KR102012690B1 - Nested hybridization pna probe system having parallel binding structure - Google Patents
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Abstract
The present invention relates to a PNA (Peptide Nucleic Acid) probe system for amplifying two real-time nucleic acids of a parallel binding structure capable of double hybridization to a target nucleotide, a composition comprising the probe system, and a detection method using the same. By using the composition comprising the PNA probe system according to the present invention, not only the presence of nucleotides to be detected can be confirmed or quantitatively determined, but also sequence mutations, in particular, single nucleotide sequence mutations that have been difficult to detect by conventional techniques. Single Nucleotide Polymorphism (SNP) can be detected with excellent sensitivity, and at least two or more kinds of nucleotides present in a sample can be detected simultaneously.
Description
The present invention relates to two PNA (Peptide Nucleic Acid) probe systems having a parallel binding structure capable of double hybridization to a target nucleotide, a composition comprising the probe system, and a detection method using the same. The composition comprising the PNA probe system can not only confirm the presence of the nucleotide to be detected or quantitatively determine the amount of the nucleotide to be detected, but also sequence variations, in particular, single nucleotide sequence mutations that were difficult to detect by conventional methods. Nucleotide Polymorphism) can be detected with excellent sensitivity, and it has the advantage of simultaneously multi-detecting at least two or more kinds of nucleotides present in a sample.
Molecular diagnostics is an effective method for analyzing human genes (DNA or RNA) to diagnose disease infection or to identify sequencing or mutations of genes to predict and identify disease outbreaks. In particular, it is considered as the best technology among the existing disease diagnosis methods, and it is one of the technologies currently attracting attention in the medical field.
Various detection methods are used for molecular diagnosis. Typical examples include methods using real-time PCR, methods using DNA-based probes, methods using PNA-based probes, and the like. The features of each method are briefly described below.
Molecular diagnostics using real-time PCR : Real-time PCR analysis involves the PCR amplification product generation process through polymerase chain reaction (PCR) and the intensity of the fluorescence signal in real time by combining with primers or probes labeled with fluorescent material. By showing, more accurate quantitative analysis is possible. The target nucleotide detection method used for real time PCR can be divided into two types. The first is primer-based detection, which has the disadvantage of difficulty in design and quantitative analysis. The second method is a probe-based detection method, which is convenient in design and can be applied to both quantitative and qualitative analysis. The advantages and disadvantages of the two detection methods are shown in Table 1 [Meti Buh Ga, et al., Anal. Bioanal. Chem. 396, 2023, 2010].
Detection of target nucleotides using DNA- based probes : There are two main types of DNA- based probes that are widely used. TaqMan probes are linear probes that combine a reporter molecule and a quencher molecule at the ends of a DNA sequence capable of complementarily binding to a target nucleotide, and enzymatic cleavage of the probe sequence bound to the target nucleotide. It is a method of detecting a signal of a fluorescent (reporter) material that deviates from [Holland, PM, et al. Nat'l Acad. Sci. USA, 88, 7276-7280, 1991; Livak, KJ, et al., PCR Methods Appl., 4, 357-362.
This method has the disadvantage of lowering the discrimination ability of single nucleotide sequence mutations, and thus, MGB tags have been shortened by introducing a minor groove binder (MGB) with a matte material at the 3 'end for the purpose of improving the discrimination ability of single nucleotide sequences. TaqMan has also been developed by Igor VK, et al, Nucl. Acids Res. 25, 3718-3723, 1997; Igor V. K., et al, Nucl. Acids Res. 28 (2): 655-661, 2000; I. A. Afonina, ea al, BioThechniques, 32, 940-949, 2002; I. A. Afonina, ea al, Nucleic Acids Research, 25, 2657-2660, 1997].
Molecular Beacons (MB, Molecular Beacon) is a new type of probe consisting of a stem (stem) structure to form a loop and hairpin structure of the base sequence complementary to the target nucleotide. While this method has the advantage of distinguishing single nucleotide sequence variation, it is difficult to design and synthesize probes [US 20080064033 A; S. Tyagi, et al., Nat. Biotechnol., 16, 49, 1998; Stryer, L., Ann. Rev. Biochem., 47, 819-846, 1987; S. Tyagi, et al., Nat. Biotechnol., 14, 303-308, 1996; Bonnet, G., Proc. Natl Acad. Sci. USA, 96, 61716176, 1999].
However, DNA probe-based detection methods make DNA less stable by damage by enzymes such as nucleases and proteases [Demidov et al., Biochem. Phamacol. 48, 1310-1313, 1994], as well as weak DNA-DNA binding ability due to the charge repulsion between negative charges of the DNA backbone and low single nucleotide sequence discrimination ability due to the use of long sequences to overcome it. There are disadvantages.
Detection of target nucleotides using PNA- based probes : In order to compensate for the shortcomings of using DNA probes, methods using PNA, an analog of DNA, have been studied. Since PNA has no charge in its backbone, it has less repulsion in binding to negatively charged complementary DNA oligomers, which allows it to bind to target nucleotide sequences faster and stronger than DNA probes. Egholm et al., Nature 365, 556-568, 1993 ,; Nielsen et al., Bioconjugate Chem, 5, 3-7, 1994; Demidov, et al., Biochem. Pharmacol. 48, 1310-1313, 1994].
Recently, a new method using a dual linear probe structure has been reported. This method uses short secondary probes to improve the discrimination ability of low single nucleotide sequences, which is a disadvantage of long sequence DNA probes. The two probes are designed to form anti-parallel binding to each other. James M. Coull, et al., US 6607889.
Recently, various molecular diagnostic techniques have been developed, but there is still a demand for molecular diagnostic techniques with sufficient sensitivity and specificity. In particular, single base sequence mutations can be detected within a short analysis time with high sensitivity and specificity. There is an urgent need for technological development.
The present invention is to provide a PNA-based real-time PCR analysis method that can accurately detect the target nucleotide in the sample by overcoming the disadvantages of such molecular diagnostic technology.
In the present invention, two real-time nucleic acid amplification PNA probes capable of double hybridization to a target nucleotide are used to detect the presence or amount of the target nucleotide or to detect sequence variation and present in a sample. Using a method of multiple detection of at least two or more kinds of nucleotides at the same time and excellent discrimination ability against a single nucleotide sequence, a new method for quickly and accurately detecting a target nucleotide having a plurality of single nucleotide sequences is proposed.
The PNA probe according to the present invention can be applied to all technologies that can be applied to existing TaqMan probes or molecular beacons (MB, Molecular Beacon), that is, a detection method, and can detect specific sequences, in particular, multiple detection ( multi-detection and quantification, for example, fluorescence in situ hybridization (FISH), etc. may be used, but this is just one example, but is not limited thereto and all other possible methods may be used. Will be apparent to those of ordinary skill in the art.
Other objects and advantages of the present invention will become apparent from the following description, the means for solving the problems and the detailed description of the invention.
In the present invention, in order to overcome the limitations of the stability of the existing DNA-based real-time PCR probes have developed a detection system using a PNA having thermal and biological stability, superior to the ability to recognize and bind to target nucleotides than DNA . As is already known, probe-based detection generally has many advantages over primer-based detection in detecting target nucleotides. Probes used at this time are largely divided into two types, and the pros and cons of each probe are compared in Table 2.
In Table 2, the structured probe is known to have excellent detection specificity against single nucleotide sequence mutations, but unless designed to have a stable hairpin structure due to the binding force of the stem, quenching may be incomplete and generate nonspecific fluorescence. Can be.
Therefore, design and synthesis are difficult because the probe must be manufactured in consideration of the binding energy difference between the binding energy of the stem and the target nucleotide. On the other hand, the linear probe has various advantages including convenience of fabrication, but has a disadvantage in that the detection ability against a single nucleotide sequence variation is poor due to the absence of a stem.
Therefore, the inventors of the present invention have attempted to fabricate a PNA probe system having both the advantages of a linear probe, which is easy to design and synthesize, and a molecular beacon, which exhibits high detection of single nucleotide sequences.
PNAs can hybridize with PNAs having complementary sequences in two forms, anti-parallel binding and parallel binding [FIG. 1], and the binding energy between them is shown in [FIG. 2] [Stefano Sforza, Eur. J. Org. Chem., 197-204, 1999]. Due to this difference in binding energy, the dual linear PNA probes having parallel binding sequences in the absence of target nucleotides in the sample do not fluoresce through complementary binding to each other.However, if the target nucleotides are present, the detection probes can achieve more stable complementary binding with the target nucleotides. In addition, fluorescence is generated by dissociation between the existing PNA-PNA probes.
Therefore, in the present invention, the first PNA probe is synthesized according to the target Tm, and the binding strength between the two PNA probes is completely complementary to each other in the sequence of the PNA-DNA by using the parallel binding, which is relatively weak in binding strength and easy to control. The second PNA probe was designed and synthesized such that some of the sequences of perfect-match and PNA-DNA were intermediate between different incomplete complementary mismatches. By using these two PNA linear probes to have a stem function, a detection system that is convenient for design and synthesis is developed because the sequence is not limited to improve the detectability of single nucleotide sequence mutations (see FIG. 3). .
The PNA probe according to the present invention has a form in which a reporter material and a quencher material are bound to one or both ends of a PNA oligomer having a predetermined sequence.
PNA probe according to the invention is preferably in the form of a combination of the physical properties control site and / or reporter material and the matting material at both ends, such as the structure of formula (1), but is not limited to this, to achieve the object of the present invention It will be apparent to those skilled in the art that any PNA probe structure having any structure can be used.
In Formula 1, P is a PNA base moiety having a sequence complementary to a target nucleotide, and N in the subscript is the number of PNA bases, preferably an integer of 7 to 25, more preferably an integer of 8 to 18. It is a part which forms parallel binding or anti-parallel binding to a target nucleotide. A and A ′ may be the same or different materials as reporter molecules or quencher molecules, or only one of them may be present. X and X 'may be the same or different materials as the physical property control site, none may be included, and one or more may be included. N ' and C' mean N -terminal and C -terminal, respectively.
Particularly, the PNA base portion P may have a structure as shown in
In
The PNA probe developed in the present invention is a dual linear structure that forms parallel bonds, and is easy to design and synthesize, and makes accurate detection by fast complementary binding with a target nucleotide without a non-specific signal. It is possible to form a double helix structure, which makes it relatively easy to design a sequence for detecting a single nucleotide sequence mutation and to simultaneously analyze adjacent single nucleotide sequences.
In addition, the detection method according to the present invention can increase the detection efficiency by introducing a reporter material into not only a PNA probe for detection but also a parallel binding PNA probe, and is applicable to a quantitative method for detecting a single nucleotide sequence mutation, genotyping, In addition to quantitative and single nucleotide sequence detection, it is easily applicable to detection of target nucleotides using multiplex PCR.
The PNA probe according to the present invention can be applied to all technologies that can be applied to existing TaqMan probes or molecular beacons (MB, Molecular Beacon), that is, a detection method, and can detect specific sequences, in particular, multiple detection ( multi-detection and quantification, for example, fluorescence in situ hybridization (FISH), etc. may be used, but this is just one example, but is not limited thereto and all other possible methods may be used. Will be apparent to those of ordinary skill in the art.
1 is a diagram showing a parallel binding and anti-paralle binding structure between PNA-PNA.
2 shows the relative binding strength of PNA-PNA and PNA-DNA.
3 shows a method for detecting a target nucleotide using a dual hybridized PNA probe system.
(a) When a first PNA probe is used as a detection probe in the detection method of the target nucleotide using the double hybridized PNA probe system of a parallel binding structure.
(b) The second PNA probe is used as a detection probe in the detection method of the target nucleotide using the double hybridized PNA probe system of a parallel binding structure.
4 shows the stability of the probe.
(a) PCR results immediately after mixing the first and second probes.
(b) PCR results after 6 months of storage at room temperature with the first and second probes mixed.
5 shows the increase in fluorescence intensity through double fluorescent labeling.
(a) PCR was performed by mixing the first probe with the second unfluorescent label.
(b) PCR was performed by mixing the first and second fluorescently labeled probes.
6 is a view showing a calibration curve and a result of performing PCR for confirming the detection sensitivity of warfarin single nucleotide sequence detection and the applicability of the quantitative method in relation to Example 8 using the method of the present invention.
7 is a view showing a calibration curve and a result of performing PCR for confirming the ITS gene detection sensitivity and the applicability of the quantitative assay in relation to Example 9 using the method of the present invention.
8 shows PCR results using DNA probes.
(a) PCR results using wild type and single sequence variants of the warfarin metabolism related gene CYP2C9 430 gene using a DNA probe (○ indicates a wild type gene detection line and × indicates a detection line of a single nucleotide sequence variant).
(b) PCR results using wild type and single nucleotide sequence variants of the warfarin metabolism related gene VKORC1 3730 gene using a DNA probe (○ indicates a wild type gene detection line, and × indicates a detection line of a single nucleotide sequence variant).
(c) PCR results using wild-type and single nucleotide variants of the warfarin metabolism-related gene CYP2C9 430 gene using a PNA probe (○ indicates a wild-type gene detection line, and a straight line indicates a detection line of a single nucleotide sequence variant).
(d) PCR results using wild type and single nucleotide sequence variants of the warfarin metabolism related gene VKORC1 3730 gene using a PNA probe (○ indicates a wild type gene detection line, and a straight line indicates a single nucleotide sequence detection line).
9 is Mycobacterium tuberculosis using different fluorescently labeled PNA probes. tuberculosis, MTB) and the non-tuberculosis mycobacteria (Non - tuberculous mycobacteria, a view showing a PCR result of NTM) for each specific simultaneously detected (○ is a detection line of Mycobacterium tuberculosis, △ means a detection line of the non-tuberculosis mycobacterial ).
10 is a diagram showing the change in melting temperature for a single nucleotide variant by a second probe (right peak represents the melting temperature of the wild type, the left peak represents the melting temperature of the single sequence variant).
Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are intended to illustrate the present invention in more detail, it is clear that the scope of the present invention is not limited to the examples.
The present invention relates to a method for detecting the presence or amount of a target nucleotide present in a target sample or detecting sequence variation using a PNA-based real-time PCR probe.
Definitions of terms and abbreviations used in the present invention are as follows.
Hybridization: A state in which complementary base pairs form a double helix structure through hydrogen bonding.
Parallel binding: N-terminal (N-terminal) forms a complementary relationship in the same direction when a pair of PNAs are hybridized. In the case of DNA, 5'-end is It means the form of complementary binding in the same direction, and when the PNA and DNA are hybridized, it means the form in which the N-terminal of the PNA and the 5'-terminal of the DNA form a complementary relationship in the same direction.
Anti-parallel binding: N-terminal forms complementary binding in opposite directions when a pair of PNAs are hybridized. In the case of DNA, the 5'-terminal complementary relationship is opposite to each other. When the PNA and DNA are hybridized, the N-terminus of the PNA and the 3'-end of the DNA form a complementary bond in the same direction.
Complementary bond: refers to a bond in which the base (A, T, G, C) forms a double strand structure through hydrogen bonding, and in the present invention, 5 'of a single strand forming a double strand. In addition to the antiparallel bonds in which the bases in the complementary relationship are hydrogen-bonded in the opposite directions, the parallel bonds in which the bases in the complementary relationship are hydrogen-bonded in the state in which the 5'-ends face the same direction. It also means.
Double hybridization: means that two PNA probes bind to sense and anti-sense DNA, respectively, to form two double helix structures.
Reporter molecule (reporter molecule): A material that absorbs and emits light of a specific wavelength and emits light, and refers to a material capable of labeling a probe and confirming whether hybridization between the target nucleic acid and the probe has been performed.
Quencher molecule: A material that absorbs light generated by a reporter material and reduces fluorescence intensity.
Physical property control site: means a material for controlling the solubility of a probe, such as a linker or a spacer, or for labeling a reporter material or a quenching material, such as between a PNA and a fluorescent or quenching material Linkers to facilitate linkage, spacers to control distance, materials for improving solubility and binding to target nucleotides known in the art, and the like. Linkers are described in Akira Kishimoto, Chem. Commun., 742 743, 2003; Peter E. Nielsen, Chem Bio Chem, 6668, 2005; Vladimir Guelev, JACS, 2864-2865, 2002; Ethan A. Englund and Daniel H. Appella, Organic Lett., 3465-3467, 2005 and the like can be used, but are not limited to such spacers, OlafKchler, ChemBioChem, 6977, 2005; Liisa D., J. Med. Chem., 2326-2340, 2007 and the like can be used, but not limited to, materials used for controlling solubility and binding strength include Irina V. Smolina, Vadim V. Demidov, Nucleic Acids Research, e146, 2005; I.S. Blagbrough, Biochemical
Isomer Substituents: Compounds that have the same molecular formula and method of linking members but have different spatial arrangements between atoms are called isomers, and are usually present in the case of carbon compounds in which all four atomic groups linked to carbon have different asymmetric carbons. That is, two different kinds of isomers are formed according to the three-dimensional arrangement of the substituents, and the isomeric substituents in the present invention mean substituents that form only one isomer in one direction. In
Single nucleotide sequence variation (SNP): One DNA sequence of a particular gene means different, and includes both germline and somatic mutations.
Structured probe: refers to a probe that forms a secondary structure.
Linear probe (linear probe): oligonucleotide labeled 5 'end with a fluorescent material, 3' end with a matting material, means a probe that does not form a secondary structure because there is no stem.
Double linear probe: A type of probe in which two linear oligonucleotides in which a reporter material and a quencher material are bonded to each other form a complementary bond with each other.
Perfect match: When two strands of DNA or PNA hybridize, it means that the base pairs in complementary relationship are perfectly matched.
Incomplete Complementary Mismatch (mis-match): When two strands of DNA or PNA hybridize, one or more base pairs in complementary relationship do not match.
FAM: 6-Carboxyfluorescein
Dabcyl: 4,4-Dimethylamino-azobenzene-4'-carboxylic acid
Black Hole Quencher (BHQ TM ): A matte material sold by Biosearch Technologies Inc. (USA), classified into BHQ1, BHQ2 and BHQ3 according to the structure and wavelength difference.
Blackberry Quencher: A matting material sold by Berry & Associates, USA, having the following structure:
Fluorescence in situ hybridization (FISH): To determine the presence or absence of specific nucleotides, the cells are plated on slides without culturing or extracting nucleic acids and retaining their chromosome or nucleus form. Fluorescence of chromosomes or genes is identified by reacting different types of probes with fluorescent materials to gene recognition materials (DNA, PNA, or other modified DNA) complementary to specific sequences of the target gene. It means the method of observing with a microscope. A test procedure for attaching a probe to a chromosome is called hybridization, and a probe attached to a chromosome fluoresces when exposed to ultraviolet light, and indicates a presence and location of a target nucleotide.
[ Example One] PNA Of probe Design and build
The PNA probe of the present invention was designed to specifically bind to the IS6110 gene of Mycobacterium tuberculosis and the ITS gene of Non-TB tuberculosis. In addition, it was designed and manufactured to perfectly bind to warfarin metabolism related genes CYP2C9 430 and VKORC1 3730 wild type gene and single nucleotide sequence gene.
For example, the PNA probe of the present invention may be composed of any one of SEQ ID NOs: 1 to 14 shown in Table 3 below. It will be appreciated that all of the PNA probe sequences within the range that can be easily modified by those skilled in the art from the above nucleotide sequences are within the scope of the present invention. As long as the PNA probe system capable of parallel binding can detect a target nucleotide using PNA real-time PCR according to the present invention, it is included within the scope of the present invention.
In Table 3, O is a linker, bold letters and underlined letters are γ-lysine (γ-lysine) or γ-glutamic acid-PNA monomer (monomer), K is lysine (lysine), (+) is aeg [ N- (β-alanine)], (-) means aeg [ N- (succinicacid)].
PNA probes were synthesized by solid phase synthesis from a PNA monomer protected with benzothiazolesulfonyl (Bts) and functionalized resin according to the method described in Korean Patent No. 464,261 [Lee et al. , Org. Lett., 2007, 9, 3291-3293]. In addition to this method, PNA can also be synthesized using known 9-fluorenylmetholoxycarbonyl (Fmoc: 9-flourenylmethloxycarbonyl) or t-Boc (t-butoxycarbonyl) synthesis methods [Kim L. et al., J. Org. . Chem. 59, 5767-5773, 1994; Stephen A. et al., Tetrahedron, 51, 6179-6194, 1995]. Reporter materials and quenching materials were labeled on the PNA probe according to methods well known in the art.
[ Example 2]: CYP2C9 430, VKORC1 3730 and tuberculosis / Non-tuberculosis To amplify the target nucleic acid primer Synthesis and Use
In the present invention, warfarin metabolism-related genes CYP2C9 430, VKORC1 3730 and Mycobacterium tuberculosis, MTB) and the non-tuberculosis mycobacteria (Non - analyze the region of each gene to a target nucleic acid amplification of the tuberculous mycobacteria, NTM) gene to prepare a primer so that the specific amplification achieved. The primer sets SEQ ID NO: 15, 16 for the CYP2C9 430 gene identification and the primer set SEQ ID NO: 17, 18 for the VKORC1 3730 gene identification were designed. In addition, M. tuberculosis IS6110 sequence number to the identification of gene (Mycobacterium tuberculosis, MTB) 19, 20 primer sets and non-tuberculosis mycobacteria (Non - tuberculous To identify the mycobacteria (NTM) gene ITS (Internal transcribed spacer), a primer set of SEQ ID NOs: 21 and 22 was designed. The designed primers were used by Synthetic Co., Ltd. (Korea).
In Table 4, Y means a mixed base of C and T.
[ Example 3] Warfarin Metabolic genes CYP2C9 430 and VKORC1 3730 clones acquired
To obtain clones of CYP2C9 430 and VKORC1 3730, amplification products were purified using a combination of SEQ ID NOs: 23, 24, and 25 and 26, respectively, using Labopass TM PCR purification kit (Cosmogenetech, Korea), and then pGEM-T. A large amount of DNA was obtained by binding to Easy Vector (Promega, USA) and transforming E. coli JM109 cells. In order to obtain a single nucleotide sequence variant, using a normal clone prepared by the above method, using a site-specific mutagenesis kit (stratazine, USA) to obtain a clone with a mutant gene and confirm the mutation by sequencing It was. The genotype confirmed clone was used as a standard in gene amplification of the present invention.
[ Example 4] Mycobacterium tuberculosis gene IS6110 And Non-tuberculosis Antibacterial bacteria gene ITS Secure clones
Mycobacterium to secure clones for target nucleic acids tuberculosis {ATCC 25177, US} and Mycobacterium asiaticum {KCTC 9503, Korea Life Resource Center, Korea}. DNA was extracted from InstaGene Matrix (BioRad, USA) from the strains that were distributed, and Mycobacterium was combined with the combinations of SEQ ID NOs: 27, 28, and 29, 30, respectively. tuberculosis (MTB) amplified IS6110 gene and ITS gene of non -tuberculous mycobacteria (NTM). The amplification product was purified using Labopass ™ PCR purification kit (Cosmogenetech, Korea), then bound to pGEM-T easy vector (Promega, USA) and transformed into E. coli JM109 to bulk DNA. Secured.
[ Example 5] real time PCR reaction
Using the clones obtained by the method of Examples 3 and 4 above, a real-time detection method using a PNA probe was established. 2 μl of template DNA (10 5 copies / μl), 10 μl of the mixed solution of the first probe (5 pmoles / μl) and the second probe (10 pmoles / μl), primer set (SEQ ID NOs: 15, 16 sets or 17, 18 Set or 19, 20 or 21, 22 sets,
[ Example 6] parallel coupling PNA Based real time PCR Of probe stability
In order to test the storage stability of the first probe (SEQ ID NO: 2) and the second probe (SEQ ID NO: 5), real-time detection PCR is performed using the first probe and the second probe mixed solution according to the method of Example 5, respectively. After storage at room temperature for 6 months, real-time detection PCR was performed in the same manner and the effects were compared. The results are shown in FIG. As a result, it was confirmed that the fluorescence intensity did not decrease even when stored for 6 months at room temperature.
[ Example 7] the second On the probe Through the introduction of additional reporter materials Signal strength increase
Method of Example 5 using a mixed solution of the first probe of SEQ ID NO: 4 and the second probe of SEQ ID NO: 6 to measure the change in fluorescence intensity when the reporter material is introduced into the second probe as well as the first probe Real time detection PCR was performed. The results are shown in FIG. When the reporter material was introduced into the second probe, the fluorescence intensity increased by about 50% compared to the case where the reporter material was introduced only into the first probe.
[ Example 8] The first Of probe Single Base Sequence Variation Check sensitivity for detection and applicability of quantitation
A test was conducted to confirm the sensitivity and application of the quantitative method for the detection of a single nucleotide sequence mutation using a first PNA probe (SEQ ID NO: 1) and a second PNA probe (SEQ ID NO: 5). Detection limits were determined by diluting CYP2C9 430 wild-type and single nucleotide variant clones 10-fold copies from 10 9 copies / μl to 10 1 copies / μl, respectively, and detected up to 10 1 copies / μl. In addition, as a result of analyzing the correlation between the C T (cycle threshold) according to the number of copies of the single nucleotide sequence mutation gene, the detection C T value increases as the concentration of the standard decreases. It was confirmed that it can also be applied to quantification. The results are shown in FIG.
[ Example 9] The first Of probe Confirmation of Sensitivity and Detection of Target Nucleotide
A test was conducted to confirm the sensitivity and applicability of the target nucleotide detection using a first PNA probe (SEQ ID NO: 12) and a second PNA probe (SEQ ID NO: 13). The limit of detection was determined by diluting the
[ Example 10] PNA Probe And DNA Of probe Single Base Sequence Variation Detectability compare
Like PNA probes, DNA probes (tagman probes) are used to detect single nucleotide sequence mutations of the warfarin metabolism related genes CYP2C9 430 and VKORC1 3730 of SEQ ID NOs: 30 and 31. Was used by Bioneer Co., Ltd. (Korea) for synthesis (see Table 5). The corresponding PNA probes used SEQ ID NOs: 8, 9. The results of comparing the detection of single nucleotide sequence mutations using the respective probes are shown in FIG. 8. While both DNA probes for detecting a target gene did not detect a single nucleotide sequence, the PNA probe could reliably detect a single nucleotide sequence of two different target genes.
In bold font in Table 5 indicates a single nucleotide sequence position.
[Example 11] to each other mycobacteria using other reporter substance (Mycobacterium tuberculosis, MTB) and non-tuberculosis mycobacteria (Non - tuberculous simultaneous detection of mycobacteria (NTM)
PNA probes labeled with different reporter materials were used to detect whether two different types of target nucleic acids could be detected simultaneously. Mycobacterium according to SEQ ID NOs: 10 and 14 tuberculosis, MTB) specific PNA probe and SEQ ID NO: 11 and the non-tuberculosis mycobacteria according to 13 (Non - tuberculous mycobacteria, using the specific PNA probe NTM), was detected at the same time, the M. tuberculosis and non-tuberculosis mycobacteria are each specific, and the results are shown in Fig.
[ Example 12] Single nucleotide sequence For detection specificity
The first probe (SEQ ID NO: 3), which uses the warfarin metabolism related gene CYP2C9 430 as a target gene to confirm that the specificity of a single nucleotide sequence mutation can be increased when the first probe and the second probe are mixed, is used. After performing a real-time PCR reaction according to the method of Example 5 using a probe (SEQ ID NO: 7), the melting curve analysis to measure the fluorescence while increasing by 0.5 ℃ from 25 ℃ to 95 ℃. The results are shown in FIG. There was no change in melting temperature for the wild type with and without the second probe. However, when the second probe was mixed together, it was confirmed that the melting temperature of the single nucleotide sequence variation decreased by about 4 to 6 ° C. That is, when the second probe is used together due to the melting temperature drop for the single nucleotide sequence variation, it was confirmed that the detection specificity for the single nucleotide sequence variation was improved.
Sequence List Free Text
SEQ ID NOS: 1-14 are sequences of PNA probes according to the present invention.
SEQ ID NOs: 15 to 30 are sequences of primers according to the invention.
SEQ ID NOs: 31 and 32 are sequences of DNA probes according to the invention.
<110> PANAGENE INC. <120> Parallel binding structured PNA probe system <160> 32 <170> KopatentIn 1.71 <210> 1 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> IMB-3 PNA probe <400> 1 acacggtcct caa 13 <210> 2 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> IMB-1 PNA probe <400> 2 acacggtcct caa 13 <210> 3 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> DFP-0207-26 PNA probe <400> 3 acacggtcct caak 14 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DFP-0207-22 PNA probe <400> 4 kkacacggtc ctcaakkk 18 <210> 5 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Q-B-13 PNA probe <400> 5 tgtgccagga gtt 13 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IMB-0125-2 PNA probe <400> 6 kktgtgccag gagttkkk 18 <210> 7 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> DFP-0208-10 PNA probe <400> 7 tgtgccagga gttk 14 <210> 8 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Warfarin-W-4 PNA probe <400> 8 aacacggtcc tc 12 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> 3730-M1-2 PNA probe <400> 9 atgtgtgggt 10 <210> 10 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> TB-10608-2 PNA probe <400> 10 ttcgcctacg tg 12 <210> 11 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> ITS-20608-1 PNA probe <400> 11 gtgtggtgtt tga 13 <210> 12 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> ITS-20608-3 PNA probe <400> 12 gtggtgtttg a 11 <210> 13 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> ITS-20608-4 PNA probe <400> 13 cacaccacaa actkkk 16 <210> 14 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> TB-10616-1 PNA probe <400> 14 agcggatgca ck 12 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 15 gctgcggaat tttgggatgg 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 16 gatgtggggc ttctagatta cc 22 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 gatgtggggc ttctagatta cc 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 tgtaaaaaag agcgagcgtg tg 22 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 19 cgcttcggac caccagca 18 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 20 caggatcctg cgagcgtag 19 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 21 ccacctccyt tctaaggagc acc 23 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 22 gggatgctcg caaccactat yca 23 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 23 gaagcctgtg tggctgaata 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 24 ccattcccac catgttgact 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 25 gcaaggctaa gaggcactga 20 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 26 accacagtcc atggcagac 19 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 27 tcgtccagcg ccgcttcg 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 28 cgggtccaga tggcttgc 18 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 29 gattgggacg aagtcgtaac aag 23 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 30 agcctcccac gtccttcatc ggc 23 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9-430W DNA probe <400> 31 cctcttgaac acggtcctca atgct 25 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> VKORC1-3730M DNA probe <400> 32 cattgtcatg tgtgggtatg gcagg 25
Claims (22)
Each PNA probe further comprises a physical control site at one or more ends.
Wherein each PNA probe further comprises at least one material selected from a reporter molecule and a quencher molecule.
The reporter material or the quencher is directly bonded to the PNA terminal, or a composition characterized in that it is bound through a physical control site connected to the PNA terminal.
The reporter material is fluorescein (fluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, rhodamine red, tetramethylrhodamine, FITC, oregon green (Oregon green), Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine-based dyes and thiadicarbocyanine dyes At least one fluorescent material selected from the group consisting of a composition.
The matting material is dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa Black RQ, IRDye QC- At least one selected from the group 1;
The physical control region bound to the PNA probe terminal is selected from the group consisting of hydrophilic residues, hydrophobic residues, ionic residues and hydrogen bond residues.
The physical control region bound to each of the PNA probe ends is an ionic moiety, characterized in that it comprises a positive charge, a negative charge or a zwitter ion.
Wherein said first PNA probe comprises at least two identical or different reporter materials.
Wherein each PNA probe further comprises one or more materials selected from reporter materials and matting materials.
Wherein each of the PNA probes further comprises a property control site between the PNA and the reporter material or the quencher material.
The reporter material or the quencher is bound directly to the PNA end, or detection method characterized in that it is bound through a physical control site connected to the PNA end.
The reporter material is fluorescein (fluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, rhodamine red, tetramethylrhodamine, FITC, oregon green (Oregon green), Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine-based dyes and thiadicarbocyanine dyes At least one fluorescent material selected from the group consisting of a detection method.
The matting material is dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa Black RQ, IRDye QC- At least one selected from the group 1.
The physical property control region bound to each of the PNA probe terminal is selected from the group consisting of hydrophilic residues, hydrophobic residues, ionic residues and hydrogen bond residues.
The physical property control region bound to each PNA probe terminal is an ionic moiety, and includes a positive charge, a negative charge, or a zwitter ion.
Wherein the first PNA probe comprises two or more identical or different reporter materials.
Detection of the target nucleotides in the sample is characterized in that the detection using fluorescence in situ hybridization (FISH).
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| PCT/KR2012/005458 WO2013009070A2 (en) | 2011-07-12 | 2012-07-10 | Nested hybridization pna probe system having parallel binding structure |
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| KR102008454B1 (en) * | 2012-08-28 | 2019-08-08 | 주식회사 파나진 | Composition for detecting Hepatitis C Virus(HCV) genotypes comprising parallel binding structured PNA probe system and detection method using thereof |
| EP3126511B1 (en) | 2014-04-04 | 2019-06-19 | Panagene Inc. | Melting curve analysis using pna probe, method and kit for analyzing nucleotide polymorphism using melting curve analysis |
| WO2016122198A1 (en) * | 2015-01-30 | 2016-08-04 | 주식회사 시선바이오머티리얼스 | Method for solving problem of nucleic acid detection due to threshold cycle (ct) of real-time polymerase chain reaction using pna probe |
| EP3498844B1 (en) * | 2016-08-09 | 2022-09-28 | Seasun Therapeutics | Peptide nucleic acid complex having improved cell permeability and pharmaceutical composition comprising same |
| KR20200045212A (en) * | 2018-10-22 | 2020-05-04 | (주)바이오니아 | A Probe linked with Octamine or Octamine Derivatives and Use of the Same |
| EP4284945A1 (en) * | 2021-01-26 | 2023-12-06 | 10X Genomics, Inc. | Nucleic acid analog probes for in situ analysis |
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|---|---|---|---|---|
| WO1997045539A1 (en) | 1996-05-31 | 1997-12-04 | Mikael Kubista | Probe for analysis of nucleic acids |
| US6432642B1 (en) | 1999-01-15 | 2002-08-13 | Pe Corporation (Ny) | Binary probe and clamp composition and methods for a target hybridization detection |
| KR100537979B1 (en) | 1999-12-21 | 2005-12-21 | Ingeneus Corp | Fluorescent intensity assay for duplex and triplex nucleic acid hybridization in solution utilizing fluorescent intercalators |
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| AU1366299A (en) * | 1997-10-27 | 1999-05-17 | Boston Probes, Inc. | Methods, kits and compositions pertaining to pna molecular beacons |
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Patent Citations (3)
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|---|---|---|---|---|
| WO1997045539A1 (en) | 1996-05-31 | 1997-12-04 | Mikael Kubista | Probe for analysis of nucleic acids |
| US6432642B1 (en) | 1999-01-15 | 2002-08-13 | Pe Corporation (Ny) | Binary probe and clamp composition and methods for a target hybridization detection |
| KR100537979B1 (en) | 1999-12-21 | 2005-12-21 | Ingeneus Corp | Fluorescent intensity assay for duplex and triplex nucleic acid hybridization in solution utilizing fluorescent intercalators |
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| Biosensors and Bioelectronics (2009) 25:803-808* |
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| KR20140053945A (en) | 2014-05-08 |
| WO2013009070A9 (en) | 2013-05-16 |
| WO2013009070A3 (en) | 2013-03-28 |
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