Double-stranded PNA probe

Abstract

To provide PNA probes having separable quencher and reporter so as to achieve high fluorescence intensity on hybridization of its PNA moiety with a target DNA.SOLUTION: Disclosed is a double-stranded PNA probe 1 comprising a quencher part 20 comprising a quenching group 22, a DNA region 26, and a quencher linker 24 linking the quenching group and the DNA region, and a reporter part 10 comprising a light emitting group 12, a PNA region 16 and a reporter linker 14 linking the light emitting group and the PNA region, where the quenching group and the light emitting group come on the same side when the DNA region and the PNA region are complementarily bound, where the reporter linker contains oxygen atoms less than two atoms in the main chain, and the reporter linker is shorter than the quencher linker by linear carbon bond conversion.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a peptide nucleic acid probe that can be used for detecting a target base sequence of a mutant gene or mutant cell in a cell, and particularly relates to a PNA probe of a type in which a quencher and a reporter are separated.

Background Art

[0002] When the detection target has a DNA structure, such as in real-time PCR or detection of a target gene, probes for detecting its base sequence have been developed. This probe has a structure in which a luminescent group (reporter) and a quenching group (quencher) are linked to a probe sequence having a base sequence complementary to the target base sequence. When the base sequence portion in the probe binds to the target base sequence, the luminescent group and the quenching group are separated from each other, and the luminescent group emits light. Such a probe is called a DNA probe.

[0003] Patent Document 1 discloses a method for multiplex detection of nucleic acid molecules using a DNA probe having a stem-loop structure.

[0004] Further, Patent Document 2 describes fluorescence detection of nucleic acids using a double-stranded DNA probe having a luminescent group and a quenching group provided at both ends of an oligonucleotide and having complementary portions to each other.

[0005] On the other hand, a PNA (peptide nucleic acid) probe using PNA in a portion that binds to a target DNA has also been proposed. A PNA probe is an artificial peptide mimicking DNA by binding nucleosides with peptides, combined with a luminescent group and a quenching group, for detecting a target base sequence.

[0006] Known PNA probes include double-stranded PNA probes, which consist of PNA with a light-emitting group attached and DNA with a quenching group attached, and PNA molecular beacons, which have a light-emitting group attached to one end of the PNA and a quenching group attached to the other end. Patent Document 3 discloses a PNA molecular beacon type PNA probe in which a light-emitting group and a quenching group are attached to the N-terminus and C-terminus.

[0007] Compared to DNA probes, PNA probes are resistant to nucleases and proteases, and also have high heat resistance. Furthermore, they are linked by peptide bonds rather than phosphate bonds like DNA, and their neutral pH makes them more likely to hybridize with target genes, which are attracting attention.

[0008] Non-patent document 1 introduces double-stranded PNA probes and compares them with PNA molecular beacons. Non-patent document 1 reports that double-stranded PNA probes can achieve higher emission intensity.

[0009] Furthermore, Non-Patent Document 2 discloses a double-stranded PNA (peptide nucleic acid (PNA)-DNA) probe developed to detect gene mutations specific to the mRNA sequence of the epidermal growth factor receptor (EGFR) in lung cancer cells (mutations in exon 19del E746-A750, T790M, and L858R). [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Special Publication No. 2019-528772 [Patent Document 2] Special Publication No. 2010-505440 [Patent Document 3] Special Publication No. 2017-510301 [Non-patent literature]

[0011] [Non-Patent Document 1] Kathleen E. Mach, Aniruddha M. Kaushik, KuangwenHsieh, Pak Kin Wong, Tza-Huei Wangcand Joseph C. Liao:Analyst 2019,144,1565-1574 [Non-Patent Document 2] Hajime Shigeto, Takashi Ohtsuki, Akira Iizuka, Yasuto Akiyamac and Shohei Yamamura: Analyst 2019, 144, 4613-4621 [Overview of the project] [Problems that the invention aims to solve]

[0012] PNA probes have the characteristic of readily hybridizing with target DNA. However, when formed in a stem-loop structure, the probe is composed solely of PNA, which presents a challenge in terms of difficulty in introducing it into cells. Furthermore, because both luminescent and quenching groups exist within the same molecule, even when bound to target DNA, the fluorescence intensity is reduced due to the influence of the quenching group.

[0013] On the other hand, when PNA probes are formed using a double-stranded DNA structure, they are cationic and therefore easily introduced into cells. Furthermore, with a double-stranded structure, after the PNA portion hybridizes with the target DNA, the DNA portion separates, eliminating the presence of quenching groups within the same molecule, resulting in high fluorescence intensity. This also allows for leveraging the characteristic of easily hybridizing with target DNA.

[0014] However, the fluorescence intensity (the difference in brightness between emission and quenching) of the double-stranded PNA probe was not yet sufficient, and there was a need to improve the fluorescence intensity. [Means for solving the problem]

[0015] In order to increase the fluorescence intensity, it is required that it be sufficiently dark during quenching and not become dark during emission. Generally, in order to quench fluorescence during quenching, it is necessary to bring the quenching group closer to the emission group, and in order to brighten during emission, it is considered necessary to separate the quenching group from the emission group.

[0016] Therefore, in a double-stranded PNA probe, a linker having the same structure is arranged between the portion hybridizing to the target DNA and the quenching group and the emission group so that the quenching group and the emission group of each other are close to each other (Patent Document 2).

[0017] However, the inventor of the present invention found that by changing the lengths of the linker on the reporter side and the linker on the quencher side, the fluorescence intensity rather increases, and thus completed the present invention.

[0018] More specifically, the double-stranded PNA probe according to the present invention is a quenching group, a DNA region, a quencher part including a quencher linker connecting between the quenching group and the DNA region having, an emission group, a PNA region, a reporter part including a reporter linker connecting between the emission group and the PNA region and when the DNA region and the PNA region are complementarily bound, the double-stranded PNA probe in which the quenching group and the emission group are arranged on the same side, wherein the reporter linker does not contain two or more elements of oxygen in the main chain, the quencher linker has a linear carbon bond conversion value of 13 and the reporter linker has a linear carbon bond conversion value of 3 to 11. That s

Advantages of the Invention

[0019] ​The double-stranded PNA probe according to the present invention selects a structure in which the structure of the linker portion connecting the luminescent group and PNA is different from the structure of the linker portion connecting the quenching group and DNA, and is made shorter in terms of linear carbon bond conversion. As a result, the fluorescence intensity (luminance difference between luminescence and quenching) was higher than when the lengths of the two linkers were made uniform.

[0020] Further, when the double-stranded PNA probe using this linker was made to detect the gene mutation of T790M or L858R of the epidermal growth factor receptor (EGFR), it was possible to detect with higher sensitivity than before. That is, the double-stranded PNA probe according to the present invention can detect a single nucleotide polymorphism of the target mRNA.

Brief Description of Drawings

[0021] [Figure 1] It is a schematic diagram showing the structure of the double-stranded PNA probe according to the present invention. [Figure 2] It is a diagram showing the structure in the vicinity of the luminescent group and the quenching group of the double-stranded PNA probe according to the present invention. [Figure 3] It is a diagram illustrating linear carbon bond conversion. [Figure 4] It is a diagram showing the result of detecting T790M with the double-stranded PNA probe according to the present invention. [Figure 5] It is a diagram showing the result of detecting L858R with the double-stranded PNA probe according to the present invention.

Modes for Carrying Out the Invention

[0022] Hereinafter, the double-stranded PNA probe according to the present invention will be described with reference to the drawings and examples. The following description illustrates one embodiment and one example of the present invention, and the present invention is not limited to the following description. The following description can be modified without departing from the gist of the present invention.

[0023] Figure 1 illustrates the structure and principle of the double-stranded PNA probe according to the present invention. The double-stranded PNA probe 1 according to the present invention consists of a reporter part 10 and a quencher part 20. The reporter part 10 consists of a light-emitting group 12, a reporter linker 14, a PNA region 16, and a terminal portion 18 (see Figure 1(a)). Here, the reporter linker 14 is described as binding to the N-terminal side of the PNA region 16. However, it may also be described as binding to the C-terminal side.

[0024] The quencher part 20 consists of a quenching group 22, a quencher linker 24, and a DNA region 26. The quencher part 20 may also have terminal ends attached. Here, the quencher linker 24 is described as binding to the 3' end of the DNA region 26. However, it may also be described as binding to the 5' end.

[0025] PNA region 16 is a region made up of a so-called nucleic acid peptide structure, in which nucleosides are linked by peptide bonds. PNA region 16 is designed to hybridize with the base sequence of target DNA 52.

[0026] On the other hand, DNA region 26 is a region made up of a polynucleotide structure. DNA region 26 is designed to hybridize with PNA region 16.

[0027] The luminescent group 12 is a fluorescent substance, and Fam(6-carboxyfluorescein) is preferably used. However, other known substances can also be used for the luminescent group 12. For example, Texas red, JOE, TAMRA, CY5, CY3, etc.

[0028] The quenching group 22 is a substance that quenches the luminescence of the light-emitting group 12, and DABCYL is preferably used. However, other known substances can also be used for the quenching group 22. For example, TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2, etc.

[0029] The reporter linker 14 connects the PNA region 16 to the light-emitting group 12. The quencher linker 24 connects the DNA region 26 to the quenching group 22.

[0030] The terminal portion 18 is bound to the end opposite the light-emitting group 12 and the PNA region 16, and is a portion bound to the double-stranded PNA probe 1 in order to impart some function to it. The function to be imparted here may be to improve solubility or to facilitate hybridization with target DNA. In addition to hybridization with target DNA, it may also have a function that facilitates the uptake of the double-stranded PNA probe 1 into cells.

[0031] Furthermore, as already explained, the terminal portion 18 may be attached to the opposite end of the quencher part 20, with the quenching group 22 and the DNA region 26 in between. Also, the terminal portions 18 of the reporter part 10 and the quencher part 20 may have exactly the same structure or may have different structures.

[0032] The reporter part 10 and the quencher part 20 are synthesized separately and then mixed. Upon mixing, the PNA region 16 and the DNA region 26 hybridize according to a pre-designed structure (see Figure 1(b)). At this time, the light-emitting group 12 and the quenching group 22 approach each other and are quenched. At least before use, the double-stranded PNA probe 1 is hybridized and quenched in this way, with the reporter part 10 and quencher part 20 being the same.

[0033] In this example, the luminescent group 12 is positioned on the N-terminal side of the PNA region 16 and the quenching group 22 is positioned on the 3' terminal side of the DNA region 26. However, the luminescent group 12 may also be positioned on the C-terminal side of the PNA region 16 and the quenching group 22 on the 5' terminal side of the DNA region 26. Furthermore, when the PNA region 16 and the DNA region 26 hybridize and the quenching group 22 and luminescent group 12 are on the same side, it can be said that "the quenching group 22 and luminescent group 12 are positioned on the same side."

[0034] Next, when the double-stranded PNA probe 1 is introduced into the test sequence 50 containing the target base sequence 52, the double-stranded PNA probe 1 separates, and the PNA region 16 of the reporter part 10 hybridizes with the target base sequence 52. At this time, the quencher part 20 separates from the reporter part 10, causing the light-emitting group 12 to emit light (see Figure 1(c)).

[0035] Now, in order to increase the fluorescence intensity when the double-stranded PNA probe 1 detects the target base sequence 52, it is important to keep the fluorescence emission (amount of emission during quenching) when the reporter part 10 and the quencher part 20 hybridize low. Considering the mechanism of quenching, it is generally thought that the light-emitting group 12 and the quenching group 22 should be brought as close together as possible.

[0036] To bring the light-emitting group 12 and the quenching group 22 as close together as possible, it is considered best to use the same reporter linker 14 and quencher linker 24. However, as shown in the examples described later, in practice, intentionally using reporter linker 14 and quencher linker 24 with different structures (different lengths) resulted in a decrease in the amount of light emitted during quenching and a higher fluorescence intensity (difference between quenched and emitted states) when detecting the target base sequence 52.

[0037] Figure 2 is a structural diagram showing the vicinity of the linker of the double-stranded PNA probe 1 used in the example. In reporter part 10, the luminescent group 12 is Fam. The PNA region 16 of reporter part 10 consists of a series of peptide bonds, with a base attached to each peptide portion. The reporter linker 14 connects the amino group of Fam to the carboxyl group at the C-terminus of the PNA region 16. Here, it is a linear structure with seven carbon atoms.

[0038] In quencher part 20, the quenching group 22 is DABCYL. In the DNA region 26 of quencher part 20, nucleosides are linked together by phosphate. The quencher linker 24 of quencher part 20 is bonded to the amino group of DABCYL and the amino group of the base immediately before the 3' end of the DNA region 26 (5-methylcytosine in Figure 2). Here, this is the basic structural framework of diethylene glycol dipropyl ether.

[0039] In the double-stranded PNA probe 1 according to the present invention, there is no nucleic acid peptide corresponding to the last base of the DNA region 26 on the side to which the quenching group 22 of the quencher part 20 binds. In this sense, the DNA region 26 may have unused bases that do not bind to the reporter part 10. The DNA region 26 and the PNA region 16 are designed to hybridize with each other. Here, it is shown that the linkage portion 30 between the reporter part 10 and the quencher part 20 is composed of TA and CG.

[0040] Next, we will explain the "linear carbon bond equivalent value," an index used when evaluating the linker portion in this invention. The linear carbon bond equivalent value is the number of linking elements counted by considering the section from the light-emitting group 12 (or quenching group 22) to the PNA region 16 (or DNA region 26) in the linker structure as a linear carbon bond. The linear carbon bond equivalent value is denoted by the symbol "egC".

[0041] For example, in Figure 2, reporter linker 14 has a linear structure of 7 carbon atoms, so it is denoted as "egC7" or "7 in terms of linear carbon bond count." Quencher linker 24 has linear carbon bonds and three ether bonds, but the oxygen atoms are replaced with carbon atoms. Therefore, the linear carbon bond count of quencher linker 24 is 13 (egC13).

[0042] Furthermore, reporter linker 14 does not contain more than two oxygen atoms in its main chain. Also, if there is a benzene ring in the middle of the linker, the element that connects the two ends in the shortest distance is replaced with carbon to calculate the linear carbon bond.

[0043] Refer to Figure 3. For example, if the linker is 1,2-diethylbenzene (Figure 3(a)), the shortest linkage between ethyl groups is (-C=C-), so the number of straight-chain carbon bonds is 6 (e.g., C6). If it is 1,4-diethylbenzene (Figure 3(b)), the linkage between ethyl groups is (-C=CC-), so the number of straight-chain carbon bonds is 8 (e.g., C8). Also, if there are side chains in the middle of the linker, the side chains are not counted.

[0044] In the double-stranded PNA probe 1 of the present invention, the reporter linker 14 has 90% to 10% of the linear carbon bond ratio of the quencher linker 24. That is, when the linear carbon bond ratio of the reporter linker 14 is divided by the linear carbon bond ratio of the quencher linker 24, the value is between 0.1 and 0.9. Furthermore, when comparing the two linear carbon bond ratios, the smaller one can be said to be "shorter in terms of linear carbon bond ratio". [Examples]

[0045] The following examples demonstrate the effectiveness of the double-stranded PNA probe 1 according to the present invention. The examples involved determining a target DNA, fabricating a double-stranded PNA probe 1 to detect that target DNA, and measuring the fluorescence intensity when the same concentration of target DNA was detected using the double-stranded PNA probe 1.

[0046] <Target base sequence> The target sequences were two gene mutations specific to the mRNA sequence of the epidermal growth factor receptor (EGFR) in lung cancer cells: T790M and L858R. Table 1 shows the respective target sequences. Both wild-type and mutant strains were created for each target. Note that the target gene corresponds to the target DNA labeled 52 in Figure 1.

[0047] [Table 1]

[0048] <Double-stranded PNA probe> A double-stranded PNA probe consists of two types of sequences: a reporter part and a quencher part. The end to which the light-emitting group and quenching group are bound is called the "leading end."

[0049] (1) Reporter parts The fluorescent dye Fam was used as the luminescent group 12 of the reporter part. Five types of reporter linkers 14 were prepared: Sp2, C7, bAla, C11, and Ambz. Table 2 shows their respective compositional formulas and linear carbon bond equivalent values ​​(egC). Structures containing two or more oxygen atoms are denoted with "(2O)". The reporter linker is the structure shown in parentheses in Table 2. "N" indicates the end of the luminescent group (the beginning of the reporter part), and "CO" indicates the beginning of the PNA region (the end of the reporter part). The structure of Sp6, which is used in the terminal portion described later, is also shown. The structure used in the terminal portion 18 may contain two or more oxygen atoms.

[0050] In the case of Sp6, "N" is the beginning of Sp6 and "CO" is the end of Sp6. An amino group is attached to the end of Sp6. Sp2 is a conventional reporter linker as shown in Non-Patent Document 1.

[0051] [Table 2]

[0052] The PNA regions used to detect target base sequences (mutant strains) were those described in Non-Patent Document 2. These PNA regions hardly react (hybridize) to wild strains but react well to mutant strains. Tables 3 and 4 show the composition of the reporter parts for each target base sequence. Note that the text in parentheses after "PNA" is the nucleic acid peptide (PNA region), and is represented from right to left from the N-terminus to the C-terminus. These are not polypeptides composed of phosphate bonds.

[0053] [Table 3]

[0054] Referring to Table 3, "Fam" is the light-emitting group 12, "Sp2, C7, bAla, C11, Ambz" are the reporter linker, "PNA (CTGCATGATTG)" is the PNA region for T790M, and "KK-Sp6-NH2" is the terminal portion 18.

[0055] [Table 4]

[0056] Referring to Table 4, "Fam" is the light-emitting group 12, "Sp2, C7, bAla, C11, Ambz" is the reporter linker, "PNA(TGGCCCGCCC)" is the PNA region for L858R, and "KK-Sp6-NH2" is the terminal portion 18.

[0057] As shown in Tables 3 and 4, the sequence names were determined by adding the first two or three letters of the reporter linker to the first two letters of the target DNA. In all sequences, the reporter linker is bound to the luminescent group (Fam), followed by the formation of the PNA region for detection. Subsequently, a series of lysine (K), Sp6 (see Table 2), and an amino group are bound as terminals. In Tables 3 and 4, the left side is the beginning of the sequence.

[0058] (2) Quencher parts The quencher part consists of a quenching group, a quencher linker, and a DNA region. DABCYL was used as the quenching group. The quencher linker 24 used is the one shown in Figure 2. Its linear carbon bond equivalent (egC) is 13. The DNA region of the quencher part corresponds to the PNA region of the reporter part. Therefore, it was designed separately for each reporter part. Table 5 shows the sequences of the quencher parts for each target base sequence. The quenching group and quencher linker are bound to the 3' end of the DNA region. Therefore, in Table 5, the right side is the beginning.

[0059] [Table 5]

[0060] Referring to Table 5, "DAB" is the quenching group 22, and "Linker" is the quencher linker 22. Here, it is the structure labeled 22 in Figure 2. "TCATGCAG" is the DNA region for T790M, and "GCGGGCCA" is the DNA region for L858R. No terminals were added to the quencher parts.

[0061] Refer to Table 5. Quencher parts are named by following the initial letter "Q" with the initial letter of the target gene.

[0062] <Detection Procedure> (1) Preparation of double-stranded PNA probe solution Double-stranded PNA probe solutions were prepared by mixing the reporter parts from Tables 3 and 4 and the quencher parts from Table 5 in the compositions shown in Table 6. Specifically, reporter parts T7Sp, T7C7, T7bAl, T7C11, and T7Am were mixed with quencher part QT, and reporter parts L8Sp, L8C7, L8bAl, L8C11, and L8Am were mixed with quencher part QL, respectively.

[0063] [Table 6]

[0064] The specific procedure involved thawing the reporter and quencher parts in a 1:1 ratio in PBS buffer. Next, to prevent nonspecific binding, the mixture was incubated at 95°C for 5 minutes, and then the temperature was gradually decreased using a gradient of -0.3°C / sec to 30°C.

[0065] (2) Reaction with the target base sequence 10 μL of the prepared double-stranded PNA probe solution (5 μM), 10 μL of the target gene sequence (mRNA in this case) (5 μM) from Table 1, and 80 μL of PBS were mixed (total volume 100 μL). After incubation at 37°C for 2 hours, fluorescence was measured using a fluorescence spectrometer.

[0066] (3) Fluorescence Spectrum Measurement After the reaction between the double-stranded PNA probe and the target gene sequence, the entire amount was immediately transferred to a quartz cell, and the fluorescence spectrum was measured using a Hitachi High-Tech Science F-7000 spectrofluorometer. The measurement equipment and conditions were as follows.

[0067] [Table 7]

[0068] (4) Measurement results Figures 4 and 5 show the measurement results. In Figure 4, the tested nucleotide sequence (target gene sequence) is T790M, and in Figure 5, the tested nucleotide sequence (target gene sequence) is L858R. In both graphs, the horizontal axis shows the type of target nucleotide sequence and the type of reporter linker, and the vertical axis shows the fluorescence intensity. The fluorescence intensity is calculated by setting the emission amount for "no target" to 1 and taking the ratio of the emission amount when hybridized with the target DNA. The control is Sp2.

[0069] Referring to Figures 4 and 5, there are three types of target sequences: "no target," "wild type," and "mutant." "No target" is the case where the target mRNA is not included; instead of mixing 10 μL of target RNA (5 μM) with the double-stranded PNA probe solution, 10 μL of PBS is added. "Wild type" and "mutant" are shown in Table 1.

[0070] Refer to Figure 4. For the T790M base sequence, a double-stranded PNA probe using the reporter part (Table 3) and the quencher part QT (see Table 5) was used. With reporter linkers C7, bAla, C11, and Ambz, the fluorescence intensity was very high for the mutant. This indicates that the detection sensitivity for the mutant is very high. Furthermore, the brightness difference between emission and quenching was orders of magnitude higher compared to the conventional example (control) Sp2.

[0071] In this case, the linear carbon bond equivalent value for Sp2 is 7, which is not significantly different from C7 or C11. However, the fluorescence intensity of the reporter linker Sp2 was clearly lower than that of the other linkers. This is because, when the reporter linker is Sp2, the luminescence is not sufficiently suppressed during quenching. This is thought to be because the reporter linker Sp2 has two oxygen atoms in its main chain.

[0072] On the other hand, Ambz, which has a linear carbon bond equivalent value of 5, also showed very high fluorescence intensity. This contradicts the conventional understanding that bringing the luminescent group and the quenching group closer together contributes to sufficient quenching.

[0073] Refer to Figure 5. For the L858R base sequence, a double-stranded PNA probe using the reporter part and quencher part QL (see Table 5) from Table 4 was used. The fluorescence intensity was higher for the mutant, indicating high sensitivity for detecting the mutant. Furthermore, the brightness difference between emission and quenching was orders of magnitude higher compared to the conventional example (control) Sp2.

[0074] Here too, high fluorescence intensity was observed for linear carbon bond equivalent values ​​egC between 2 and 11. Furthermore, in the case of L858R, the fluorescence intensity was higher for egC7, egC2, and egC5 than for C11, which is closer to the quencher linker (egC=13).

[0075] As described above, in the case of double-stranded PNA probes, it was found that intentionally differentiating the structures of the reporter linker and quencher linker increases the fluorescence intensity when hybridizing with target DNA.

[0076] Furthermore, the double-stranded PNA probe for T790M and L858R according to the present invention can improve detection sensitivity by changing the reporter linker type from the conventionally used Sp2, thereby increasing the fluorescence intensity when reacting with the target base sequence compared to the quenching intensity. This indicates that single nucleotide polymorphisms can be detected with higher sensitivity than before. [Industrial applicability]

[0077] The double-stranded PNA probe according to the present invention can achieve high detection power when detecting target base sequences. It is particularly effective when detecting single nucleotide polymorphisms (T790M and L858R mutations) specific to the mRNA sequence of the epidermal growth factor receptor (EGFR) in lung cancer cells. [Explanation of symbols]

[0078] 1. Double-stranded PNA probe 10 Reporter Parts 12. Luminescent group 14 Reporter Linker 16 PNA area 18 Terminal part 20 Quencher Parts 22 Quenching group 24 Quencher Linker 26 DNA region 50 DNA sequences to be tested 52 Target base sequences

Claims

1. Quenching agent, DNA region and Includes a quencher linker connecting the quenching group and the DNA region. Quencher parts, A light-emitting group, PNA region and Includes a reporter linker connecting the light-emitting group and the PNA region. Having reporter parts, A double-stranded PNA probe in which the quenching group and the light-emitting group are arranged on the same side when the DNA region and the PNA region are complementaryly bound, A double-stranded PNA probe characterized in that the reporter linker does not contain two or more oxygen elements in the main chain, the quencher linker has 13 linear carbon bond values, and the reporter linker has 3 to 11 linear carbon bond values.

2. The DNA targeted by the PNA region is characterized in that, as described in 1, The double-stranded PNA probe shown.

3. The DNA targeted by the PNA region is the epidermal growth factor receptor (EGFR) of lung cancer cells. The characteristic of being a variant of T790M or L858R, whichever is correct. A double-stranded PNA probe as described in claim 1.

4. The reporter linker is C7(N-[-(CH ２ ) ７ -]-CO), bAla(N -[-CH ２ CH ２ CO-]-CO)、C11(N-[-(CH ２ ) １１ -]-CO)、 Ambz (N-[-CH ２ C ６ H ４ -]-CO) is any of the claims characterized by A double-stranded PNA probe as described in any one of claims 1 to 3.

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