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WO2017171385A2 - Procédé d'analyse de caractère génétique polymorphe mononucléotidique à base d'hydrogel et appareil associé - Google Patents

Procédé d'analyse de caractère génétique polymorphe mononucléotidique à base d'hydrogel et appareil associé Download PDF

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WO2017171385A2
WO2017171385A2 PCT/KR2017/003392 KR2017003392W WO2017171385A2 WO 2017171385 A2 WO2017171385 A2 WO 2017171385A2 KR 2017003392 W KR2017003392 W KR 2017003392W WO 2017171385 A2 WO2017171385 A2 WO 2017171385A2
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adp
snp
gene
single nucleotide
double
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Korean (ko)
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WO2017171385A3 (fr
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최낙원
이수현
강지윤
최웅선
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Korea Institute of Science and Technology KIST
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Korea Institute of Science and Technology KIST
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Priority claimed from KR1020170039414A external-priority patent/KR101873858B1/ko
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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

Definitions

  • Single nucleotide polymorphisms are base modifications at specific loci in the human genome and are generally found at a rate of one per 1000 bp on average. Since monobasic polymorphism has a very important meaning as a biomarker genetically and medically in disease sensitivity and drug sensitivity, many methods for single nucleotide polymorphism genotyping have been studied.
  • single base polymorphism detection based on hydrogels allows the detection environment to be made in a two-dimensional to three-dimensional matrix, thereby increasing the probe density and increasing the sensitivity of the detection.
  • Hydrogel-based bioassay technology has been actively conducted since 10 years ago, and multiple detection of DNA fragments, miRNAs, and proteins among multiple biomarkers by fluorescence through photocrosslinking of hydrogels It is applied to biological quantitative analysis.
  • Single base polymorphisms using hydrogels include temperature-dependent detection and pH-dependent detection. Temperature-dependent detection is based on the thermodynamic difference between the two alleles that bind to the probe.At a certain temperature, only the mutant gene binds to the probe to produce fluorescence, whereas the wild gene fails to bind to the probe, resulting in fluorescence. Use inconspicuous points. (Jung, Yun Kyung, Jungkyu Kim, and Richard A. Mathies. "Microfluidic hydrogel arrays for direct genotyping of clinical samples.” Biosensors and Bioelectronics 79 (2016): 371-378) pH-dependent methods also likewise mutated and wild genes. Take advantage of the fact that the binding with a specific probe depends on the pH environment.
  • An object of the present invention is to provide a device and method capable of detecting a single nucleotide polymorphism for several types of genes simultaneously by using a hydrogel.
  • One aspect of the present invention to achieve the above object comprises a polymerase chain reaction (PCR) reaction chip comprising a plurality of hydrogel porous microparticles, at least one hydrogel porous microparticles in a single base polymorphism of the gene of analysis (SNP)
  • a first ADP (Anchored Double-labled Probe) comprising a double-labeled nucleotide (double-labeled nucleotide) consisting of a sequence complementary to the base is fixed, and at least one hydrogel porous microparticles a fluorescent chromophore
  • a second ADP comprising a double-labeled nucleotide consisting of a quencher and a sequence complementary to the wild gene to be analyzed is immobilized, a fluorophore at the 5 'end of each of the double-labeled nucleotides, and a quench at the 3' end.
  • SNP single nucleotide polymorphism
  • Another aspect of the present invention provides a single base polymorphism (SNP) genotyping method using the single base polymorphism (SNP) genotyping apparatus.
  • the PCR reaction and the single nucleotide polymorphism (SNP) detection are performed at the same time, so that the analysis is faster and simpler than existing methods, and false information due to contamination can be minimized.
  • the present invention can be easily distinguished from the SNP inclusion of the gene of analysis using the probe (first ADP and second ADP) containing the different double-labeled nucleotides by the on / off of the fluorescent signal, such as conventional CYBR green, etc.
  • the fluorescence detection method using the dsDNA-binding dye of the remarkably excellent selectivity shows an improved sensitivity than other conventional methods.
  • hydrogel porous microparticles are used, SNPs of various types of genes may be simultaneously and multiplely detected without a separate encoding / decoding technique.
  • the use of the hydrogel porous microparticles allows the existing two-dimensional analysis to be analyzed three-dimensionally, which further improves the density and sensitivity of the probe and is suitable for biological analysis because the hydrogel has biocompatibility.
  • Figure 1 is an illustration of two types of hydrogel porous microparticles contained in the PCR reaction chip according to an embodiment of the present invention.
  • Figure 2 is a diagram showing a state in which the gene amplification is generated at the beginning of the PCR reaction according to an embodiment of the present invention is diffused into the hydrogel porous microparticles to bind to each ADP.
  • Figure 3 is a homozygous gene (Homozygous SNP) in the middle of the PCR reaction according to an embodiment of the present invention, the perfect complement with the one of the two types of ADP (luminescence) in the fluorophore (F) It is a figure which shows.
  • FIG 4 is a diagram showing that when the heterozygous gene (Heterozygous SNP) in the middle of the PCR reaction according to an embodiment of the present invention at the same time having both the mutant gene and wild gene both types of ADP luminescence in the fluorophore (F).
  • Heterozygous SNP Heterozygous SNP
  • FIG. 5 is a diagram illustrating whether fluorescence is generated as a result of real-time polymerase chain reaction using hydrogel porous microparticles containing ADP having a length of 12T as a T-spacer.
  • SNP target (+) means a case where the target gene of the ADP is put into a PCR solution of a real-time polymerase chain reaction
  • SNP target (-) means a case where the target gene of the ADP is not inserted.
  • FIG. 6 is an embodiment of the present invention using a PCR reaction chip containing a hydrogel porous microparticles (SNP target (+)) containing an ADP having a T-spacer of 30T length real-time polymerase chain reaction The result confirms whether or not fluorescence is generated.
  • SNP target (+) hydrogel porous microparticles
  • FIG. 7 is a graph illustrating fluorescence intensities according to lengths of the T-spacers identified in FIGS. 5 and 6.
  • FIG. 8A to 8C are graphs comparing the fluorescence intensity according to the molecular weight of the covalent polymer and the type of the connection part included in the ADP when preparing the hydrogel porous microparticles as an embodiment of the present invention.
  • the molecular weight size of the induction polymer is 600
  • FIG. 8B is a graph of the molecular weight size of the covalent polymer is 4000
  • FIG. 8C is comparing the fluorescence intensity according to the molecular weight of the covalent polymer and the type of the connection part included in the ADP when preparing the hydrogel porous microparticles as an embodiment of the present invention.
  • the molecular weight size of the induction polymer is 600
  • FIG. 8B is a graph of the molecular weight size of the covalent polymer is 4000
  • Figure 9 shows the comparison of the amplification efficiency according to the conditions of the annealing (annealing) step of the PCR as an embodiment of the present invention
  • Condition # 1 is 60 °C and 15 sec
  • Condition # 2 is 60 °C and 25 sec
  • Condition # 3 is 55 °C and 15 sec
  • Condition # 4 refers to 55 °C and 25 sec
  • post is a hydrogel porous microparticles
  • sol is a conventional solution of qPCR in solution.
  • An embodiment of the present invention provides a single base polymorphism (SNP) genotyping apparatus including a polymerase chain reaction (PCR) reaction chip including a plurality of hydrogel porous microparticles.
  • SNP single base polymorphism
  • PCR polymerase chain reaction
  • the at least one hydrogel porous microparticle included in the PCR reaction chip includes a double-labeled nucleotide consisting of a sequence complementary to a single nucleotide polymorphism (SNP) sequence of the gene of interest.
  • a first ADP (Anchored Double-labled Probe) is immobilized, and the second ADP including a double-labeled nucleotide consisting of a sequence complementary to the wild gene of interest may be immobilized on one or more of the other hydrogel porous microparticles.
  • a fluorescent colorant may be connected to the 5 'end of each of the double-labeled nucleotides of the first ADP and the second ADP, and a quencher may be connected to the 3' end.
  • the fluorescent color developer according to one embodiment 6-FAM (6-carboxyfluorescein), TET (tetrachlorofluorescein) and 6-HEX (6-carboxy-2,4,4,5,7,7-hexachlorofluorescein succinimidyl ester) It may include a dye selected from the group consisting of, and the like, the matting agent may include one selected from the group consisting of tetramethylrhodamine (TAMRA), Iowa Black TM FQ, BHQ TM and the like, but is not limited thereto.
  • TAMRA tetramethylrhodamine
  • the gene to be analyzed includes a polynucleotide such as DNA, and more specifically, may be human genomic DNA.
  • the gene to be analyzed may be a homozygous or heterozygous conjugate according to an embodiment of the genome.
  • the first ADP and the second ADP may include a connection part consisting of 1 to 72 T bases or 1 to 50 A bases, and the connection part may be fixed to the hydrogel porous microparticles.
  • the linking portion may be more specifically composed of 12 to 50 T bases or A bases.
  • the connection part provides a degree of freedom so that the first ADP and the second ADP fixed to the hydrogel porous microparticles are easily complementary to the gene to be analyzed.
  • the connecting portion consisting of the T base is referred to as a thymine spacer or a T-spacer
  • the connecting portion consisting of the A base is also referred to as an adenine spacer or an A-spacer. .
  • the T or A base included in the linking portion is less than 12, it is not easy to bind to the analyte gene, and when the T base is 72 and the A base is more than 50, the hydrogel porous microparticles ADP longer than the pore size is synthesized, so that the reaction is not smooth or ADP synthesis is impossible.
  • the first ADP and the second ADP may be physically and chemically fixed to the hydrogel porous microparticles.
  • it may be chemically immobilized by an acrylate functional group, an avidin-biotin bond, or a thiol-ene bond, but is not limited thereto if the ADP can be immobilized on the microparticle. Do not.
  • the PCR reaction chip may include two types of hydrogel porous microparticles (Hydrogel scaffold), each microparticle complementary to a single nucleotide polymorphism (SNP) sequence of the gene (Human DNA).
  • a first ADP comprising a double-labeled nucleotide consisting of a Mutant-specific sequence and a second ADP comprising a double-labeled nucleotide consisting of a wild-specific sequence complementary to the wild gene to be analyzed are immobilized.
  • the gene to be analyzed includes a single nucleotide polymorphism (SNP) sequence as an example, it is completely complementary to the double-labeled nucleotide of the first ADP, and fluoresces the color of the second ADP. Incomplete complementary binding with double label nucleotides prevents fluorescence from developing.
  • the gene to be analyzed is a wild gene that does not include a single nucleotide polymorphism (SNP) sequence, it may be incompletely complementary to the double-labeled nucleotide of the first ADP, thereby preventing the fluorescence from developing. Fluorescence is developed by full complementary binding to the double label nucleotides of.
  • Hydrogel porous microparticles according to an embodiment of the present invention is not limited as long as it is a three-dimensional structure, for example, plate-like (plate type), columnar (post type), spherical (spherical type), hemispherical (hemispherical type) and the like.
  • the hydrogel porous microparticles may have, for example, an average particle size of 10 ⁇ m to 5000 ⁇ m, and more specifically, an average particle size of 100 ⁇ m to 3000 ⁇ m.
  • the material of the hydrogel porous microparticles can be used without limitation as long as it is a polymer that can be solidified. Specifically, polyethylene glycol-diacrylate (PEG-DA) or polyacrylamide (PAAM) can be used. Hydrophilic polymers such as
  • the hydrogel porous microparticles may be a porous structure including pores.
  • the porosity of the microparticles may be 10% to 80% by volume, more specifically 20% to 70% by volume with respect to the total volume of the microparticles. If it falls outside the above range, the porosity may drop or the stability of the fine particles may become unstable.
  • Hydrogel porous microparticles as an embodiment of the present invention comprises the steps of preparing a prepolymer solution by mixing a prepolymer (pre-polymer), a povalent copolymer (porogen), a photoinitiator and purified water; Mixing the prepolymer solution with a solution comprising the first ADP or the second ADP; And after loading the mixed solution in the PCR reaction chip, it can be prepared by a manufacturing method comprising the step of molding the mixed solution to the desired shape.
  • the volume ratio of the solution containing the prepolymer solution and the nucleic acid primer may be 20 to 5: 1.
  • the term "pre-polymer” refers to a prepolymer obtained by stopping a polymerization or polycondensation reaction at an appropriate stage in order to facilitate molding of the polymer, and in the present invention, molding before solidification. It means the polymer of the state which is easy to process.
  • the solution including the first ADP or the second ADP may further include a T-spacer and / or a functional group for immobilizing the respective ADP on the microparticles. have.
  • the step of preparing the prepolymer solution may further include adjusting the size of pores formed in the microparticles by modifying the molecular weight of the pore polymer included in the solution.
  • the covalent polymer may be, for example, polyethylene glycol (PEG), and specifically, PEG200, PEG300, PEG400, PEG600, PEG1000, PEG1500, PEG2000, PEG3000, PEG3350, PEG4000, PEG6000, PEG8000, PEG10000, PEG12000, PEG20000, PEG35000, PEG40000 and the like (manufactured by Sigma Aldrich) can be used.
  • Single base polymorphism (SNP) genotyping efficiency may vary depending on the molecular weight of the covalent polymer.
  • the manufacturing method may further include washing the molded hydrogel porous microparticles.
  • the cleaning step may include removing ADP that is not fixed in the pores of the porous structure through cleaning, and may further include removing the covalent polymer.
  • Forming the mixed solution into a desired shape may include forming a desired shape through a mask or a mold, or forming a droplet into a desired shape by photocrosslinking. This can be produced in particles of various shapes and sizes.
  • the present invention can manufacture a plurality of hydrogel porous microparticles injected by varying the type of the first ADP and the second ADP according to the type of the gene to be analyzed when the hydrogel porous microparticles are prepared as an embodiment.
  • an analysis apparatus capable of analyzing two or more different single nucleotide polymorphisms (SNP) genotypes without a separate label in one reaction chip.
  • a plurality of hydrogel porous microparticles injected by varying the types of the first ADP and the second ADP according to the type of the gene to be analyzed may use the color of each microparticle, such as a quantum dot.
  • SNP single nucleotide polymorphisms
  • One embodiment of the present invention can provide a single base polymorphism genotyping method using the above-described single base polymorphism (SNP) genotyping apparatus.
  • the method comprises the steps of injecting a PCR solution containing the analyte gene, forward primer, reverse primer to the PCR reaction chip of a single nucleotide polymorphism (SNP) genotyping apparatus; A PCR step of forward and backward primers included in the PCR solution to amplify the gene to be analyzed to generate a gene amplification (amplicon); Dispersing the gene amplification product into the hydrogel porous microparticles and binding the first ADP or the second ADP; And after completion of the PCR may include determining whether the fluorescent color of the hydrogel porous microparticles contained in the PCR reaction chip.
  • the forward primer or reverse primer may be a polynucleotide consisting of 10 to 50 consecutive DNA sequences, but the sequence type and sequence length of the primer may be modified without limitation depending on the gene to be analyzed.
  • the gene amplification means a PCR amplification of a polynucleotide, such as DNA or RNA, oligonucleotides.
  • the gene amplification is not limited to the number of gene amplification, and is the broadest to include all amplification of the gene, for example, may have a size of about 100-200 bp.
  • PCR polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • Multiplex polymerase chain reaction Multiplex polymerase chain reaction
  • Real time PCR real time quantitative polymerase chain reaction
  • qPCR quantitative polymerase chain reaction
  • Multiplex PCR multiplex polymerase chain reaction
  • Real time PCR real time polymerase chain reaction
  • real-time quantitative polymerase chain reaction One or more of a real time qPCR and a real time multiplex PCR.
  • the single nucleotide polymorphism genotyping method will be described in detail with reference to FIGS. 2 to 4.
  • Figure 2 is an illustration of the initial cycle of the real-time multiplex polymerase chain reaction, the forward primer and the reverse primer contained in the PCR solution in the PCR reaction chip amplifies the nucleotide sequence around the specific monobasic polymorphism of the gene of analysis Gene amplification (amplicon) can be generated.
  • the amplification product since the amplification product has a relatively short base sequence of several hundred base pairs and several nm in size, it may diffuse into the hydrogel porous microparticles.
  • Spreading fragments may have a sequence of wild gene or variant gene (SNP) if homozygous, depending on the nature of the genome.
  • SNP wild gene or variant gene
  • Heterozygous has a sequence of wild and mutant genes at the same time.
  • the fragments diffused into the microparticles are complementarily bound to the first ADP or the second ADP according to the complementary base sequence.
  • the omnidirectional primer may bind to the gene amplification complementarily to polymerize a replication strand complementary to a single stranded fragment with the help of Taq DNA polymerase.
  • Figure 3 is an illustration of the late cycle of the real-time multiplex polymerase chain reaction for homozygous genes. Since the fragments, ie, amplifiers, which are cloned in FIG. 2 have a sequence of one of a nucleotide sequence of a mutant gene or a nucleotide sequence of a wild gene, are included in each of two types of hydrogel porous microparticles according to an embodiment of the present invention. Perfect matching with only one type of ADP, either the first ADP or the second ADP.
  • the step of the gene amplification product according to an embodiment of the present invention is diffused into the hydrogel porous microparticles and combined with the first ADP or the second ADP, so that the gene of analysis is a single nucleotide polymorphism (SNP) base.
  • Including the sequence may include full complementary binding to the double label nucleotide of the first ADP, and incomplete complementary binding to the double label nucleotide of the second ADP.
  • the gene of interest is a wild gene that does not include a single nucleotide polymorphism (SNP) sequence, it may be incompletely complementary binding with the double label nucleotide of the first ADP, and with the double label nucleotide of the second ADP. Completely complementary binding.
  • the ADP is completely complementary to the gene amplification, so that all base sequences are matched with each other, so that the forward primer is amplified and the ADP is separated.
  • the forward primer is fragmented according to the 5'-3 'nucleic acid terminal hydrolase activity of Taq DNA polymerase in a chain polymerization reaction in which a single strand fragment forms a double strand fragment.
  • fluorescence resonance energy transfer FRET
  • the fluorophore stays in the hydrogel porous microparticles because it is connected to the connecting portion, that is, thymine spacer.
  • the connecting portion that is, thymine spacer.
  • the step of confirming whether the fluorescence is developed according to an embodiment of the present invention can distinguish whether it is completely complementary or incomplete complementary binding depending on whether fluorescence is present in the hydrogel porous microparticles of the present invention.
  • the step of confirming whether the fluorescence coloration according to an embodiment of the present invention if the fluorescent color appears in the hydrogel porous microparticles to which the first ADP is fixed the gene of analysis is a single base polymorphism (SNP) base Comprising a sequence, and if the fluorescent color appears on the hydrogel porous microparticles fixed to the second ADP may include determining that the gene to be analyzed is a wild gene that does not contain a single nucleotide polymorphism (SNP) nucleotide sequence .
  • SNP single base polymorphism
  • Figure 4 is an illustration of the late cycle of the real-time multiplex polymerase chain reaction for heterozygous genes.
  • the gene of analysis may have a mutant gene and a wild gene at the same time, and thus the sequence complementary to the first ADP and the wild gene of analysis comprising a double-labeled nucleotide consisting of a sequence complementary to the SNP sequence of the gene of analysis. Fluorescence may be developed in both types of hydrogel porous microparticles including a second ADP including a double-labeled nucleotide.
  • the PCR step may include a denaturation step, an annealing step, and an elongation step.
  • the annealing step may obtain a high PCR amplification efficiency. In order to be made for 10 to 60 seconds at 50 to 70 °C.
  • a PCR reaction chip was prepared in which a plurality of hydrogel porous microparticles were loaded at different positions.
  • PEG 700DA poly (ethylene glycol) diacrylate
  • Mn 700 PEG 600 (poly (ethylene glycol); Mn 600): DNase-free water: Irgacure 1173 was mixed in a volume ratio of 20: 40: 35: 5 100 ⁇ M of CYP2C9 mutant-specific ADP was mixed in a 9: 1 volume ratio to prepare an ADP-prepolymer mixed solution.
  • ADP-prepolymer mixed solution was prepared in the same manner as above except that ADP was used, respectively, CYP2C9 wild-specific ADP, VKORC1 mutant-specific ADP and VKORC1 wild-specific ADP.
  • double-labeled nucleotides included in each ADP were purchased from IDT (Integrated DNA Technologies). At the 5 'end of each double-labeled nucleotide, i6 FAMK was used as Fluorophore at the 5' end, and Iowa Boack FQ was used as the quencher at the 3 'end.
  • the ADP-prepolymer mixed solution mixed with the four different ADPs was loaded in different positions in the PCR reaction chip.
  • the PCR reaction chip was photocrosslinked by irradiating UV (365 nm, 728 mW / cm 2 ) for 100 ms through a mask. Subsequently, the ADP in the non-crosslinked solution state was washed by injecting DNase-free water to prepare a PCR reaction chip including four post-type hydrogel porous microparticles having a total of four types of ADP. It was.
  • PCR conditions were performed to repeat 40 cycles of the first denaturation (95 ° C., 8 sec), followed by the second denaturation (95 ° C., 3 sec), annealing and elongation (60 ° C., 15 sec). It was.
  • a plurality of hydrogel porous microparticles were prepared to have different colors to prepare a PCR reaction chip loaded with the same.
  • the prepolymer mixed solution for colorless cord microparticles was prepared by mixing PEG 700DA: PEG 600: DNase-free water: Irgacure 1173 in a volume ratio of 20: 40: 35: 5.
  • the tubes containing the blue cord prepolymer solution were connected.
  • the final seventh inlet connected a tube containing one of the four ADP solutions, and the outlet of the PDMS channel connected a collection tube containing 1 ⁇ TET (Tris-EDTA-Tween) solution.
  • One of the six inlets to which the color coded polymer solution was connected and the inlet to which the ADP solution was connected were moved by applying a pressure of about 1: 3.
  • the flow time and stop time of the pressure actuator were set to about 500 ms. After setting the mask size according to the width of the PDMS channel, an exposure time was set to 100 ms and a hold time was set to 200 ms.
  • hydrogel porous microparticles in the form of discs each having the four colors were prepared through SFL (stop flow lithography), and then stored at 4 ° C.
  • CYP2C9 mutant-specific ADP has a color code
  • CYP2C9 wild-specific ADP has a red code
  • VKORC1 mutant-specific ADP has a green code
  • VKORC1 wild-specific ADP has a blue code.
  • PCR conditions were performed to repeat 40 cycles of the first denaturation (95 ° C., 8 sec), followed by the second denaturation (95 ° C., 3 sec), annealing and elongation (60 ° C., 15 sec). It was.
  • the single base polymorphism (SNP) genotyping efficiency according to the length of T-spacer, that is, the connection portion of each ADP in the hydrogel porous microparticles of the single base polymorphism (SNP) genotyping apparatus Compared.
  • PCR reaction chip included two kinds of hydrogel porous microparticles, and the two kinds of microparticles included an ADP including a double-labeled nucleotide having the following nucleotide sequence.
  • each of the ADP was to include a junction consisting of 12 or 30 T bases respectively (length of T-spacer: 12T, 30T).
  • Primer sequence information of the PCR solution included in the real-time polymerase chain reaction is as follows.
  • Reverse primer (SEQ ID NO: 3): 5'-GGT CAG TGA TAT GGA GTA GGG TCA-3 '
  • FIG. 5 shows the results of real-time polymerase chain reaction of the hydrogel porous microparticles having a T-spacer length of 12T (ADP sequence: 5Acryd / 12T / i6 FAMK / probe (27nt) / 3BHQ_1).
  • SNP target (+) when the target gene of the ADP was added to the PCR solution of the real-time polymerase chain reaction (SNP target (+)), fluorescence was developed in the hydrogel porous microparticles, and the target gene was not added. In the case of (SNP target (-)), fluorescence was not developed in the hydrogel porous microparticles.
  • FIG. 6 shows the results of real-time polymerase chain reaction of hydrogel porous microparticles having a T-spacer length of 30T (ADP sequence: 5Acryd / 30T / i6 FAMK / probe (27nt) / 3IABkFQ).
  • ADP sequence 5Acryd / 30T / i6 FAMK / probe (27nt) / 3IABkFQ
  • FIG. 7 is a graph comparing fluorescence intensities according to the lengths of the T-spacers identified in FIGS. 5 and 6, and when the length is 30T, the fluorescence color is more clearly seen, indicating that the analysis efficiency is excellent.
  • the single base polymorphism (SNP) genotyping efficiency was compared according to the pore size of the hydrogel porous microparticles of the single base polymorphism (SNP) genotyping apparatus and the length of the junction included in the ADP. .
  • 8A is a prepolymer mixed solution obtained by mixing PEG 700DA: PEG 600: DNase-free water: Irgacure 1173 in a volume ratio of 20: 40: 35: 5 (v / v), and each of the connecting portions included in ADP is 30T, Except that 50A, 50T, 72T is shown the results of real-time polymerase chain reaction using a PCR reaction chip prepared in the same manner as in [Test Example 1].
  • 30T is a connection portion consisting of 30 T bases
  • 50A is a connection portion consisting of 50 A bases
  • 50T means a connection portion consisting of 50 T bases
  • 72T means a connection portion consisting of 72 T bases.
  • 8B is a prepolymer mixed solution containing PEG 700DA: PEG4000 (50% w / v): DNase-free water: Irgacure 1173 in a volume ratio of 20: 65: 10: 5 (v / v), and includes ADP. Except that the connecting portion is 30T, 50A, 50T, 72T, respectively, shows the results of real-time polymerase chain reaction using a PCR reaction chip prepared in the same manner as in [Test Example 1].
  • FIG. 8C shows a prepolymer mixed solution containing PEG 700DA: PEG6000 (50% w / v): DNase-free water: Irgacure 1173 in a volume ratio of 18: 58: 19: 5 (v / v), and includes ADP. Except that the connecting portion is 30T, 50A, 50T, 72T, respectively, shows the results of real-time polymerase chain reaction using a PCR reaction chip prepared in the same manner as in [Test Example 1].
  • the fluorescence color development of the hydrogel porous microparticles including the mutant-specific ADP increased, and the single base polymorphism (SNP) detection efficiency also increased.
  • the length of the linkage included in the ADP increased, the degree of freedom of photocrosslinked primer was increased, and the single nucleotide polymorphism (SNP) detection efficiency was increased.
  • the number of T bases was more than a certain number, the difference in efficiency was not large.
  • PCR amplification efficiency according to PCR condition change was compared in single nucleotide polymorphism (SNP) genotyping.
  • SNP single nucleotide polymorphism

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Abstract

La présente invention concerne un appareil d'analyse de caractère génétique polymorphe mononucléotidique et un procédé d'analyse associé utilisant une puce de réaction de PCR qui comprend une pluralité de microparticules poreuses d'hydrogel dans lesquelles une sonde à double marqueur ancrée (ADP) comprenant un nucléotide à double marqueur constitué de séquences complémentaires d'une séquence nucléotidique d'un polymorphisme mononucléotidique (SNP) d'un gène à analyser ou un nucléotide à double marqueur constitué de séquences complémentaires d'un gène de type sauvage à analyser est immobilisée. La présente invention permet une analyse rapide et facile car la réaction de PCR et la détection de polymorphisme mononucléotidique sont effectuées simultanément, et elle présente une excellente sélectivité et une excellente sensibilité. De plus, des SNP de divers types de gènes peuvent être détectés simultanément et en multiples.
PCT/KR2017/003392 2016-03-30 2017-03-29 Procédé d'analyse de caractère génétique polymorphe mononucléotidique à base d'hydrogel et appareil associé Ceased WO2017171385A2 (fr)

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KR20160038413 2016-03-30
KR10-2016-0038413 2016-03-30
KR1020170039414A KR101873858B1 (ko) 2016-03-30 2017-03-28 하이드로겔 기반 단일염기 다형성 유전형질 분석 방법 및 장치
KR10-2017-0039414 2017-03-28

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WO2017171385A2 true WO2017171385A2 (fr) 2017-10-05
WO2017171385A3 WO2017171385A3 (fr) 2018-09-07

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