complementary sequences on the opposite sides to each other, so that the adjacent nucleic acid detection oligonucleotides are present. The ends of 5 are attached to each other. As a result, more nucleic acid detection oligonucleotides 5 can be bound to the template nucleic acid 100a as compared with the case where nucleic acid detection oligonucleotides having no complementary sequences on the opposite sides are bound to the template nucleic acid. .. Therefore, a larger amount of the fluorescent substance 10 can be added to the template nucleic acid 100a, and fluorescence can be detected with high sensitivity even with a small amount of the target nucleic acid.

[A-2-2. Tm value of oligonucleotide for nucleic acid detection]
In order to control the binding position and binding order of the nucleic acid detection oligonucleotide 5, the nucleic acid detection oligonucleotide 5 may be designed based on the Tm value. Hereinafter, the Tm value of the nucleic acid detection oligonucleotide 5 according to the present embodiment will be described with reference to FIGS. 2 and 3.

In the nucleic acid detection oligonucleotide 5 according to the present embodiment, the Tm values of the first oligonucleotide 1, the second oligonucleotide 2, and the third oligonucleotide 3 are based on the Tm value of the primer bound to the template nucleic acid 100a. May also be large. Thereby, the nucleic acid detection oligonucleotide 5 can bind to the template nucleic acid 100a before the primer binds to the template nucleic acid 100a.

Here, the Tm value means the Tm value (melting temperature) of a standard double-stranded nucleic acid. The Tm value can be obtained from the melting curve by a conventional method. The melting curve shows, for example, the absorbance and fluorescence intensity of a solution over time (in real time) when the temperature of a solution containing only standard double-stranded nucleic acids is changed from a high temperature that causes thermal denaturation to a low temperature. It can be obtained by measuring. In the obtained melting curve, the Tm value is the temperature at which the average rate of change of the absorbance and the fluorescence intensity with respect to the temperature or the differential value is maximum.

Moreover, you may use the calculated value as the Tm value. For example, the Tm value can be calculated from the base sequence information of a standard double-stranded nucleic acid by using general-purpose primer / probe design software or the like. That is, the factor that determines the Tm value depends on the binding strength of the double-stranded DNA of the PCR amplification product, and depends on the GC content, the amplification length, etc., so that the Tm value can be changed depending on the target nucleic acid. It is possible.

The Tm value (Tm (n)) of the nucleic acid detection oligonucleotide 5 according to the present embodiment may be larger than the Tm value (Tm (p)) of the primer, for example, when the Tm (p) value is 60 ° C. In some cases, the Tm (n) value is 70 ° C. or higher and 80 ° C. or lower.

Further, in the nucleic acid detection oligonucleotide 5 according to the present embodiment, the Tm values of the first oligonucleotide 1, the second oligonucleotide 2, and the third oligonucleotides 3a to 3d are set from the upstream side of the template nucleic acid 100a. It may become smaller in the order of bonding toward the downstream side.

That is, among the nucleic acid detection oligonucleotides 5, the Tm value of the first oligonucleotide 1 (Tm (n1))> the Tm value of the third oligonucleotide 3a in the order of binding from the upstream side to the downstream side of the template nucleic acid 100a. (Tm (n3a))> Tm value of the third oligonucleotide 3b (Tm (n3b))> Tm value of the third oligonucleotide 3c (Tm (n3c))> Tm value of the third oligonucleotide 3d (Tm (n3d)) )> The Tm value of the second oligonucleotide 2 (Tm (n2)) may be obtained.

As a result, the nucleic acid detection oligonucleotide 5 binds to the template nucleic acid 100a in this order from the binding region side of the primer toward the downstream side of the template nucleic acid 100a. Therefore, even if the prima binds before all of the nucleic acid detection oligonucleotides 5 bind to the template nucleic acid 100a, as many nucleic acid detection oligonucleotides 5 as possible bind to the template nucleic acid 100a during the DNA extension reaction. be able to.

Hereinafter, the process of binding of the first oligonucleotide 1, the second oligonucleotide 2, the third oligonucleotides 3a to 3d, and the primer to the template nucleic acid 100a in the present embodiment will be described with reference to FIG.

For example, as shown in FIG. 3A, the first oligonucleotide 1 binds to the most upstream side of the template nucleic acid 100a among the nucleic acid detection oligonucleotides 5 that bind to the template nucleic acid 100a. At this time, since the base sequence at the 3'end of the first oligonucleotide 1 does not have a base sequence complementary to the template nucleic acid 100a, only the base sequence at the 3'end (here, the side modified with the quencher 11) is a template. It remains unbound to nucleic acid 100a.

The base sequence at the 3'end of the first oligonucleotide 1 is the 5'end of the third oligonucleotide 3a that binds to the template nucleic acid 100a adjacent to the first oligonucleotide 1 (here, the side modified with the fluorescent substance 10). ) And has a complementary base sequence. Therefore, as shown in the binding site 30a of FIG. 2 and (b) of FIG. 3, the first oligonucleotide 1 and the third oligonucleotide 3a bind to each other at adjacent ends.

The number of bases in the complementary base sequence of the 5'end (the side modified with the fluorescent substance 10) of the first oligonucleotide 1 is not particularly limited as long as the first oligonucleotide 1 can maintain binding to the template nucleic acid 100a. , At least 1 base, for example, 7 bases or more and 25 bases or less, preferably 9 bases or more and 25 bases or less, more preferably 12 bases or more and 25 bases or less, and further preferably 15 bases or more and 25 bases or less. is there.

As described above, since the first oligonucleotide 1 has a base sequence complementary to the base sequence on the upstream side of the template nucleic acid 100a at the 5'end, it binds to the most prime binding region side of the nucleic acid detection oligonucleotide 5. can do. Here, since the first oligonucleotide 1 does not have a base sequence complementary to the template nucleic acid 100a at the 3'end'(the side modified with the extinguishing substance 11), the other oligonucleotide 5 is the first oligonucleotide 1. When bound adjacent to the downstream side, it can bind to the 5'terminal side of other adjacent oligonucleotides 5.

Next, as shown in FIG. 3B, the third oligonucleotide 3a binds to the template nucleic acid 100a adjacent to the first oligonucleotide 1. Here, at least one base at the 5'end (the side modified with the fluorescent substance 10) of the third oligonucleotide 3a complements the 3'end (the side modified with the quencher 11) of the first oligonucleotide 1. Binds to a specific base sequence.

The complementary base sequences of the adjacent oligonucleotides 5 (here, the first oligonucleotide 1 and the third oligonucleotide 3a) need only be able to maintain the binding, for example, at least 1 base, and 5 bases or less. All you need is. This complementary base sequence should have a relatively high proportion of C (cytosine) and G (guanine). Since C and G are bound by hydrogen bonds and have a stronger binding force than A (adenine) and T (thymine), a higher proportion of CG can strengthen the binding in the complementary sequence.

Next, as shown in FIG. 3 (b), the base sequence of the 5'end (the side modified with the fluorescent substance 10) of the third oligonucleotide 3a is the 3'end (quenching substance) of the first oligonucleotide 1. It binds to the complementary base sequence of (the side modified with 11). Then, at least one base other than the 5'end and the 3'end of the third oligonucleotide 3a binds to the template nucleic acid 100a. The complementary base sequence (between the 3'end and the 5'end) other than the 5'end and the 3'end of the third oligonucleotide 3a (the base sequence of the binding site 23a in FIG. 2) is the third oligonucleotide 3a. Is not particularly limited as long as it can maintain the binding to the template nucleic acid 100a, and the preferable number of bases is the same as that of the first oligonucleotide 1 described above.

Further, as shown in FIG. 3C, the third oligonucleotide 3a is 5 of the third oligonucleotide 3b adjacent to the 3'terminal side (the side modified with the fluorescent substance 10) of the third oligonucleotide 3a. It has a sequence complementary to the'end (the side modified with the extinguishing substance 11), and at least one base at the 5'end of the third oligonucleotide 3b binds to the 3'end of the third oligonucleotide 3a. .. The complementary base sequence (between the 3'end and the 5'end) other than the 5'end and the 3'end of the third oligonucleotide 3b (the base sequence of the binding site 23b in FIG. 2) is the third oligonucleotide 3b. Is not particularly limited as long as it can maintain the binding to the template nucleic acid 100a, and the preferable number of bases is the same as that of the first oligonucleotide 1 described above.

Finally, the second oligonucleotide 2 binds to the most downstream side of the template nucleic acid 100a among the nucleic acid detection oligonucleotides 5 that bind to the template nucleic acid 100a. The base sequence of the 5'end (the side modified with the fluorescent substance 10) of the second oligonucleotide 2 is a base complementary to the 3'end (the side modified with the quencher 11) of the adjacent third oligonucleotide 3d. Due to its sequence, the second oligonucleotide 2 binds to the 3'end (the side modified with quencher 11) of the third oligonucleotide 3d (binding site 30e in FIG. 2).

As described above, in order to bind the nucleic acid detection oligonucleotide 5 to the template nucleic acid 100a in order from the upstream to the downstream of the template nucleic acid 100a, it is necessary to design so that the Tm value of each oligonucleotide 5 is different. It is preferable, and more preferably, the Tm values may be designed to differ from each other by 1 ° C. or higher, more preferably 2 ° C. or higher, and even more preferably 3 ° C. or higher.

Then, by making the Tm value of the nucleic acid detection oligonucleotide 5 larger than the Tm value of the primer, as shown in FIG. 3D, in the primer, all the nucleic acid detection oligonucleotides 5 are used as the template nucleic acid 100a. After binding, it binds to the template nucleic acid 100a.

In this way, when the primer binds to the template nucleic acid 100a, the DNA polymerase acts and the DNA elongation reaction is started. As a result, the oligonucleotide 5 for nucleic acid detection is decomposed, and a fluorescent signal is emitted.

[A-2-3. Fluorescence mechanism]
The mechanism of fluorescence in this embodiment will be described with reference to FIG. FIG. 4 is a diagram illustrating how the nucleic acid detection oligonucleotide 5 according to the present embodiment fluoresces.

In the present embodiment, as shown in FIG. 3D, after all nucleic acid detection oligonucleotides 5 are bound to the template nucleic acid 100a, the primers (forward (Fw) primer and reverse (Rv) primer) are applied. It binds to template nucleic acids 100a and 100b. As described above, when these primers are bound, the DNA polymerase reaction initiates a DNA elongation reaction.

Although the present specification shows an example in which the nucleic acid detection oligonucleotide 5 binds to the template nucleic acid 100a, the nucleic acid detection oligonucleotide 5 of the present disclosure may bind to the template nucleic acid 100b.

As shown in FIG. 4, the first oligonucleotide 1 bound to the template nucleic acid 100a in the process of elongation of the primer (referring to the elongation of DNA described above) is subjected to the 5'→ 3'exonuclease activity of the DNA polymerase. It is disassembled. At this time, the fluorescent substance 10 and the quenching substance 11 modified to the first oligonucleotide 1 are spatially separated from each other (for example, a part 1a, 1b, 1c of the first oligonucleotide), and the fluorescent substance 10 is used. It emits a fluorescent signal.

In the first oligonucleotide 1 before decomposition, since the fluorescent substance 10 and the quenching substance 11 are in close proximity to each other, the function of the fluorescent substance 10 to emit a fluorescent signal is hindered by the quenching substance 11. However, when the first oligonucleotide 1 is decomposed in the process of DNA elongation, the action of the quencher 11 is lost and the fluorescent substance 10 emits a fluorescent signal.

Since this fluorescent signal is proportional to the number of molecules (copy number) of the amplified target nucleic acid, the initial concentration of the target nucleic acid can be calculated based on the intensity of the obtained fluorescent signal.

[B. Primer]
The primer in the present embodiment can be appropriately set according to the base sequence of the target nucleic acid as described above in the description of the Tm value. Since the relatively high Tm value has a relatively high binding property to the template nucleic acid, a primer having a suitable Tm value may be designed according to the type of the target nucleic acid.

[Summary]
As described above, the nucleic acid detection oligonucleotide 5 according to the present embodiment is a nucleic acid detection oligonucleotide 5 that binds to the template nucleic acid 100a and is modified by the fluorescent substance 10 and the extinguishing substance 11, and is for nucleic acid detection. The oligonucleotide 5 contains at least the first oligonucleotide 1, the second oligonucleotide 2, and the third oligonucleotide 3, and the first oligonucleotide 1 is the most 3 of the nucleic acid detection oligonucleotides 5 that bind to the template nucleic acid 100a. Of the nucleic acid detection oligonucleotides 5 that bind to the'terminal side and at least one base at the 5'end has a complementary sequence to the template nucleic acid 100a, and the second oligonucleotide 2 binds to the template nucleic acid 100a. It binds to the most 5'end side, and at least one base at the 3'end has a sequence complementary to the template nucleic acid 100a, and the third oligonucleotide 3 is the first oligonucleotide 1 and the second oligonucleotide 2. Is bound to the region between the regions that bind to the template nucleic acid 100a, respectively, and at least one base other than the 5'end and the 3'end has a complementary sequence to the template nucleic acid 100a, and the first oligonucleotide 1, Of the second oligonucleotide 2 and the third oligonucleotide 3, the oligonucleotides that are adjacent to each other and bind to the template nucleic acid 100a are the 5'end of the oligonucleotide located on the 5'end side of the template nucleic acid 100a when viewed from one side and the other. At least one base at the 3'end of the oligonucleotide on the 3'end side of the template nucleic acid has a complementary sequence.

As described above, when the adjacent nucleic acid detection oligonucleotides 5 have complementary sequences on the opposite sides, the ends of the adjacent nucleic acid detection oligonucleotides 5 are bound to each other. As a result, more nucleic acid detection oligonucleotides 5 can be bound to the template nucleic acid 100a than in the case where a nucleic acid detection oligonucleotide having no complementary sequence on the opposite side is bound to the template nucleic acid. Therefore, a larger amount of the fluorescent substance 10 can be added to the template nucleic acid 100a, and fluorescence can be detected with high sensitivity even with a small amount of the target nucleic acid.

Further, in the nucleic acid detection oligonucleotide 5 according to the present embodiment, the third oligonucleotide 3 may contain a plurality of types of oligonucleotides 3a to 3d. By including a plurality of types of third oligonucleotides 3a to 3d having different base sequences designed based on the base sequence of the target nucleic acid (template nucleic acid 100a), many fluorescent substances 10 can be imparted to the template nucleic acid 100a. ..

Further, in the nucleic acid detection oligonucleotide 5 according to the present embodiment, the Tm values of the first oligonucleotide 1, the second oligonucleotide 2, and the third oligonucleotide 3 are the Tm of the primer that binds to the template nucleic acid 100a. May be greater than the value. Thereby, the nucleic acid detection oligonucleotide 5 can bind to the template nucleic acid 100a before the primer binds to the template nucleic acid 100a.

Further, in the nucleic acid detection oligonucleotide 5 according to the present embodiment, the Tm values of the first oligonucleotide 1, the second oligonucleotide 2, and the third oligonucleotide 3 are set from the 3'end side of the template nucleic acid 100a. It may become smaller in the order of binding toward the 5'end side. As a result, the nucleic acid detection oligonucleotide 5 binds to the template nucleic acid 100a in this order from the binding region side of the primer toward the downstream side of the template nucleic acid 100a. Therefore, even if the prima binds before all of the nucleic acid detection oligonucleotides 5 bind to the template nucleic acid 100a, as much nucleic acid detection oligonucleotide 5 as possible binds to the template nucleic acid 100a while the DNA is elongated. be able to.

The oligonucleotides for nucleic acid detection according to the present disclosure have been described above based on the embodiments, but the present disclosure is not limited to these embodiments. As long as the gist of the present disclosure is not deviated, various modifications that can be conceived by those skilled in the art are applied to the embodiment, and other forms constructed by combining some components in the embodiment are also within the scope of the present disclosure. include.

1 First oligonucleotide 2 Second oligonucleotide 3, 3a, 3b, 3c, 3d Third oligonucleotide 5 Nucleic acid detection oligonucleotide 10 Fluorescent substance 11 Quenching substance 100a, 100b Template nucleic acid

Claims (4)

A nucleic acid detection oligonucleotide modified with a fluorescent substance and a quencher that binds to a template nucleic acid.
The nucleic acid detection oligonucleotide contains at least a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide.
The first oligonucleotide binds to the most 3'terminal side of the nucleic acid detection oligonucleotide that binds to the template nucleic acid, and at least one base at the 5'end has a sequence complementary to the template nucleic acid. Ori,
The second oligonucleotide binds to the most 5'terminal side of the nucleic acid detection oligonucleotide that binds to the template nucleic acid, and at least one base at the 3'end has a sequence complementary to the template nucleic acid. Ori,
The third oligonucleotide binds to a region between the region where the first oligonucleotide and the second oligonucleotide each bind to the template nucleic acid, and contains at least one base other than the 5'end and the 3'end. It has a complementary sequence to the template nucleic acid and has a complementary sequence.
The first oligonucleotide, the second oligonucleotide, and the third oligonucleotide, which are adjacent to each other and bind to the template nucleic acid, are the oligonucleotides on the 5'terminal side of the template nucleic acid when viewed from one side. At least one base of the 5'end and the 3'end of the oligonucleotide on the 3'end side of the template nucleic acid when viewed from the other has a complementary sequence.
Oligonucleotide for nucleic acid detection. The third oligonucleotide contains a plurality of types of oligonucleotides.
The oligonucleotide for detecting nucleic acid according to claim 1. The Tm value of each of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide is larger than the Tm value of the primer that binds to the template nucleic acid.
The oligonucleotide for detecting nucleic acid according to claim 1 or 2. The Tm values of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide decrease in the order of binding from the 3'end side to the 5'end side of the template nucleic acid.
The oligonucleotide for detecting nucleic acid according to any one of claims 1 to 3.

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