WO2016104949A1 - Procédé pour garantir la qualité d'un oligo synthétique artificiel à l'aide d'une sonde à anp - Google Patents

Procédé pour garantir la qualité d'un oligo synthétique artificiel à l'aide d'une sonde à anp Download PDF

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WO2016104949A1
WO2016104949A1 PCT/KR2015/012212 KR2015012212W WO2016104949A1 WO 2016104949 A1 WO2016104949 A1 WO 2016104949A1 KR 2015012212 W KR2015012212 W KR 2015012212W WO 2016104949 A1 WO2016104949 A1 WO 2016104949A1
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target
oligo
base
pna probe
synthetic
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Korean (ko)
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박재신
박희경
정진욱
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주식회사 시선바이오머티리얼스
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Publication of WO2016104949A1 publication Critical patent/WO2016104949A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/318—Chemical structure of the backbone where the PO2 is completely replaced, e.g. MMI or formacetal
    • C12N2310/3181—Peptide nucleic acid, PNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/107—Temperature of melting, i.e. Tm

Definitions

  • the present invention relates to a method for quality assurance of artificial synthetic oligos using a PNA probe, and more particularly, a melting curve analysis method using a PNA probe coupled with a reporter and a quencher to determine terminal base denaturation of a target artificial synthetic oligo.
  • This step of capping has the advantage of preventing the deletion (deletion) of the synthetic oligo to be produced as a result.
  • the synthesis is performed based on the 3 'end, it is substantially impossible to replace the base of the 3' end of the first oligo synthesized.
  • the sequence of the sequence is controlled by the program of the machine, and when the problem occurs, the probability of abnormal synthesis is very high.
  • a QC for identifying the number of synthetic bases polyacrylamide gel electrophoresis, HPLC analysis, and MALDI-TOF analysis are used.
  • QC to identify the correct sequencing can be confirmed by amplifying the synthetic oligo (PCR: Polymerase Chain Reaction, amplification), cloning it back into a vector, and finally sequencing. have.
  • accurate sequencing of synthesized oligonucleotides is impossible and has many problems in cost, time, and procedures.
  • the work for checking the number of bases of the synthetic oligo also has the same problem in terms of time and cost.
  • PNA Protein Nucleic Acid
  • PNA Peptide Nucleic Acid
  • PNA's biggest feature is that it is very stable in temperature, pH and the like, unlike other synthetic oligos. This advantage can be used as a good raw material to confirm the base polymorphism.
  • PNA has high specificity because it enables high temperature stability and short length synthesis.
  • the present inventors have made diligent efforts to effectively detect terminal base denaturation of target synthetic oligos.
  • a target curve of the synthetic synthetic oligos is determined by dissolution curve analysis. It was confirmed that terminal base denaturation can be effectively detected, and the present invention was completed.
  • An object of the present invention is to provide a melting curve analysis method using a PNA probe combined with a reporter and a quencher to determine terminal base degradation of a target artificial synthesis oligo.
  • Another object of the present invention is to provide a method and kit for discriminating the base sequence difference between a PNA probe and a terminal base-modified oligonucleotide.
  • Still another object of the present invention is to provide a method for detecting and detecting a base mutation of a target artificial synthesis oligo.
  • the present invention is a fusion for determining the difference between the PNA probe using a reporter and a quencher PNA probe and the target sequence synthesis oligonucleotide of the terminal base degradation (degradation) Provide a curve analysis method.
  • the present invention also provides a method comprising the steps of: (a) mixing a PNA probe and a primer combined with a reporter and a quencher with a target artificial oligo to hybridize the PNA probe with the target artificial oligo; (b) melting the hybridized product with varying temperature to obtain a melting curve; And (c) analyzing the obtained fusion curves to determine base sequence differences between the PNA probe and the target synthetic oligos, wherein the base sequence differences between the PNA probe and the terminal base oligonucleotide degraded are determined.
  • a method comprising the steps of: (a) mixing a PNA probe and a primer combined with a reporter and a quencher with a target artificial oligo to hybridize the PNA probe with the target artificial oligo; (b) melting the hybridized product with varying temperature to obtain a melting curve; And (c) analyzing the obtained fusion curves to determine base sequence differences between the PNA probe and the target synthetic oligos, wherein the base sequence differences
  • the present invention also includes a PNA probe in which a reporter and a quencher are coupled to each other, and a nucleotide sequence difference between the PNA probe and a target artificial oligo oligonucleotide degraded by a terminal base using a melting curve analysis method is determined.
  • a PNA probe in which a reporter and a quencher are coupled to each other, and a nucleotide sequence difference between the PNA probe and a target artificial oligo oligonucleotide degraded by a terminal base using a melting curve analysis method is determined.
  • the present invention also provides a method comprising the steps of: (a) mixing a PNA probe and a primer combined with a reporter and a quencher with a target artificial oligo to hybridize the PNA probe with the target artificial oligo; (b) melting the hybridized product with varying temperature to obtain a melting curve; And (c) analyzing the obtained melting curve to determine the quality of the target artificial synthesis oligo, thereby providing a quality test method of the target artificial synthetic oligo which is estimated to be degraded.
  • the present invention also includes a PNA probe in which a reporter and a quencher are bound, and a kit for testing the quality of a target artificial synthesis oligo which is estimated to denature the terminal base using a melting curve analysis method. to provide.
  • the present invention also provides a method comprising the steps of: (a) mixing a PNA probe and a primer combined with a reporter and a quencher with a target artificial oligo to hybridize the PNA probe with the target artificial oligo; (b) melting the hybridized product with varying temperature to obtain a melting curve; And (c) analyzing the obtained fusion curve to detect the presence or absence of a base mutation of the target synthetic oligo. .
  • the present invention also includes a PNA probe in which a reporter and a quencher are coupled to each other, and a kit for detecting base mutation of a target artificial synthetic oligo, which is estimated to be mutated from a base using a melting curve analysis method. To provide.
  • Figure 1 shows a schematic diagram that can determine whether the synthetic oligo oligosynthesis using artificial oligo and PNA.
  • Figure 2 shows a schematic diagram according to the binding position of the synthetic oligo oligo PNA probe.
  • Figure 3 shows a schematic diagram for performing PCR using CFX96 TM Real-Time for melting curve analysis of artificial synthetic oligos and PNA probe to determine whether denaturation.
  • Figure 4 shows the melting curve temperature conditions for confirming artificial synthetic oligodenaturation.
  • FIG. 5 is a graph of artificially synthesizing oligos simulating denaturation and analyzing melting curves and melting temperatures thereof in order to determine whether the synthetic oligonucleotides are denatured or mutated.
  • FIG. 6 is a graph of melting curves and melting temperatures using artificially synthesized oligomers in which the reporter and the quencher are denatured to determine whether the reporter and the quencher are denatured or modified. to be.
  • FIG. 7 is a graph analyzing the melting curve and the melting temperature of each artificial oligo oligonucleotide having two bases added at 3 'and 5' ends and a PNA probe.
  • FIG. 8 is a graph of a melting curve and a melting temperature analysis using a PNA probe while changing the ratio of a normal artificial oligo oligonucleotide modified synthetic oligo oligonucleotide.
  • FIG. 9 is a graph of artificially synthesizing oligos simulating denaturation and analyzing melting curves and melting temperatures using them to determine whether the synthetic oligonucleotides are denatured or mutated according to the machine type of the real-time gene amplifier.
  • the present invention was measured the change in the melting curve according to the base mutation position and the number of mutant bases of the base sequence region to which the PNA probe and the target synthetic oligos bind, the base sequence that binds to the target synthetic oligos and PNA probe
  • the melting temperature (Tm) was sequentially reduced according to the number of deleted bases, and the center of the base sequence that binds to the PNA probe
  • Tm melting temperature
  • the present invention provides a melting curve for determining a sequence difference between a PNA probe using a PNA probe coupled with a reporter and a quencher and a target artificial oligo oligonucleotide degraded with a terminal base. It relates to an analysis method.
  • the PNA probe has a perfect match with the base sequence of the target synthetic oligo oligonucleotide (perfect match) to show the expected melting temperature (Tm) value, and incomplete hybridization with the target synthetic oligo oligonucleotide having a base sequence difference ( mismatch) and lower melting temperature (Tm) than expected, wherein the terminal base is located at the 5 'end or 3' end of the target synthetic oligo. It may be characterized in that the base.
  • the denaturation may be characterized in that it occurs in one or two or more bases, the denaturation may be characterized in that the deletion, substitution or insertion, the denaturation is to distribute or distribute the target artificial synthesis oligo It may be characterized in that it occurs in the storage step.
  • the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5 may be at least one selected from the group consisting of have.
  • TAMRA 6-carboxytetramethyl-rhodamine
  • the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5 may be at least one selected from the group consisting of 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 oligo' is not different from the term 'target artificial oligo' as used herein and is used interchangeably herein.
  • Hybridization' of the present invention is meant that complementary single stranded nucleic acids form a double-stranded nucleic acid.
  • Hybridization can occur when the complementarity between two nucleic acid strands is perfect or even when some mismatch base is present.
  • the degree of complementarity required for hybridization may vary depending on the hybridization conditions, and may be particularly controlled by temperature.
  • 'base degradation' of the present invention refers to degeneration of the base sequence of a target oligo or a target artificial synthesis oligo, exogenous factors (the distribution and storage state of oligo) or endogenous factors (mutation of oligo) Can be caused by)).
  • 'Base denaturation' is not different from the term 'denaturation' as used herein, and is used interchangeably herein.
  • the base mutation or mutation is characterized in that the denaturation occurs by deletion, substitution or insertion of the oligo base
  • the PNA probe of the present invention is characterized in that the base of the target artificial synthesis oligo Mutation resulting from deletion, substitution or insertion can be analyzed by melting curve analysis.
  • the PNA probe including the reporter and quencher of the present invention hybridizes with the target synthetic oligo, and generates a fluorescent signal. As the temperature increases, the PNA probe rapidly melts with the target artificial oligo at the proper melting temperature of the probe, thereby extinguishing the fluorescent signal.
  • the presence or absence of base denaturation of the target artificial synthetic oligo may be detected by high resolution fluorescence melting curve analysis (FMCA) obtained from the fluorescence signal according to the temperature change.
  • FMCA fluorescence melting curve analysis
  • the probe of the present invention may combine a reporter and a fluorescent material of a quencher capable of quenching reporter fluorescence at both ends.
  • the reporter is reported as FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7', -tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5.
  • the quencher may be one or more selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, but is not limited thereto, preferably Dabcyl You can use (FAM-labeled).
  • the PNA probe has a perfect match with the base sequence of the target synthetic oligo oligonucleotide (Tm) value, the expected melting temperature (Tm) value, the target artificial synthetic oligonucleotide in the presence of a base mutation incomplete mismatch (mismatch) It can be characterized by showing a lower melting temperature (Tm) value than expected.
  • Tm melting temperature
  • the base mutation position of the target synthetic oligo is located at the end or the center of the PNA probe. Can be designed to come.
  • the artificial synthetic oligonucleotide having a complete hybridization is deleted, substituted or inserted into four kinds of probes (FAM-, HEX-, texas Red-, Cy5-labeled), and the melting curves were analyzed, and the target synthetic oligos with the missing or substituted bases were completely hybridized regardless of the base denaturing position of the entire synthetic sequence.
  • the length of the artificial oligo is produced differently to predict the binding range of the artificial oligo and PNA probe and each melting curve
  • Tm melting temperature
  • the melting temperature difference was measured while changing the ratio of the target synthetic oligo (perfect match) and the modified target artificial oligo showing complete hybridization, The higher the ratio of denatured target synthetic oligos, the greater the difference in melting temperature.
  • the method of determining base denaturation of target synthetic oligos using the PNA of the present invention can determine the denaturation ratio of the target synthetic oligos. It was confirmed that it can (Table 5).
  • the present invention provides a method for preparing a hybrid nanoparticle comprising: (a) mixing a PNA probe and a primer combined with a reporter and a quencher with a target artificial oligo to hybridize the PNA probe with the target synthetic oligo; (b) melting the hybridized product with varying temperature to obtain a melting curve; And (c) analyzing the obtained fusion curves to determine base sequence differences between the PNA probe and the target synthetic oligos, wherein the base sequence differences between the PNA probe and the terminal base oligonucleotide degraded are determined.
  • a method for determining a difference between a PNA probe comprising a method and a reporter and a quencher, and a difference between a PNA probe using a fusion curve analysis method and a target artificial synthetic oligonucleotide sequence in which a terminal base is degraded will be.
  • the PNA probe has a perfect match with the base sequence of the target synthetic oligo oligonucleotide (perfect match) to show the expected melting temperature (Tm) value, and incomplete hybridization with the target synthetic oligo oligonucleotide having a base sequence difference ( mismatch) and lower melting temperature (Tm) than expected, wherein the terminal base is located at the 5 'end or 3' end of the target synthetic oligo. It may be characterized in that the base.
  • the denaturation may be characterized in that it occurs in one or two or more bases, the denaturation may be characterized in that the deletion, substitution or insertion, the denaturation is to distribute or distribute the target artificial synthesis oligo It may be characterized in that it occurs in the storage step.
  • the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5 may be at least one selected from the group consisting of have.
  • TAMRA 6-carboxytetramethyl-rhodamine
  • the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5 may be at least one selected from the group consisting of have.
  • two or more target synthetic oligos may be used, and the reporter labeled on the PNA probe may be different for each target synthetic oligo, thereby determining the difference in the nucleotide sequence of the two or more target synthetic oligos. have.
  • kits of the present invention may optionally include reagents necessary to conduct target nucleic acid amplification reactions (eg, PCR reactions) such as buffers, DNA polymerase cofactors and deoxyribonucleotide-5-triphosphates.
  • reagents necessary to conduct target nucleic acid amplification reactions eg, PCR reactions
  • the kits of the present invention may also include various polynucleotide molecules, reverse transcriptases, various buffers and reagents, and antibodies that inhibit DNA polymerase activity.
  • the optimum amount of reagent used in a particular reaction of the kit can be easily determined by those skilled in the art having learned the disclosure herein.
  • kits of the present invention can be made in a separate package or compartment containing the aforementioned components.
  • the present invention provides a method for preparing a hybrid nanoparticle comprising: (a) mixing a PNA probe and a primer combined with a reporter and a quencher with a target artificial oligo to hybridize the PNA probe with the target synthetic oligo; (b) melting the hybridized product with varying temperature to obtain a melting curve; And (c) analyzing the obtained melting curve, and determining a quality of the target artificial synthesis oligo, which is estimated to be degraded, including a step of determining the quality of the target synthetic oligo.
  • the present invention relates to a kit for testing the quality of a target artificial synthetic oligo including a quencher-coupled PNA probe and estimated to denature the terminal base using fusion curve analysis.
  • the PNA probe has a perfect match with the base sequence of the target synthetic oligo oligonucleotide (perfect match) to show the expected melting temperature (Tm) value, and incomplete hybridization with the target synthetic oligo oligonucleotide having a base sequence difference ( mismatch) and lower melting temperature (Tm) than expected, wherein the terminal base is located at the 5 'end or 3' end of the target synthetic oligo. It may be characterized in that the base.
  • the denaturation may be characterized in that it occurs in one or two or more bases, the denaturation may be characterized in that the deletion, substitution or insertion of the oligo base, the denaturation target artificial synthesis oligo It may be characterized in that it occurs in the distribution or storage step.
  • the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5 may be at least one selected from the group consisting of have.
  • TAMRA 6-carboxytetramethyl-rhodamine
  • the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5 may be at least one selected from the group consisting of have.
  • the reporter labeled on the PNA probe is different for each target synthetic oligo, by analyzing the sequence differences of the two or more target synthetic oligos It may be characterized by determining the quality.
  • the present invention provides a method for preparing a hybrid nanoparticle comprising: (a) mixing a PNA probe and a primer combined with a reporter and a quencher with a target artificial oligo to hybridize the PNA probe with the target synthetic oligo; (b) melting the hybridized product with varying temperature to obtain a melting curve; And (c) analyzing the obtained fusion curve to detect the presence or absence of a base mutation of the target synthetic oligo, and a method for detecting and detecting a base mutation of the target synthetic oligo which is estimated to be mutated.
  • the present invention relates to a kit for detecting a base mutation of a target synthetic oligo oligonucleotide comprising a PNA probe coupled with a reporter and a quencher, and in which the base is estimated to be mutated using a melting curve analysis method.
  • the PNA probe is in perfect hybridization with the base sequence of the target artificial synthesis oligonucleotide (perfect match), showing the expected melting temperature (Tm) value, and incomplete hybridization (mismatch with the target artificial synthesis oligonucleotide having a mutation) And a lower melting temperature (Tm) than expected, and the base is located at the 5 'end or 3' end of the target synthetic oligo. It can be characterized by.
  • the base mutation may be characterized in that it occurs in one or two or more bases, the base mutation may be characterized in that the deletion, substitution or insertion of the oligo base, the base mutation is a target It may be characterized in that it occurs in the step of circulating or storing the artificial oligos.
  • the quencher may be at least one selected from the group consisting of TAMRA (6-carboxytetramethyl-rhodamine), BHQ1, BHQ2 and Dabcyl, the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5 may be at least one selected from the group consisting of have.
  • TAMRA 6-carboxytetramethyl-rhodamine
  • the reporter is FAM (6-carboxyfluorescein), Texas red, HEX (2 ', 4', 5 ', 7',-tetrachloro-6-carboxy-4,7-dichlorofluorescein), JOE, CY3 and CY5 may be at least one selected from the group consisting of have.
  • two or more target synthetic oligos may be used, and the reporter labeled on the PNA probe may be different for each target synthetic oligo, to detect the presence or absence of a base mutation of the two or more target synthetic oligos. have.
  • oligos disguised as degradation In anticipation of the results during the preparation and use of artificial synthetic oligos, oligos disguised as degradation, oligos disguised as substitution, and oligos disguised as deletion were prepared.
  • oligos having denatured, mutated or deleted fluorescence specifically used as probes were used.
  • primers are specifically made of 18mer ⁇ 25mer
  • probes are made of 25mer ⁇ 35mer
  • artificial oligos were prepared by varying the binding positions of the PNA probe and oligo.
  • it was intended to produce a complementary PNA probe that can confirm all abnormalities of the produced artificial synthetic oligo (Table 1).
  • the PNA probe was designed directly to confirm the abnormality of the artificial oligos.
  • All PNA probes (FAM-labeled, Dabcyl) used in the present invention were synthesized by HPLC purification method in Panagene (Panagene, Korea), and the purity of all synthesized probes was confirmed by mass spectrometry. Unnecessary secondary structures of the probes were avoided for more effective binding of.
  • the fluorescent reporter and the quencher were crossed when the PNA probe was manufactured.
  • the melting temperature (Tm) of the mutated portion of the base was produced so that a difference could occur due to complementary binding of the artificial oligos based on the prepared PNA probe.
  • Table 1 below shows PNA probes and oligo sequences for synthesizing oligos.
  • O means a linker and K means lysine.
  • PCR was performed using a CFX96 TM Real-Time System (Bio-Rad, USA) to analyze PNA probes and melting curves to determine the abnormality of the synthetic oligos synthesized above.
  • the mixture was analyzed using a dissolution curve.
  • Conditions for the dissolution curve analysis were as follows; 2X EyeBio Real-Time MeltingArray TM Buffer (SeaSunBio Real-Time MeltingArray TM buffer, EyeBio, Korea), 1 ⁇ l / 2.5 pmol artificial oligo, 0.5 ⁇ l / 10 pmol PNA probe, sterile distilled water (DW) It was performed by adding 8.5 ⁇ l.
  • the dissolution curve analysis was performed for 3 minutes at 90 ° C. and 1 minute at 75 ° C., followed by a 0.5 ° C. increase from 40 ° C. to 80 ° C. to perform fluorescence measurement. It was kept stationary for 5 seconds between each step ( Figure 4).
  • the melting curve was analyzed.
  • degradation of the base may occur under various conditions during use, and in addition, when the 3 ′ end degeneration occurs, it significantly affects gene amplification.
  • substitutions and deletions may occur due to machine errors, incorrect input, etc. during the synthesis process. Therefore, the experiment was carried out by artificially synthesizing oligos disguised as denaturation, substitution, and deletion of oligos (FIG. 5).
  • Table 2 shows the melting temperature for checking the abnormality of the artificial oligos.
  • PCR was performed using the CFX96 TM Real-Time System (Bio-Rad, USA) to analyze PNA probes and melting curves for the analysis of the synthetic oligos and the abnormalities synthesized above.
  • the mixture was analyzed using a dissolution curve.
  • Conditions for the dissolution curve analysis were as follows; 2X EyeBio Real-Time MeltingArray TM Buffer (SeaSunBio Real-Time MeltingArray TM buffer, EyeBio, Korea), 1 ⁇ l / 2.5 pmol artificial oligo, 0.5 ⁇ l / 10 pmol PNA probe, sterile distilled water (DW) It was performed by adding 8.5 ⁇ l.
  • the dissolution curve analysis was performed for 3 minutes at 90 ° C. and 1 minute at 75 ° C., followed by a 0.5 ° C. increase from 40 ° C. to 80 ° C. to perform fluorescence measurement. It was kept stationary for 5 seconds between each step ( Figure 4).
  • the melting curves were analyzed by predicting and producing a form that may occur during synthesis and use.
  • synthetic oligos in which a reporter and a quencher are combined are used as probes to confirm genetic variation.
  • the reporter and the quencher may be crossed in position depending on the experimental conditions. Therefore, in order to identify the artificial oligo oligonucleotide combined with the reporter and the quencher, the experiment was further performed by crossing the fluorescence and the quencher (FIG. 6).
  • PCR was performed using the CFX96 TM Real-Time System (Bio-Rad, USA) to analyze PNA probes and melting curves for the analysis of the synthetic oligos and the abnormalities synthesized above.
  • the mixture was analyzed using a dissolution curve.
  • Conditions for the dissolution curve analysis were as follows; 2X EyeBio Real-Time MeltingArray TM buffer (SeaSunBio Real-Time MeltingArray TM buffer, EyeBio, Korea), 1 ⁇ l / 2.5 pmol artificial oligo, 0.5 ⁇ l / 10 pmol PNA probe, sterile distilled water (DW) It was performed by adding 8.5 ⁇ l.
  • the dissolution curve analysis was performed for 3 minutes at 90 ° C. and 1 minute at 75 ° C., followed by a 0.5 ° C. increase from 40 ° C. to 80 ° C. to perform fluorescence measurement. It was kept stationary for 5 seconds between each step ( Figure 4).
  • the lengths of the synthetic oligonucleotides were different from each other according to the characteristics of the individual or nucleotide sequences.
  • Table 4 shows the melting temperature according to the binding position of the artificial oligo oligo PNA probe.
  • PCR was performed using the CFX96 TM Real-Time System (Bio-Rad, USA) to analyze PNA probes and melting curves for the analysis of the synthetic oligos and the abnormalities synthesized above.
  • the mixture was analyzed using a dissolution curve.
  • Conditions for the dissolution curve analysis were as follows; 2X EyeBio Real-Time MeltingArray TM buffer (SeaSunBio Real-Time MeltingArray TM buffer, EyeBio, Korea), 1 ⁇ l / 2.5 pmol artificial oligo, 0.5 ⁇ l / 10 pmol PNA probe, sterile distilled water (DW) It was performed by adding 8.5 ⁇ l.
  • the dissolution curve analysis was performed for 3 minutes at 90 ° C. and 1 minute at 75 ° C., followed by a 0.5 ° C. increase from 40 ° C. to 80 ° C. to perform fluorescence measurement. It was kept stationary for 5 seconds between each step ( Figure 4).
  • Example 6 Analysis of the melting curve for each machine to confirm the abnormality of the artificial oligo
  • PCR was performed using the CFX96 TM Real-Time System (Bio-Rad, USA) to analyze PNA probes and melting curves for the analysis of the synthetic oligos and the abnormalities synthesized above.
  • the mixture was analyzed using a dissolution curve.
  • Conditions for the dissolution curve analysis were as follows; 2X EyeBio Real-Time MeltingArray TM buffer (SeaSunBio Real-Time MeltingArray TM buffer, EyeBio, Korea), 1 ⁇ l / 2.5 pmol artificial oligo, 0.5 ⁇ l / 10 pmol PNA probe, sterile distilled water (DW) It was performed by adding 8.5 ⁇ l.
  • the dissolution curve analysis was performed for 3 minutes at 90 ° C. and 1 minute at 75 ° C., followed by a 0.5 ° C. increase from 40 ° C. to 80 ° C. to perform fluorescence measurement. It was kept stationary for 5 seconds between each step ( Figure 4).
  • Synthesis of synthetic oligos and DNA or RNA structures of synthetic oligos in the case of using the quality test method of artificial synthetic oligos using a PNA probe combined with a reporter and a quencher according to the present invention Abnormal synthesis results such as sequence abnormalities that can be caused by problems can be confirmed quickly, so it is very convenient to check the quality of artificial oligos and to manage genetic diagnostic products, and is useful for monitoring the quality of synthetic oligos. Can be.

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Abstract

La présente invention concerne un procédé permettant de garantir la qualité d'un oligo synthétique artificiel à l'aide d'une sonde à ANP. Plus spécifiquement, l'invention concerne : un procédé d'analyse d'une courbe de fusion à l'aide d'une sonde à ANP, un rapporteur étant combiné à un extincteur pour déterminer la dénaturation de nucléotides terminaux d'un oligo synthétique artificiel cible ; un procédé permettant de déterminer la dénaturation de nucléotides d'un oligo synthétique artificiel cible l'utilisant ; un kit pour détecter la dénaturation de nucléotides d'un oligo synthétique artificiel cible, le kit comprenant une sonde à ANP ; un procédé permettant de vérifier la qualité d'un oligo synthétique artificiel à l'aide du kit ; et un procédé de détection de la mutation de nucléotides d'un oligo synthétique artificiel. L'application du procédé de contrôle de la qualité d'un oligo synthétique artificiel à l'aide d'une sonde à ANP, dans laquelle un rapporteur et un extincteur sont combinés l'un avec l'autre conformément à la présente invention, permet de vérifier, dans un temps court, une anomalie ou un oligonucléotide synthétique d'un produit de diagnostic génique et des résultats de synthèse anormaux, par exemple une anomalie de séquences nucléotidiques qui peut se produire en raison de problèmes lors de la synthèse de l'oligo synthétique artificiel avec une structure d'ADN ou d'ARN. Il est ainsi possible de vérifier, de manière très commode, la qualité de la synthèse d'un oligo artificiel et de gérer le produit de diagnostic génique. La présente invention peut de ce fait être avantageusement utilisée dans le cadre de la surveillance de la qualité de l'oligo synthétique artificiel.
PCT/KR2015/012212 2014-12-24 2015-11-13 Procédé pour garantir la qualité d'un oligo synthétique artificiel à l'aide d'une sonde à anp WO2016104949A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008109823A2 (fr) * 2007-03-08 2008-09-12 Idaho Technology Amorces pour analyse de fusion
WO2009135832A1 (fr) * 2008-05-06 2009-11-12 Qiagen Gmbh Détection simultanée de multiples séquences d'acide nucléique dans une réaction
WO2011050173A1 (fr) * 2009-10-21 2011-04-28 Brandeis University Procédés, kits et mélanges réactionnels destinés à l'analyse de séquences d'acide nucléique monocaténaire
US8691504B2 (en) * 2009-05-26 2014-04-08 Xiamen University Method for detecting variations in nucleic acid sequences
KR20140046688A (ko) * 2012-10-10 2014-04-21 주식회사 파나진 리포터 및 소광자가 결합된 pna 프로브를 이용한 융해곡선 분석방법, 융해곡선 분석을 통한 염기다형성 분석방법 및 염기다형성 분석 키트.
KR20150028063A (ko) * 2013-09-05 2015-03-13 주식회사 시선바이오머티리얼스 리포터 및 소광자가 결합된 프로브를 이용한 액상형 어레이 및 이를 이용한 표적핵산 또는 돌연변이 검출방법

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008109823A2 (fr) * 2007-03-08 2008-09-12 Idaho Technology Amorces pour analyse de fusion
WO2009135832A1 (fr) * 2008-05-06 2009-11-12 Qiagen Gmbh Détection simultanée de multiples séquences d'acide nucléique dans une réaction
US8691504B2 (en) * 2009-05-26 2014-04-08 Xiamen University Method for detecting variations in nucleic acid sequences
WO2011050173A1 (fr) * 2009-10-21 2011-04-28 Brandeis University Procédés, kits et mélanges réactionnels destinés à l'analyse de séquences d'acide nucléique monocaténaire
KR20140046688A (ko) * 2012-10-10 2014-04-21 주식회사 파나진 리포터 및 소광자가 결합된 pna 프로브를 이용한 융해곡선 분석방법, 융해곡선 분석을 통한 염기다형성 분석방법 및 염기다형성 분석 키트.
KR20150028063A (ko) * 2013-09-05 2015-03-13 주식회사 시선바이오머티리얼스 리포터 및 소광자가 결합된 프로브를 이용한 액상형 어레이 및 이를 이용한 표적핵산 또는 돌연변이 검출방법

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