KR102266987B1 - Method for Ensuring Quality of Synthetic Oligo Using Peptide Nucleic Acid Probe - Google Patents

Abstract

The present invention relates to a quality assurance method of an artificial synthetic oligo using a PNA probe, and more particularly, a melting curve analysis method using a PNA probe coupled with a reporter and a quencher for determining the terminal base denaturation of a target artificial synthetic oligo, A method for determining base denaturation of a target artificial synthetic oligo using the kit, a kit for detecting base denaturation of a target artificial synthetic oligo including a PNA probe, a quality inspection method of an artificial synthetic oligo using the kit, and a base mutation of an artificial synthetic oligo using the kit It relates to a detection method.
In the case of using the quality inspection method of artificially synthesized oligos using a PNA probe coupled with a reporter and a quencher according to the present invention, whether there is an abnormality in the synthetic oligos of the genetic diagnosis product and the synthesis of artificially synthesized oligos of DNA or RNA structure Since abnormal synthesis results such as nucleotide sequence abnormalities that can occur due to problems can be checked within a short time, it is very convenient to inspect the quality of artificially synthesized oligos and to manage genetic diagnostic products, and is useful for monitoring the quality of artificially synthesized oligos. can be

Description

Method for Ensuring Quality of Synthetic Oligo Using Peptide Nucleic Acid Probe

The present invention relates to a quality assurance method of an artificial synthetic oligo using a PNA probe, and more particularly, a melting curve analysis method using a PNA probe coupled with a reporter and a quencher for determining the terminal base denaturation of a target artificial synthetic oligo, A method for determining terminal nucleotide denaturation of a target artificial synthetic oligo using the kit, a kit for detecting terminal nucleotide denaturation of a target artificial synthetic oligo including a PNA probe, a quality inspection method of an artificially synthesized oligo using the kit, and a mutation of an artificially synthesized oligo base ) to the detection method.

Starting with the confirmation of the DNA structure by James D. Watson & Francis Crick in 1950, in the year 2000, it was used in various fields of molecular biology based on a large-capacity automatic DNA synthesizing machine. For example, it is used for DNA hybridization probes, primers for DNA sequencing, DNA ampification, and Oligos for gene synthesis using oligos. Among them, primers and probes are the most actively used and studied among artificially synthesized oligos, and are being utilized and used in various diagnostic fields. In the case of artificially synthesized oligos, they are all manufactured on an automatic DNA synthesis machine, and capping is performed using acetic anhydride and N-methylimidazole to control abnormal synthesis during synthesis. ) step in progress. This capping step has the advantage of preventing deletion of the synthetic oligo to be produced as a result. In addition, since synthesis proceeds based on the 3' end of all automatic DNA synthesis machines used up to now, it is practically impossible to replace the base at the 3' end of the initially synthesized oligo. However, the sequence of the nucleotide sequence is controlled by the machine's program, and the probability of abnormal synthesis in the event of a machine problem is very high.

Among the DNA used in diagnosis, probes and primers play a very important role in reading the results. In the case of a probe, it is used to check the presence or absence of a specific gene, and in the case of a primer, it is necessary for amplification of a specific gene. However, when abnormal synthesis occurs during the production of synthetic DNA oligos (probes and primers), there is a problem in reading the results. In addition, in the case of synthetic DNA oligos, degradation occurs depending on the effects of storage temperature, acidity (pH), etc. according to long-term experiments, which also causes a problem in reading the results. In order to solve this problem, QC data (Quality Control data) of various raw materials used to read results is requested or repeated experiments are conducted.

QC (Quality Control) of the artificially synthesized DNA oligo consists of a step of confirming the number of synthetic bases and a step of confirming the exact base sequence of the synthesized base. As QC for checking the number of synthetic bases, polyacrylamide gel electrophoresis, HPLC analysis, and MALDI-TOF analysis are used. However, as a QC for confirming the correct nucleotide sequence, it is possible to amplify the synthetic oligo (PCR: Polymerase Chain Reaction, amplification), clone it again into a vector, and finally confirm it through sequencing. have. In fact, accurate sequencing of the synthesized oligo is impossible, and there are many problems in cost, time, and procedure. In addition, the operation for confirming the number of bases in the synthetic oligo also has the same problem in terms of time and cost.

In the case of PNA (Peptide Nucleic Acid) used for nucleotide polymorphism analysis, which is being studied recently, it is an artificial nucleotide and has excellent complementary binding ability with DNA or RNA. In addition, it has excellent biological stability against nucleolytic enzymes and proteolytic enzymes, and has superior chemical stability and water solubility compared to other artificially synthesized oligos.

Among them, the biggest characteristic of PNA is that it is very stable to temperature and pH, unlike other synthetic oligos, so that once synthesized PNA is structurally almost unchanged. This advantage can be used as a good raw material for confirming nucleotide polymorphism.

Moreover, in the case of PNA, it has excellent specificity for discriminating single base mismatches because of its high temperature stability and short-length synthesis. This advantage can effectively distinguish denaturation of bases that may occur during synthesis, that is, deletion, substitution, and insertion.

However, diagnostic products in various fields using synthetic oligos are expanding, but methods of providing accurate information on errors in the synthesis process are stagnant, and moreover, 1 to 2 at the 5' or 3' ends of the synthetic oligos. There is no way to detect the degradation of the bases of dogs.

Accordingly, the present inventors have made diligent efforts to effectively detect the terminal base denaturation of the target artificial synthetic oligo. As a result, when a PNA probe coupled with a reporter and a quencher is used, the target artificial synthetic oligo is analyzed by dissolution curve analysis. It was confirmed that terminal base denaturation can be effectively detected, and the present invention has been completed.

An object of the present invention is to provide a method for analyzing a melting curve using a PNA probe coupled with a reporter and a quencher in order to determine the terminal base degradation of a target artificial synthetic oligo.

Another object of the present invention is to provide a method and kit for discriminating the nucleotide sequence difference between a PNA probe and a target artificially synthesized oligo having denatured terminal bases.

Another object of the present invention is to provide a method and a kit for testing the quality of an artificially synthesized target oligo.

Another object of the present invention is to provide a method and a kit for detecting a base mutation of a target artificially synthesized oligo.

In order to achieve the above object, the present invention provides a PNA probe using a PNA probe coupled with a reporter and a quencher and a fusion target artificial synthetic oligo in which the terminal base is denatured (degradation) for nucleotide sequence discrimination A curve analysis method is provided.

The present invention also comprises the steps of (a) hybridizing the PNA probe and the target artificial oligo by mixing the PNA probe and the primer to which the reporter and the quencher are bound with the synthetic target oligo; (b) melting the hybridized product while changing the temperature to obtain a melting curve; And (c) analyzing the obtained melting curve to determine the base sequence difference between the PNA probe and the target artificial synthetic oligo in which the terminal base is denatured (degraded) comprising the step of determining the base sequence difference between the PNA probe and the target artificial synthetic oligo provide a way

The present invention also includes a PNA probe to which a reporter and a quencher are bound, and a PNA probe using a melting curve analysis method and a target artificial synthetic oligo in which the terminal base is denatured (degradation) for nucleotide sequence discrimination kit is provided.

The present invention also comprises the steps of (a) hybridizing the PNA probe and the target artificial oligo by mixing the PNA probe and the primer to which the reporter and the quencher are bound with the synthetic target oligo; (b) melting the hybridized product while changing the temperature to obtain a melting curve; and (c) analyzing the obtained melting curve to determine the quality of the target artificially synthesized oligo. It provides a method for inspecting the quality of the target artificially synthesized oligo, which is estimated to be degraded at the terminal base.

The present invention also includes a PNA probe to which a reporter and a quencher are bound, and a kit for quality inspection of the target artificial synthetic oligo, which is estimated that the terminal base is denatured using a melting curve analysis method. to provide.

The present invention also comprises the steps of (a) hybridizing the PNA probe and the target artificial oligo by mixing the PNA probe and the primer to which the reporter and the quencher are bound with the synthetic target oligo; (b) melting the hybridized product while changing the temperature to obtain a melting curve; and (c) analyzing the obtained melting curve to detect the presence or absence of base mutation in the target artificial synthetic oligo. It provides a method for detecting base mutation of the target artificial synthetic oligo, which is estimated to be base mutated. .

The present invention also includes a PNA probe to which a reporter and a quencher are bound, and a kit for detecting base mutations in a target artificial synthetic oligo that is presumed to be base mutated using a melting curve analysis method provides

In the case of using the quality inspection method of artificially synthesized oligos using a PNA probe coupled with a reporter and a quencher according to the present invention, whether there is an abnormality in the synthetic oligos of the genetic diagnosis product and the synthesis of artificially synthesized oligos of DNA or RNA structure Since abnormal synthesis results such as nucleotide sequence abnormalities that can occur due to problems can be checked within a short time, it is very convenient to inspect the quality of artificially synthesized oligos and to manage genetic diagnostic products, and is useful for monitoring the quality of artificially synthesized oligos. can be

1 shows a schematic diagram that can confirm whether the artificially synthesized oligo is denatured using the artificially synthesized oligo and PNA.
Figure 2 shows a schematic diagram according to the binding position of the synthetic oligo and the PNA probe.
Figure 3 shows a schematic diagram for performing ^{PCR using CFX96 TM} Real-Time for the analysis of the melting curve of the artificially synthesized oligo and the PNA probe to determine whether it is denatured.
Figure 4 shows the melting curve temperature conditions for confirming the denaturation of the synthetic oligo.
5 is a graph analyzing a melting curve and a melting temperature by artificially synthesizing an oligo masquerading as denaturation in order to check whether the artificially synthesized oligo is denatured or mutated.
6 is a graph analyzing a melting curve and melting temperature using artificially synthesized oligos simulating denaturation in which a reporter and a quencher are bonded in order to check whether the denaturation and base variation of the artificially synthesized oligo having a reporter and a quencher bonded. to be.
7 is a graph analyzing each of the melting curves and melting temperatures by predicting the binding range of each artificially synthesized oligo and PNA probe with two bases added to the 3' and 5' ends.
8 is a graph showing a melting curve and melting temperature analysis using a PNA probe while changing the ratio of a normal artificially synthesized oligo to a denatured artificially synthesized oligo.
9 is a graph of analyzing a melting curve and a melting temperature by artificially synthesizing a denatured oligo in order to check whether the artificially synthesized oligo is denatured or mutated according to the machine type of the real-time gene amplifier.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

In an embodiment of the present invention, the change of the melting curve according to the nucleotide mutation position and the number of mutated nucleotides of the nucleotide sequence site to which the PNA probe and the target artificial synthetic oligo bind are measured, and the nucleotide sequence binding to the target artificial synthetic oligo and the PNA probe When the bases corresponding to the middle and end of the nucleotides were deleted, it was confirmed that as the number of deleted bases increased, the melting temperature (Tm) sequentially decreased according to the number of deleted bases, and the middle part of the nucleotide sequence binding to the PNA probe When the base of the mutation was confirmed, the melting temperature (Tm) difference was larger (Table 2).

Therefore, in one aspect, the present invention provides a melting curve for discriminating the nucleotide sequence difference between a PNA probe using a PNA probe coupled with a reporter and a quencher and a target artificial synthetic oligo in which the terminal base is denatured (degradation) It is about the analysis method.

In the present invention, the PNA probe shows an expected melting temperature (Tm) value by perfectly matching the nucleotide sequence of the target artificial oligo, and incomplete hybridization with the synthetic target oligo having a nucleotide sequence difference ( It may be characterized in that it forms a mismatch) and exhibits a lower melting temperature (Tm) value than expected, and the terminal base is located at the 5' end (5' end) or 3' end (3' end) of the target artificial synthetic oligo. It may be characterized in that it is a base.

In the present invention, the denaturation may be characterized in that it occurs in one or two or more bases, and the denaturation may be characterized by deletion, substitution or insertion, and the denaturation may be characterized in that the target artificially synthesized oligo is distributed or It may be characterized in that it occurs in the storage step.

In the present invention, the quencher may be characterized in that at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, and the reporter is FAM (6-carboxyfluorescein), Texas Red, HEX (2',4',5',7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, can be characterized in that at least one selected from the group consisting of CY3 and CY5 have.

The 'target artificial oligo' of the present invention refers to a nucleic acid sequence to be detected or not, and is annealed or hybridized with a primer or probe under hybridization, annealing or amplification conditions. 'Target artificial synthetic oligo' is not different from the term 'target artificial synthetic oligo' used herein, and is used interchangeably herein.

'Hybridization' in the present invention means that complementary single-stranded nucleic acids form double-stranded nucleic acids. Hybridization may occur when the complementarity between two nucleic acid strands is perfect (perfect match) or even if some mismatched bases are present. The degree of complementarity required for hybridization may vary depending on hybridization conditions, and in particular may be controlled by temperature.

As used herein, the term 'base denaturation (degradation)' refers to denaturation of the nucleotide sequence of a target oligo or target artificial oligo, extrinsic factors (distribution and storage status of oligos) or intrinsic factors (mutation of oligos) )) can be caused by The term 'base modification' is not different from the term 'modification' used herein, and is used interchangeably herein. The base mutation or mutation is characterized in that it includes denaturation due to deletion, substitution, or insertion of an oligo base, and the PNA probe of the present invention is a base of an artificially synthesized target oligo. Deletion, substitution, or insertion of mutations can be analyzed through melting curve analysis.

The PNA probe containing the reporter and the quencher of the present invention generates a fluorescence signal after hybridization with the target artificial oligo, and as the temperature rises, the fluorescence signal is quenched by rapidly fusion with the target artificial oligo at the appropriate melting temperature of the probe. , it is possible to detect the presence or absence of base denaturation of the target artificial synthetic oligo through high-resolution fluorescence melting curve analysis (FMCA) obtained from the fluorescence signal according to the temperature change.

In the probe of the present invention, a fluorescent substance of a reporter and a quencher capable of quenching the reporter fluorescence may be bound to both ends. The reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2',4',5',7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5. It may be at least one selected from the group consisting of, and the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto, preferably Dabcyl (FAM-labeled) can be used.

In the present invention, the PNA probe shows the expected melting temperature (Tm) value by making a perfect match with the nucleotide sequence of the target artificial oligo, and the synthetic target oligo having base mutations is incompletely hybridized (mismatch) It can be characterized in that it shows a lower melting temperature (Tm) value than expected. In addition, in the PNA probe of the present invention, for the difference in melting temperature (Tm) between the target artificial oligo and the target artificial oligo having base denaturation, the base mutation position of the target artificial oligo is at the end or the middle of the PNA probe. You can design it to come.

In another embodiment of the present invention, in order to determine the base modification of the target artificial synthetic oligo through melting curve analysis, four types of probes (FAM-, HEX-, texas Red-, Cy5-labeled) were prepared to hybridize and the melting curve was analyzed, and the target artificial synthetic oligo in which the base was deleted or substituted regardless of the base modification position of the entire base sequence was completely hybridized. It was confirmed that the visible target artificial synthetic oligo (perfect match) and the melting temperature (Tm) difference occurred, and the target artificial synthetic oligo showing complete hybridization, the target artificial synthetic oligo with a deletion of the base, and the base-substituted targeted artificial synthetic oligo sequence As a result, it was confirmed that the melting temperature was low (Table 3).

In another embodiment of the present invention, in order to analyze the melting curve according to the position of the artificially synthesized oligo and the PNA probe, the length of the artificially synthesized oligo is prepared differently from each other to predict the binding range of the artificially synthesized oligo and the PNA probe, and each melting curve And as a result of analyzing the melting temperature, it was confirmed that in the case of the synthetic oligo having two bases added, the difference between the target artificial oligo (perfect match) and the melting temperature (Tm) showing complete hybridization was not large (Table 4).

In another embodiment of the present invention, in order to determine the defective ratio of the target artificial synthetic oligo, the melting temperature difference was measured while changing the ratio of the target artificial synthetic oligo showing complete hybridization and the modified target artificial synthetic oligo, It was confirmed that the higher the ratio of the denatured target artificial synthetic oligo, the greater the difference in melting temperature appeared, and the method of determining the base denaturation of the target artificial synthetic oligo using the PNA of the present invention can determine the denaturation rate of the target artificial synthetic oligo. It was confirmed that it can be (Table 5).

Accordingly, the present invention, in another aspect, (a) a reporter (reporter) and a quencher (quencher) is combined with the PNA probe and the primer is mixed with the synthetic target oligo, hybridizing the PNA probe and the target artificial oligo; (b) melting the hybridized product while changing the temperature to obtain a melting curve; And (c) analyzing the obtained melting curve to determine the base sequence difference between the PNA probe and the target artificial synthetic oligo in which the terminal base is denatured (degraded) comprising the step of determining the base sequence difference between the PNA probe and the target artificial synthetic oligo A kit for discriminating the difference between a PNA probe using a melting curve analysis method and a target artificially synthesized oligonucleotide in which a terminal base is denatured (degradation), including a method and a PNA probe coupled with a reporter and a quencher will be.

In the present invention, the PNA probe shows an expected melting temperature (Tm) value by perfectly matching the nucleotide sequence of the target artificial oligo, and incomplete hybridization with the synthetic target oligo having a nucleotide sequence difference ( It may be characterized in that it forms a mismatch) and exhibits a lower melting temperature (Tm) value than expected, and the terminal base is located at the 5' end (5' end) or 3' end (3' end) of the target artificial synthetic oligo. It may be characterized in that it is a base.

In the present invention, the denaturation may be characterized in that it occurs in one or two or more bases, and the denaturation may be characterized by deletion, substitution or insertion, and the denaturation may be characterized in that the target artificially synthesized oligo is distributed or It may be characterized in that it occurs in the storage step.

In the present invention, the quencher may be characterized in that at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, and the reporter is FAM (6-carboxyfluorescein), Texas Red, HEX (2',4',5',7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, can be characterized in that at least one selected from the group consisting of CY3 and CY5 have.

In the present invention, it can be characterized in that two or more target artificial synthetic oligos are used, and the reporter labeled on the PNA probe is different for each target artificial synthetic oligo, thereby discriminating the nucleotide sequence difference between two or more target artificial synthetic oligos. have.

The kit of the present invention may optionally include reagents necessary for performing a target nucleic acid amplification reaction (eg, PCR reaction), such as a buffer, a DNA polymerase cofactor, and deoxyribonucleotide-5-triphosphate. Optionally, the kit of the present invention may also include various polynucleotide molecules, reverse transcriptases, various buffers and reagents, and antibodies that inhibit DNA polymerase activity. In addition, in the kit, the optimal amount of reagents used in a particular reaction can be easily determined by a person skilled in the art having the disclosure herein. Typically, the kit of the present invention may be manufactured as a separate package or compartment comprising the aforementioned components.

In another aspect, the present invention comprises the steps of: (a) mixing a PNA probe and a primer to which a reporter and a quencher are bound with a target artificial oligo, hybridizing the PNA probe and the target artificial oligo; (b) melting the hybridized product while changing the temperature to obtain a melting curve; and (c) analyzing the obtained melting curve, and determining the quality of the target artificial synthetic oligo. A method and a reporter for the quality inspection of the target artificial synthetic oligo presumed to be degrading the terminal base comprising the step of determining the quality of the target artificial synthetic oligo; and It relates to a kit for inspecting the quality of an artificially synthesized target oligo comprising a PNA probe to which a quencher is bound, and the terminal base using a melting curve analysis method is estimated to be denatured (degradation).

In the present invention, the PNA probe shows an expected melting temperature (Tm) value by perfectly matching the nucleotide sequence of the target artificial oligo, and incomplete hybridization with the synthetic target oligo having a nucleotide sequence difference ( It may be characterized in that it forms a mismatch) and exhibits a lower melting temperature (Tm) value than expected, and the terminal base is located at the 5' end (5' end) or 3' end (3' end) of the target artificial synthetic oligo. It may be characterized in that it is a base.

In the present invention, the denaturation may be characterized in that it occurs in one or two or more bases, and the denaturation may be characterized by deletion, substitution or insertion of an oligo base, and the denaturation may be a target artificial synthetic oligonucleotide. It may be characterized in that it occurs in the stage of distribution or storage.

In the present invention, the quencher may be characterized in that at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, and the reporter is FAM (6-carboxyfluorescein), Texas Red, HEX (2',4',5',7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, can be characterized in that at least one selected from the group consisting of CY3 and CY5 have.

In the present invention, two or more target artificial synthetic oligos are used, and the reporter labeled on the PNA probe is different for each target artificial synthetic oligo, and the base sequence difference between two or more targeted artificial synthetic oligos is analyzed to determine the target artificially synthesized oligos. It may be characterized by determining the quality.

In another aspect, the present invention, (a) a reporter (reporter) and the quencher (quencher) is combined with the PNA probe and the primer to the target artificial oligo and hybridization of the PNA probe and the synthetic target oligo; (b) melting the hybridized product while changing the temperature to obtain a melting curve; and (c) analyzing the obtained melting curve to detect the presence or absence of base mutation in the target artificial synthetic oligo. A method and reporter ( reporter) and a quencher-bound PNA probe, and relates to a kit for detecting nucleotide mutations in a target artificial synthetic oligo that is presumed to be nucleotide mutated using a melting curve analysis method.

In the present invention, the PNA probe perfectly matches the nucleotide sequence of the target artificial oligo and shows the expected melting temperature (Tm) value, and is incompletely hybridized with the synthetic target oligo having a base mutation. ) and may be characterized in that it shows a lower melting temperature (Tm) value than expected, and the base is a base located at the 5' end (5' end) or 3' end (3' end) of the target artificial synthetic oligo. It can be characterized as

In the present invention, the base mutation may be characterized in that it occurs in one or two or more bases, and the base mutation may be characterized by deletion, substitution or insertion of an oligo base, and the base mutation is the target. It may be characterized in that it occurs in the stage of distribution or storage of the artificially synthesized oligo.

In the present invention, the quencher may be characterized in that at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, and the reporter is FAM (6-carboxyfluorescein), Texas Red, HEX (2',4',5',7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, can be characterized in that at least one selected from the group consisting of CY3 and CY5 have.

In the present invention, two or more It may be characterized in that the presence or absence of base mutations in two or more target artificial synthetic oligos is detected by using a target artificial synthetic oligo and different reporters labeled on the PNA probe for each of the target artificial synthetic oligos.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: For identification of synthetic oligos probe making

By predicting the results that may occur during the production and use of artificial synthetic oligos, oligos simulating degradation, oligos simulating substitution, and oligos simulating deletions were prepared. In addition, in the case of artificially synthesized oligos, oligos having denaturation, mutation or deletion marked with fluorescence specifically used as probes were additionally prepared. In addition, in the case of a primer, an artificially synthesized oligo was prepared by changing the binding position of the PNA probe and the oligo because the individual-specific 18mer to 25mer and the 25mer to 35mer probe were produced. In addition, it was attempted to prepare a complementary PNA probe capable of confirming all abnormalities in the artificially synthesized oligos (Table 1).

First, the PNA probe was designed directly to check the abnormality of the artificially synthesized oligo. All PNA probes (FAM-labeled, Dabcyl) used in the present invention were synthesized by HPLC purification in Panagene (Korea), and the purity of all synthesized probes was confirmed using mass spectrometry, and target artificially synthesized oligos and For more effective binding of the probe, unnecessary secondary structures of the probe were avoided. In addition, the fluorescent reporter and quencher positions were crossed to prepare the PNA probe due to the structural characteristics of the fluorescence-labeled artificial oligo.

The melting temperature (Tm) of the mutated portion of the base was prepared so that a difference could occur due to the complementary binding of the artificially synthesized oligo to the prepared PNA probe. Table 1 below shows PNA probes and oligo sequences for identification of synthetic oligos.

name (sequence number) sequence (5'->3') type PROBE-1
(SEQ ID NO: 1) (Dabcyl)TCATGCCGCTGCT-O-K(HEX) PNA probe PROBE-2
(SEQ ID NO: 2) (HEX)TCATGCCGCTGCT-O-K(Dabcyl) PNA probe PM
(SEQ ID NO: 3) AGTACGGCGACGA perfect match PM1
(SEQ ID NO: 4) CGAGTACGGCGACGA Perfect match (5` end 2 base more) PM2
(SEQ ID NO: 5) AGTACGGCGACGACG Perfect match (3` end 2 base more) 5`-1dg
(SEQ ID NO: 6) GTACGGCGACGA 5`end 1 base degradation 5`-2dg
(SEQ ID NO: 7) TACGGCGACGA 5`end 2 base degradation 3`-1dg
(SEQ ID NO: 8) AGTACGGCGACG 3`end 1 base degradation 3`-2dg
(SEQ ID NO: 9) AGTACGGCGAC 3`end 2 base degradation MID-mu
(SEQ ID NO: 10) AGTACGCCGACGA Inter Mutation MID-de
(SEQ ID NO: 11) AGTACGCGACGA Inter deletion 5`-mu
(SEQ ID NO: 12) ACTACGGCGACGA 5' end mutation 5`-de
(SEQ ID NO: 13) ATACGGCGACGA 5` end deletion 3`-mu
(SEQ ID NO: 14) AGTACGGCGACCA 3' end mutation 3`-de
(SEQ ID NO: 15) AGTACGGCGACA 3` end deletion PM-F
(SEQ ID NO: 16) FAM-AGTACGGCGACGA-BHQ1 report and quenching tagging perfect match 5`-1dg-F
(SEQ ID NO: 17) FAM-GTACGGCGACGA-BHQ1 report and quenching tagging 5` end degradation 3`-1dg-F
(SEQ ID NO: 18) FAM-AGTACGGCGACG-BHQ1 report and quenching tagging 3` end degradation MID-mu-F
(SEQ ID NO: 19) FAM-AGTACGCCGACGA-BHQ1 report and quenching tagging inter mutation MID-de-F
(SEQ ID NO: 20) FAM-AGTACGCGACGA-BHQ1 report and quenching tagging inter deletion

In Table 1, O denotes a linker and K denotes lysine.

^{PCR was performed using the CFX96 TM} Real-Time system (Bio-Rad, USA) for the analysis of the fusion curve of the artificially synthesized oligo and the PNA probe to check for abnormality, and the synthetic oligo and PNA were performed. A dissolution curve analysis was performed by mixing the probes (FIG. 3).

Example 2: artificial synthesis of oligo substitution( substitution ), fruit ( deletion ) and denaturation ( degradation ) analysis of the dissolution curve

^{PCR was performed using the CFX96 TM} Real-Time system (Bio-Rad, USA) for the PNA probe and melting curve analysis to check whether the synthetic oligo was abnormal, and the synthetic oligo and PNA probe were mixed and analyzed using a dissolution curve. The conditions for dissolution curve analysis are as follows; The total volume to be 20㎕ 2X real-time bio-eye SSB MeltingArray ^TM buffer ^{(SeaSunBio Real-Time MeltingArray TM buffer} , gaze Bio, Korea), synthetic oligonucleotide 1㎕ / 2.5pmol, PNA probes 0.5㎕ / 10pmol, sterilized distilled water (DW) 8.5 μl was added. The dissolution curve analysis process was performed by heating at 90° C. for 3 minutes and at 75° C. for 1 minute, and then increasing the temperature from 40° C. to 80° C. by 0.5° C. to measure fluorescence. A stationary state was maintained for 5 seconds between each step (Fig. 4).

In order to confirm the denaturation and base variation of the artificially synthesized oligo, the prediction was made and then the melting curve was analyzed. In the case of artificially synthesized oligos, base degradation may occur under various conditions during use, and if 3' end denaturation occurs, gene amplification is greatly affected. In addition, substitutions and deletions may occur during the synthesis process due to machine errors or incorrect input. Therefore, an experiment was performed by artificially synthesizing an oligo simulating denaturation, substitution, and deletion of the oligo ( FIG. 5 ).

The analysis of denaturation and base variation of the artificial synthetic oligo was repeated 5 times in one tube, and the average value thereof was calculated and indicated. Table 2 below shows the melting temperature for checking whether the artificially synthesized oligo is abnormal.

name type Average temperature (℃) Melting temperature difference (℃) PM perfect match 62.5 - 5` 1dg 5` end 1 base degradation 60.0 - 2.5 5` 2dg 5` end 2 base degradation 52.0 - 10.5 3` 1dg 3` end 1 base degradation 60.0 - 2.5 3` 2dg 3` end 2 base degradation 54.5 - 8.0 MID-mu Inter Mutation 40.5 - 22.0 MID-de Inter deletion 38.5 - 24.0 5`-mu 5' end mutation 52.5 - 10.0 5`-de 5` end deletion 53.0 - 9.5 3`-mu 3' end mutation 57.0 - 5.5 3`-de 3` end deletion 56.5 - 6.0

As a result, as shown in FIG. 5 and Table 2, the change of the melting curve according to the nucleotide mutation position and the number of mutated bases of the nucleotide sequence site to which the PNA probe and the target artificial synthetic oligo are bound was measured, and the target artificial synthetic oligo and PNA When the bases corresponding to the center and the end of the base sequence binding to the probe were deleted, it was confirmed that the melting temperature (Tm) sequentially decreased according to the number of deleted bases as the number of deleted bases increased. It was confirmed that the difference in melting temperature (Tm) was larger when the base in the middle of the nucleotide sequence was mutated.

Example 3: Reporter and a quencher combined artificial synthesis of oligo Analysis of dissolution curves according to denaturation and base mutation

^{PCR was performed using the CFX96 TM} Real-Time system (Bio-Rad, USA) for the analysis of the PNA probe and the melting curve to confirm the abnormality with the synthetic oligos synthesized above, and the synthetic oligos and PNA probes were mixed and analyzed using a dissolution curve. The conditions for dissolution curve analysis are as follows; The total volume to be 20㎕ 2X real-time bio-eye SSB MeltingArray ^TM buffer ^{(SeaSunBio Real-Time MeltingArray TM buffer} , gaze Bio, Korea), synthetic oligonucleotide 1㎕ / 2.5pmol, PNA probes 0.5㎕ / 10pmol, sterilized distilled water (DW) 8.5 μl was added. The dissolution curve analysis process was performed by heating at 90° C. for 3 minutes and at 75° C. for 1 minute, and then increasing the temperature from 40° C. to 80° C. by 0.5° C. to measure fluorescence. A stationary state was maintained for 5 seconds between each step (FIG. 4).

In order to confirm the denaturation and base variation of the reporter and quencher-coupled artificially synthesized oligo, it was prepared in a form that could occur during synthesis and use, and the melting curve was analyzed. In general, an artificially synthesized oligo in which a reporter and a quencher are combined is used as a probe to confirm a genetic variation. The reporter and the quencher may cross positions depending on the experimental conditions. Therefore, in order to confirm an artificially synthesized oligo in which a reporter and a quencher are combined, an additional PNA probe also crossed a fluorescence and a quencher to conduct an experiment (FIG. 6).

When analyzing the denaturation and base variation of the reporter and the quencher-coupled artificial synthetic oligo, it was repeated 5 times in one tube, and the average value thereof was calculated and indicated. Table 3 below shows the melting temperature of the abnormal synthetic oligo in which the reporter and the quencher are combined.

name
type
Average
Temperature (℃)
Melting temperature difference (℃)
Average
Temperature (℃)
Melting temperature difference (℃) PROBE-1 PROBE-2 PM perfect match 62.5 - 62.5 - 5`-1dg-F report and quenching tagging 5` end degradation 60.0 - 2.5 60.0 - 2.5 3`-1dg-F report and quenching tagging 3` end degradation 60.0 - 2.5 60.0 - 2.5 MID-mu-F report and quenching tagging inter mutation 40.5 - 22.0 41.0 - 21.5 MID-de-F report and quenching tagging inter deletion 38.5 - 24.0 38.5 - 24.0

As a result, as shown in FIGS. 6 and 3, for the determination of base modification of the target artificial synthetic oligo through melting curve analysis, four types of artificial synthetic oligos in which the bases of the artificial synthetic oligos that form complete hybridization are deleted, substituted or inserted It was prepared to hybridize with the probes (FAM-, HEX-, texas Red-, Cy5-labeled), and the melting curve was analyzed. It was confirmed that the synthetic oligo had a difference in melting temperature (Tm) from the target artificial synthetic oligo showing complete hybridization, the target artificial synthetic oligo showing complete hybridization, the base-deleted target artificial synthetic oligo, and the base substitution It was confirmed that the melting temperature was low in the order of the target artificial synthetic oligos.

Example 4: artificial synthesis Oligowa PNA probe Analysis of melting curve according to location

^{PCR was performed using the CFX96 TM} Real-Time system (Bio-Rad, USA) for the analysis of the PNA probe and the melting curve to confirm the abnormality with the synthetic oligos synthesized above, and the synthetic oligos and PNA probes were mixed and analyzed using a dissolution curve. The conditions for dissolution curve analysis are as follows; The total volume to be 20㎕ 2X real-time bio-eye SSB MeltingArray ^TM buffer ^{(SeaSunBio Real-Time MeltingArray TM buffer} , gaze Bio, Korea), synthetic oligonucleotide 1㎕ / 2.5pmol, PNA probes 0.5㎕ / 10pmol, sterilized distilled water (DW) 8.5 μl was added. The dissolution curve analysis process was performed by heating at 90° C. for 3 minutes and at 75° C. for 1 minute, and then increasing the temperature from 40° C. to 80° C. by 0.5° C. to measure fluorescence. A stationary state was maintained for 5 seconds between each step (FIG. 4).

In the case of artificially synthesized oligos, the lengths were prepared differently depending on the characteristics of the individual or base sequence, the binding range of the artificially synthesized oligo and the PNA probe was predicted, and the melting curve and melting temperature of each were analyzed (FIG. 7).

When analyzing the melting curve and melting temperature according to the position of the artificially synthesized oligo and PNA probe, it was repeated 5 times in one tube, and the average value thereof was calculated and indicated. Table 4 below shows the melting temperature according to the binding position of the synthetic oligo and the PNA probe.

name
type
Average temperature (℃)
Melting temperature difference (℃) PM perfect match 62.5 - PM1 perfect match
(5` end 2 base more) 62.0 - 0.5 PM2 perfect match
(3` end 2 base more) 62.0 - 0.5

As a result, as shown in FIGS. 7 and 4, in order to analyze the melting curve according to the position of the artificially synthesized oligo and the PNA probe, the length of the artificially synthesized oligo was prepared differently from each other, and the binding range of the artificially synthesized oligo and the PNA probe was predicted. And as a result of analyzing the melting curves and melting temperatures of each, it was confirmed that the difference in melting temperature (Tm) from the artificial synthetic oligo with two bases added was not significantly different from the target artificial synthetic oligo showing complete hybridization (perfect match).

Example 5: Artificial synthesis to confirm denaturation of oligo Analysis of the melting curve according to the relative ratio

^{PCR was performed using the CFX96 TM} Real-Time system (Bio-Rad, USA) for the analysis of the PNA probe and the melting curve to confirm the abnormality with the synthetic oligos synthesized above, and the synthetic oligos and PNA probes were mixed and analyzed using a dissolution curve. The conditions for dissolution curve analysis are as follows; The total volume to be 20㎕ 2X real-time bio-eye SSB MeltingArray ^TM buffer ^{(SeaSunBio Real-Time MeltingArray TM buffer} , gaze Bio, Korea), synthetic oligonucleotide 1㎕ / 2.5pmol, PNA probes 0.5㎕ / 10pmol, sterilized distilled water (DW) 8.5 μl was added. The dissolution curve analysis process was performed by heating at 90° C. for 3 minutes and at 75° C. for 1 minute, and then increasing the temperature from 40° C. to 80° C. by 0.5° C. to measure fluorescence. A stationary state was maintained for 5 seconds between each step (FIG. 4).

In the case of artificially synthesized oligos, they are dissociated and acidified during long-term storage. In the case of acidification, degradation of the artificially synthesized oligo is caused. However, the degree of denaturation does not occur temporarily by a single reaction, but proceeds sequentially. Therefore, after mixing in the relative ratio of the normal synthetic oligo and the denatured artificial oligo, the melting curve and melting temperature were analyzed using a PNA probe (FIG. 8).

When analyzing the relative ratio of artificially synthesized oligos for denaturation confirmation using a PNA probe, it was repeated 5 times in one tube, and the average value thereof was calculated and indicated. Table 5 below shows the melting temperature according to the concentration of the modified artificially synthesized oligo.

PM : 3`-1 dg -F(%)
Temperature (℃)
Melting temperature difference (℃) 100 : 0 62.5 - 90:10 62.0 - 0.5 80:20 62.0 - 0.5 70:30 61.5 - 1.0 60:40 60.5 - 2.0 50:50 60.0 - 2.5 40:60 59.5 - 3.0 30:70 59.0 - 3.5 20:80 58.5 - 4.0 10:90 55.0 - 7.5 0 : 100 54.5 - 8.0

As a result, as shown in FIGS. 8 and 5, in order to determine the defective ratio of the target artificial synthetic oligo, fusion while changing the ratio of the target artificial synthetic oligo showing complete hybridization and the modified target artificial synthetic oligo (perfect match) The temperature difference was measured, and it was confirmed that the higher the ratio of the denatured target artificial synthetic oligo, the greater the difference in the melting temperature appeared, and the method for determining the base modification of the target artificial synthetic oligo using PNA of the present invention is the target artificial synthetic oligo. It was confirmed that the denaturation rate of

Example 6: artificial synthesis of oligo Melting curve analysis for each machine to check for abnormalities

^{PCR was performed using the CFX96 TM} Real-Time system (Bio-Rad, USA) for the analysis of the PNA probe and the melting curve to confirm the abnormality with the synthetic oligos synthesized above, and the synthetic oligos and PNA probes were mixed and analyzed using a dissolution curve. The conditions for dissolution curve analysis are as follows; The total volume to be 20㎕ 2X real-time bio-eye SSB MeltingArray ^TM buffer ^{(SeaSunBio Real-Time MeltingArray TM buffer} , gaze Bio, Korea), synthetic oligonucleotide 1㎕ / 2.5pmol, PNA probes 0.5㎕ / 10pmol, sterilized distilled water (DW) 8.5 μl was added. The dissolution curve analysis process was performed by heating at 90° C. for 3 minutes and at 75° C. for 1 minute, and then increasing the temperature from 40° C. to 80° C. by 0.5° C. to measure fluorescence. A stationary state was maintained for 5 seconds between each step (FIG. 4).

^{CFX96 TM} Real-Time System (Bio-Rad) and LightCycler®96 (Bio-Rad) were used to check the differences between real-time gene amplifiers (Real-Time PCR) that can be analyzed using artificially synthesized oligos simulating denaturation, substitution, and deletion. Roche) was used for analysis (FIG. 9).

When analyzing the machine-specific differences of artificially synthesized oligos using PNA probes, it was repeated 5 times in one tube, and the average values thereof were calculated and indicated. Table 6 below shows the melting temperature of each machine for checking whether the artificially synthesized oligo is abnormal.

name
type
Average
Temperature (℃)
Melting temperature difference (℃)

Average
Temperature (℃)
Melting temperature difference (℃)
CFX96 ^TM
LightCycler®96 PM perfect match 62.5 - 65.66 - 5`-1dg-F report and quenching tagging 5` end degradation 60.0 - 2.5 63.55 - 2.11 3`-1dg-F report and quenching tagging 3` end degradation 60.0 - 2.5 63.54 - 2.12 MID-mu-F report and quenching tagging inter mutation 40.5 - 22.0 46.71 - 18.95 MID-de-F report and quenching tagging inter deletion 38.5 - 24.0 44.80 - 20.86

As a result, as shown in FIGS. 9 and 6, in order to check whether the artificially synthesized oligo is denatured or mutated according to the machine type of the real-time gene amplifier, an oligo masquerading as denaturation was artificially synthesized, and the melting curve and As a result of analyzing the melting temperature, it was confirmed that the difference in the analysis results according to the machine type was not significant.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Seasunbiomaterials <120> Method for Ensuring Quality of Synthetic Oligo Using Peptide Nucleic Acid Probes <130> P14-B292 <160> 20 <170> KopatentIn 2.0 <210> 1 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> PROBE-1 <400> 1 tcatgccgct gct 13 <210> 2 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> PROBE-2 <400> 2 tcatgccgct gct 13 <210> 3 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> PM <400> 3 agtacggcga cga 13 <210> 4 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> PM1 <400> 4 cgagtacggc gacga 15 <210> 5 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> PM2 <400> 5 agtacggcga cgacg 15 <210> 6 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> 5`-1dg <400> 6 gtacggcgac ga 12 <210> 7 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> 5`-2dg <400> 7 tacggcgacg a 11 <210> 8 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> 3`-1dg <400> 8 agtacggcga cg 12 <210> 9 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> 3`-2dg <400> 9 agtacggcga c 11 <210> 10 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> MID-mu <400> 10 agtacgccga cga 13 <210> 11 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> MID-de <400> 11 agtacgcgac ga 12 <210> 12 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> 5`-mu <400> 12 actacggcga cga 13 <210> 13 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> 5`-de <400> 13 atacggcgac ga 12 <210> 14 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> 3`-mu <400> 14 agtacggcga cca 13 <210> 15 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> 3`-de <400> 15 agtacggcga ca 12 <210> 16 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> PM-F <400> 16 agtacggcga cga 13 <210> 17 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> 5`-1dg-F <400> 17 gtacggcgac ga 12 <210> 18 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> 3`-1dg-F <400> 18 agtacggcga cg 12 <210> 19 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> MID-mu-F <400> 19 agtacgccga cga 13 <210> 20 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> MID-de-F <400> 20 agtacgcgac ga 12

Claims (59)

delete delete delete delete delete delete delete delete A method for discriminating the nucleotide sequence difference between a PNA probe and a target artificial synthetic oligo in which the terminal base is denatured, comprising the following steps:
(a) hybridizing the PNA probe and the target artificial oligo by mixing the PNA probe and the primer to which the reporter and the quencher are bound with the synthetic target oligo;
(b) melting the hybridized product while changing the temperature to obtain a melting curve; and
(c) when the melting temperature (Tm) value of the hybridized product obtained by analyzing the obtained melting curve differs from the melting temperature of the normal target artificial synthetic oligo by 2° C. to 24° C., the terminal base of the target artificial synthetic oligo Determining that is denatured.
10. The method of claim 9, wherein the PNA probe completely hybridizes with the nucleotide sequence of the target artificial oligo, shows an expected melting temperature (Tm) value, and incompletely hybridizes with the synthetic target oligo having a nucleotide sequence difference. A method of discriminating a nucleotide sequence difference, characterized in that it shows a lower melting temperature (Tm) value than expected by forming a mismatch.
10. The method of claim 9, wherein the terminal base is a base located at the 5' end (5' end) or the 3' end (3' end) of the artificially synthesized target oligo.
The method of claim 9, wherein the denaturation occurs in one or two or more bases.
The method of claim 9, wherein the denaturation is a deletion, substitution, or insertion.
The method of claim 9, wherein the denaturation occurs in the step of distributing or storing the target artificial synthetic oligo.
The method of claim 9, wherein the quencher is at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2, and Dabcyl.
10. The method of claim 9, wherein the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2',4',5',7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 And CY5 base sequence difference discrimination method, characterized in that at least one selected from the group consisting of.
10. The method of claim 9, wherein two or more targets A method for discriminating a nucleotide sequence difference, comprising using an artificial synthetic oligo and discriminating the nucleotide sequence difference between two or more target artificial synthetic oligos by differentiating the reporter labeled on the PNA probe for each of the target artificial synthetic oligos.
delete delete delete delete delete delete delete delete A method for inspecting the quality of an artificially synthesized target oligo that is estimated to be denatured (degradation) of the terminal base comprising the following steps:
(a) hybridizing the PNA probe and the target artificial oligo by mixing the PNA probe and the primer to which the reporter and the quencher are bound with the synthetic target oligo;
(b) melting the hybridized product while changing the temperature to obtain a melting curve; and
(c) when the melting temperature (Tm) value of the hybridized product obtained by analyzing the obtained melting curve differs from the melting temperature of the normal target artificial synthetic oligo by 1 ° C to 8 ° C, the end of the target artificial synthetic oligo Determining that the base is 30% to 100% denatured.
The method of claim 26, wherein the PNA probe completely hybridizes with the nucleotide sequence of the target artificial oligo, showing an expected melting temperature (Tm) value, and incompletely hybridizes with the synthetic target oligo having a nucleotide sequence difference. A method of inspecting the quality of an artificially synthesized target oligo, characterized in that it shows a lower melting temperature (Tm) value than expected by forming a mismatch.
The method according to claim 26, wherein the terminal base is a base located at the 5' end or 3' end of the target artificial synthetic oligo.
The method of claim 26, wherein the denaturation occurs at one or two or more bases.
The method of claim 26, wherein the denaturation is a deletion, substitution, or insertion of an oligo base.
The method of claim 26, wherein the denaturation occurs in the step of distributing or storing the target artificial synthetic oligo.
The method of claim 26, wherein the quencher is at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2, and Dabcyl.
27. The method of claim 26, wherein the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2',4',5',7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 And CY5 quality inspection method of the target artificial synthetic oligo, characterized in that at least one selected from the group consisting of.
27. The method of claim 26, Determining the quality of the target artificial oligo by using two or more target artificial synthetic oligos, and by differentiating the reporter labeled on the PNA probe for each target artificial synthetic oligo, and analyzing the nucleotide sequence difference between two or more targeted artificial synthetic oligos. A method for quality inspection of target artificially synthesized oligos.
delete delete delete delete delete delete delete delete A method for detecting base mutation of a target artificially synthesized oligo that is presumed to be base mutated, comprising the following steps:
(a) hybridizing the PNA probe and the target artificial oligo by mixing the PNA probe and the primer to which the reporter and the quencher are bound with the synthetic target oligo;
(b) melting the hybridized product while changing the temperature to obtain a melting curve; and
(c) when the melting temperature (Tm) value of the hybridized product obtained by analyzing the obtained melting curve differs from the melting temperature of the normal target artificial synthetic oligo by 6° C. to 24° C., the base of the target artificial synthetic oligo Determining that a mutation has occurred.
44. The method of claim 43, wherein the PNA probe completely hybridizes with the base sequence of the target artificial oligo, showing the expected melting temperature (Tm) value, and incompletely hybridizing with the synthetic target oligo having a base mutation ( A method for detecting base variation of a target artificially synthesized oligo, characterized in that the melting temperature (Tm) value is lower than expected by forming a mismatch.
44. The method of claim 43, wherein the base is a base located at the 5' end or 3' end of the target artificially synthesized oligo.
44. The method of claim 43, wherein the nucleotide mutation occurs in one or two or more bases.
44. The method of claim 43, wherein the base mutation is a deletion, substitution, or insertion of an oligo base.
44. The method of claim 43, wherein the base mutation occurs in the step of distributing or storing the target artificial synthetic oligo.
44. The method of claim 43, wherein the quencher is at least one selected from the group consisting of 6-carboxytetramethyl-rhodamine (TAMRA), BHQ1, BHQ2 and Dabcyl.
44. The method of claim 43, wherein the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2',4',5',7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5. A method for detecting base mutation of a target artificially synthesized oligo, characterized in that at least one selected from the group consisting of CY5.
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