KR20170113336A - Method for hydrogel-based genotyping of single nucleotide polymorphism and the apparatus therefor - Google Patents

Abstract

The present invention relates to a double label nucleotide consisting of a sequence complementary to a single nucleotide polymorphism (SNP) nucleotide sequence of a gene to be analyzed or an ADP (double stranded probe) comprising a double-label nucleotide sequence complementary to a wild gene to be analyzed, And a method for analyzing a single nucleotide polymorphism using the PCR reaction chip comprising the plurality of immobilized hydrogel porous microparticles. The present invention demonstrates that the PCR reaction and the detection of single nucleotide polymorphism are performed simultaneously, so that the analysis is quick and simple, and exhibits excellent selectivity and sensitivity. In addition, multiple SNPs of various kinds of genes can be detected simultaneously.

Description

METHOD FOR HYDROGEL-BASED GENOTYPING OF SINGLE NUCLEOTIDE POLYMORPHISM AND THE APPARATUS THEREFOR BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

Apparatus and methods for single base polymorphic genotyping analysis are disclosed herein.

Single nucleotide polymorphisms (SNPs) are nucleotide variations at specific loci of the human genome, usually found at a rate of one per 1000 bp on average. Since single nucleotide polymorphisms are very important as biomarkers for disease susceptibility and drug sensitivity both genetically and medically, many methods for single nucleotide polymorphic genotyping have been studied.

In particular, hydrogel-based single base polymorphism detection allows detection environments to be performed in a two-dimensional, three-dimensional matrix, thereby increasing probe density while increasing sensitivity of detection. Hydrogel-based bio-essay technology has been actively conducted since the past 10 years. Through the photocrosslinking of hydrogels, DNA fragments, miRNAs, and proteins among various types of biomarkers can be detected in multiple And are applied to biological quantitative analysis.

Single-nucleotide polymorphisms using hydrogels include temperature-dependent and pH-dependent detection methods. The temperature-dependent detection method uses a thermodynamic difference between two alleles combined with a probe. At a specific temperature, only the mutated gene binds to the probe to generate fluorescence. On the other hand, the wild gene can not bind to the probe, I use something that I can not stand out. Biosensors and Bioelectronics 79 (2016): 371-378). PH-Dependent Methods Similarly, mutant genes and wild-type genes, such as wild-type genes, Lt; RTI ID = 0.0 > pH < / RTI > environment. (Zhang, Huaibin, et al., &Quot; Genotyping by alkaline dehybridization using graphically encoded particles. "Chemistry-A European Journal 17.10 (2011): 2867-2873). However, both methods require temperature and pH optimization in common. That is, there is an inconvenience that specific temperature and pH at which the wild gene and the mutant gene are distinguishable must be specifically detected for each gene, which means that there is a limit in simultaneous multiple detection. In order to increase the sensitivity, the two methods amplify a gene to be detected through a polymerase chain reaction before detection. However, the two methods amplify a gene to be detected by a polymerase chain reaction (PCR) before detection, The occurrence of contamination in the course of the process may cause false information to be generated in subsequent detection of single nucleotide polymorphisms.

Therefore, there is a need for a more effective method for overcoming the problems of the single base polymorphism detection method using the hydrogel described above, in terms of technical aspects and cost.

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 Zhang, Huaibin, et al. "Genotyping by alkaline dehybridization using graphically encoded particles." Chemistry-A European Journal 17.10 (2011): 2867-2873

It is an object of the present invention to provide an apparatus and a method capable of detecting single nucleotide polymorphisms in various kinds of genes by simultaneous multiplexing using hydrogel.

In order to accomplish the above object, one aspect of the present invention is a polymerase chain reaction (PCR) reaction chip comprising a plurality of hydrogel porous microparticles, wherein at least one of the hydrogel porous microparticles has a single base polymorphism A first ADP (Anchored Double-Labled Probe) comprising a double-labeled nucleotide consisting of a sequence complementary to the SNP base sequence is immobilized, and at least one of the hydrogel- , A quencher and a second ADP comprising a double-labeled nucleotide consisting of a sequence complementary to the wild gene to be analyzed are immobilized. A fluorophore is attached to the 5 'end of each double label nucleotide, and a quencher (SNP) genetic trait analysis device to which a Quencher is linked All.

Another aspect of the present invention provides a single nucleotide polymorphism (SNP) genotyping analysis method using the single nucleotide polymorphism (SNP) genetic analysis apparatus.

The analysis apparatus and method of the present invention can perform quick and simple analysis and minimize false information due to contamination because PCR reaction and single nucleotide polymorphism (SNP) detection are simultaneously performed. The present invention can easily distinguish SNPs of a gene to be analyzed by on / off of a fluorescent signal by using probes (first ADP and second ADP) containing different double label nucleotides, Of dsDNA-binding dyes, and exhibits improved sensitivity based on PCR based on other analytical methods. In addition, by matching the annealing temperature to the probe design, it is possible to detect several kinds of genes simultaneously at the same temperature. In an embodiment, when a plurality of hydrogel porous microparticles are used, SNPs of various kinds of genes can be simultaneously detected without encoding / decoding. By using the hydrogel porous microparticles, it is possible to analyze the existing two-dimensional analysis three-dimensionally, which shows the improved density and sensitivity of the probe, and the hydrogel is biocompatible and thus suitable for biological analysis.

1 is an illustration of two types of hydrogel porous microparticles contained in a PCR reaction chip according to an embodiment of the present invention.
FIG. 2 is a diagram showing a state in which gene amplifications are generated in the early stage of a PCR reaction according to an embodiment of the present invention and diffused into the hydrogel microparticles to bind to ADPs.
FIG. 3 is a graph showing the fluorescence intensity of a homozygous SNP in the mid-PCR reaction according to an embodiment of the present invention. Fig.
FIG. 4 is a graph showing that both types of ADP are emitted from a fluorescent dye (F) when a heterozygous SNP at the mid and late stage of PCR reaction according to an embodiment of the present invention has a mutant gene and a wild gene simultaneously.
FIG. 5 is a graph showing the fluorescence of a hydrogel-based microparticle containing ADP having a T-spacer length of 12 T according to an embodiment of the present invention. The SNP target (+) means a case where the target gene of the ADP is added to the PCR solution of the real-time PCR, and the SNP target (-) means the case where the target gene of the ADP is not inserted.
6 is a graph showing the results of a real-time PCR using a PCR reaction chip containing hydrogel microparticles (SNP target (+)) containing ADP having a T-spacer length of 30T Results The fluorescence intensity was checked.
FIG. 7 is a graph comparing the fluorescence intensity according to the length of the T-spacer shown in FIG. 5 and FIG.
8A to 8C are graphs showing the comparison of fluorescence intensity according to the size of the molecular weight of the porogen inducing polymer and the type of the connecting portion included in the ADP when preparing the hydrogel porous fine particles according to an embodiment of the present invention. The molecular weight of the induction polymer is 600, the size of the molecular weight of the pore-generating polymer is 4000, and the size of the pore-generating polymer is 6000.
FIG. 9 shows a comparison of the amplification efficiencies according to the conditions of the annealing step of the PCR as one embodiment of the present invention. Condition # 1 is 60 ° C. and 15 sec, Condition # 2 is 60 ° C. and 25 sec, Condition # 3 means 55 ° C and 15 sec, Condition # 4 means 55 ° C and 25 sec, post means hydrogel porous microparticle, and sol means conventional qPCR.

Hereinafter, the present invention will be described in detail.

One embodiment of the present invention provides a single nucleotide polymorphism (SNP) genotyping analysis apparatus comprising a polymerase chain reaction (PCR) chip comprising a plurality of hydrogel porous microparticles.

In one embodiment, the at least one hydrogel porous microparticle contained in the PCR reaction chip includes a double-labeled nucleotide sequence complementary to a single nucleotide polymorphism (SNP) nucleotide sequence of a gene to be analyzed A first ADP (Anchored Double-labled Probe) is immobilized, and a second ADP comprising a double-labeled nucleotide consisting of a sequence complementary to the wild gene to be analyzed may be immobilized on the other one or more of the hydrogel porous microparticles .

Also, in one embodiment, a fluorescent dye and a quencher may be connected to the 5 'end and the 3' end of each double label nucleotide of the first ADP and the second ADP. Specifically, the fluorescent coloring agent according to an embodiment may be 6-carboxyfluorescein, tetrachlorofluorescein (TET) and 6-carboxy-2,4,4,5,7,7-hexachlorofluorescein succinimidyl ester (6-FAM) The quencher may include a dye selected from the group consisting of tetramethylrhodamine (TAMRA), Iowa Black ™ FQ, and BHQ ™.

In one embodiment, the gene to be analyzed includes polynucleotides such as DNA, and more specifically, human genomic DNA. The gene to be analyzed may be a homozygote or heterozygote depending on the characteristics of the genome as an example.

In one embodiment, the first ADP and the second ADP may include a connecting portion composed of 1 to 72 T bases or 1 to 50 A bases, and the connecting portion may be fixed to the hydrogel porous microparticles. More specifically, the connecting portion may be 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 immobilized on the hydrogel porous microparticles can facilitate complementary binding with the gene to be analyzed. In the present specification, the connection made of the T base is referred to as a Thymine spacer or a T-spacer, and the connection made of the A base is also referred to as an adenine spacer or an A-spacer . In one embodiment, when the number of T or A bases contained in the connecting portion is less than 12, it is not easy to bind to the gene to be analyzed. When the number of T bases is 72 and the number of A bases is more than 50, There is a problem that the ADP synthesized longer than the pore size is synthesized, the reaction is not smooth or ADP synthesis is impossible.

In one embodiment, the first ADP and the second ADP may be physically chemically immobilized on the hydrogel porous microparticles. For example, an acrylate functional group, an Avidin-Biotin bond or a Thiol-Ene bond, but it is not limited thereto as long as the ADP can be fixed to the microparticle Do not.

The monoclonal polymorphic trait analyzing apparatus will be described in detail with reference to FIG. In one embodiment, the PCR reaction chip may comprise two types of hydrogel scaffolds, each microparticle being complementary to a single nucleotide polymorphism (SNP) nucleotide sequence of the analyte gene (Human DNA) A second ADP comprising a double-labeled nucleotide consisting of a mutant-specific sequence and a double-labeled nucleotide consisting of a wild-specific sequence complementary to the wild gene to be analyzed are immobilized. The 5 'ends of the first ADP and the second ADP are immobilized on the hydrogel porous microparticles (Gel-Bound DNA Probe), and the immobilized ADP has a free movement including a T-spacer have. Since the fluorescent colorant (quencher) connected to each double-labeled nucleotide before the PCR reaction is located at a close distance from each other, the emission of the fluorescent colorant is suppressed by FRET (Fluorescence Resonance Energy Transfer) Do not. However, after the PCR reaction, if the gene to be analyzed contains a single nucleotide polymorphism (SNP) base sequence, fluorescence is generated by completely complementary binding with the double label nucleotide of the first ADP, The fluorescence is not developed due to incomplete complementary binding with the double label nucleotide. In one embodiment, if the gene to be analyzed is a wild-type gene that does not contain a single nucleotide polymorphism (SNP) nucleotide sequence, fluorescence is not incomplete due to incomplete complementary binding with the double-labeled nucleotide of the first ADP, The double label nucleotide completely complementary to the fluorescence is generated.

The hydrogel porous fine particle according to an embodiment of the present invention is not limited as long as it is a three dimensional structure. For example, the hydrogel porous fine particle may be a plate type, a post type, a spherical type, a hemispherical type or the like. Further, in one embodiment, the hydrogel porous fine particles may have a particle size of, for example, an average of from 10 μm to 5000 μm, and more specifically, a particle size of from 100 μm to 3000 μm on average. The hydrogel porous microparticles can be used without limitation as long as they are solid polymerizable (pre-polymer) materials. Specifically, polyethylene glycol-diacrylate (PEG-DA) or polyacrylamide ) Can be used.

In one embodiment, the hydrogel porous microparticle may be a porous structure including a void. In one embodiment, the porosity of the microparticles may be from 10% by volume to 80% by volume, more specifically from 20% by volume to 70% by volume, based on the total volume of the microparticles. Outside of the above range, the porosity may be deteriorated or the stability of the fine particles may become unstable.

 In one embodiment of the present invention, the hydrogel porous fine particles as described above may be prepared by mixing a pre-polymer, a porogen, a photoinitiator and purified water to prepare a prepolymer solution; Mixing the prepolymer solution with a solution comprising the first ADP or the second ADP; And loading the mixed solution into a PCR reaction chip, and then molding the mixed solution into a desired shape. At this time, the volume ratio of the solution containing the prepolymer solution and the nucleic acid primer may be 20 to 5: 1. As used herein, the term "pre-polymer" means a prepolymer in which polymerization or polycondensation reaction is stopped at an appropriate stage in order to facilitate molding of the polymer. In the case of the present invention, Means a polymer in an easily processable state. In one embodiment, the solution comprising the first ADP or the second ADP may further comprise a first ADP or a second ADP comprising a T-spacer and / or a functional group for immobilizing each ADP to the microparticles have.

In one embodiment, the step of preparing the prepolymer solution may further include adjusting the size of the pores formed in the fine particles by modifying the molecular weight of the pore-generating polymer contained in the solution. For example, polyethylene glycol (PEG) may be used as the porogen induction polymer. Specific examples thereof include PEG200, PEG300, PEG400, PEG600, PEG1000, PEG1500, PEG2000, PEG3000, PEG3350, PEG4000, PEG6000, PEG8000, PEG12000, PEG20000, PEG35000, PEG40000, etc. (manufacturer: Sigma Aldrich) can be used. The SNP genotyping analysis efficiency may vary depending on the molecular weight of the porogen inducing polymer.

In one embodiment, the manufacturing method may further include cleaning the formed hydrogel porous fine particles. The rinsing step may include removing the unfixed ADP inside the pores of the porous structure through cleaning, and may further comprise removing the porosity-inducing polymer.

The step of molding the mixed solution into a desired shape may include forming a desired shape through a mask or a mold or forming a droplet shape to form a desired shape, Thus, particles of various shapes and sizes can be produced.

The present invention also provides a method for preparing a hydrogel porous microparticle, which comprises injecting a plurality of hydrogel porous microparticles injected with different kinds of first ADP and second ADP depending on the kind of a gene to be analyzed when preparing the hydrogel porous microparticles And an analysis device capable of analyzing two or more different single nucleotide polymorphism (SNP) genotypes in a reaction chip without separately marking can be provided by classifying the positions of the PCR reaction chip in different positions. Alternatively, in one embodiment, a plurality of hydrogel porous microparticles injected with different kinds of first ADP and second ADP depending on the type of the gene to be analyzed may be colored using a quantum dot or the like (SNP) genotypes of two or more different types of SNPs in a reaction chip.

One embodiment of the present invention can provide a method for analyzing a single base polymorphic genetic trait using the single nucleotide polymorphism (SNP) genetic trait analysis apparatus described above. Specifically, the method comprises: injecting a PCR solution containing a gene to be analyzed, a forward primer, and a reverse primer into a PCR reaction chip of a SNP genotyping apparatus; A PCR step of amplifying the gene to be analyzed by an omni-directional primer and a reverse primer included in the PCR solution to generate a gene amplicon; The gene amplification diffuses into the hydrogel porous microparticle and binds to the first ADP or the second ADP; And confirming fluorescence of the hydrogel porous microparticles contained in the PCR reaction chip after completion of the PCR.

In one embodiment, the forward primer or the reverse primer may be a polynucleotide consisting of 10 to 50 contiguous DNA sequences, but the sequence and sequence length of the primer can be varied without restriction depending on the gene to be analyzed. In one embodiment, the amplicon means a PCR amplification product such as a polynucleotide such as DNA or RNA, an oligonucleotide, or the like. The gene amplification is not limited to the number of times of gene amplification but may be a maximum of all the amplification products of the gene, for example, about 100-200 bp. In addition, as an example, the PCR (polymerase chain reaction) can be performed by using a quantitative polymerase chain reaction (qPCR), a multiplex PCR, a real time PCR, (Real time qPCR) and real time multiplex PCR (Real time multiplex PCR).

The monoclonal polymorphic trait analysis method will be described in detail with reference to FIG. 2 to FIG. 4.

FIG. 2 is an illustration of an initial cycle of a real-time PCR reaction in which the forward primer and the reverse primer contained in the PCR solution amplify the base sequence around a specific single nucleotide polymorphism of the gene to be analyzed To generate a gene amplicon. At this time, since the amplified product has a base sequence of several hundred base pairs and a relatively short base sequence of several nanometers, it can diffuse into the hydrogel microparticles. The diffused fragments may have a sequence of wild genes or mutated genes (SNP) if homozygous, depending on the characteristics of the genome. In addition, if heterozygous, it has a sequence of wild genes 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. In addition, the omnidirectional primer can be complementarily bound to the gene amplification product, and with the aid of Taq DNA polymerase, it is possible to polymerize a complementary replication strand to a single strand fragment.

3 is an example of a real-time multiplex polymerase chain reaction middle cycle in the case of a homozygous gene. The cloned fragments in FIG. 2, that is, the amplifications, have a sequence of a nucleotide sequence of a mutant gene or a nucleotide sequence of a wild gene, and thus are included in two kinds of hydrogel porous microparticles according to an embodiment of the present invention Perfect matching can be achieved only with one kind of ADP among the first ADP or the second ADP. That is, when the gene amplification product according to an embodiment of the present invention diffuses into the hydrogel porous microparticles and binds to the first ADP or the second ADP, the step of analyzing the target gene may be performed using a single nucleotide polymorphism (SNP) Sequence complementary to the double-labeled nucleotide of the first ADP, and incomplete complementarity with the double-labeled nucleotide of the second ADP. In one embodiment, if the gene to be analyzed is a wild gene that does not contain a SNP base sequence, it is incompletely complementary to the double label nucleotide of the first ADP, and the double label nucleotide of the second ADP Fully complementary combining.

In one embodiment, the ADPs that are completely complementary to the gene amplification are matched with all the base sequences, so that the ADP is separated as the omnidirectional primer amplifies. Specifically, the forward primer is fragmented according to the exonuclease activity of the 5'-3 'nucleic acid of the Taq DNA polymerase in a chain polymerization in which a single-stranded section forms a double-stranded section. In this process, since the distance between the fluorescent colorant and the quencher is distant from each other, the fluorescence of the fluorescent coloring agent can be developed because FRET (fluorescent resonance energy transfer) phenomenon no longer occurs. At this time, since the fluorescent coloring agent is connected to the connecting portion, that is, the thymine spacer, the fluorescent gel stays in the porous fine particles of the hydrogel. On the other hand, in the case of incomplete complementary mismatching ADP, that is, mismatching ADP, the amplification of the omnidirectional primer does not cause light emission because ADP is not degraded by Taq DNA polymerase.

From the above viewpoint, the step of confirming the fluorescence coloring according to an embodiment of the present invention can distinguish whether complementary or incomplete complementary binding is complete, depending on whether fluorescence appears in the hydrogel porous fine particles of the present invention . In the step of confirming the fluorescence coloring according to an embodiment of the present invention, when fluorescence color develops in the hydrogel porous microparticles to which the first ADP is immobilized, the analysis target gene is expressed as a single nucleotide polymorphism (SNP) And determining that the analyzed gene is a wild gene that does not contain a single nucleotide polymorphism (SNP) nucleotide sequence when fluorescence color develops in the hydrogel porous microparticles having the second ADP immobilized thereon .

4 is an illustration of a real-time multiplex polymerase chain reaction intermediate cycle in the case of a heterozygous gene. In the case of the heterozygote, since the gene to be analyzed can have the mutation gene and the wild gene at the same time, the first ADP including the double label nucleotide consisting of the sequence complementary to the SNP nucleotide sequence of the gene to be analyzed and the sequence complementary to the wild gene to be analyzed The fluorescence can be developed in both types of hydrogel porous microparticles including a second ADP comprising a double-labeled nucleotide consisting of a single nucleotide.

In the analysis method according to an embodiment of the present invention, the PCR step may include a denaturation step, an annealing step, and an elongation step. Specifically, the annealing step may include a step of obtaining a high PCR amplification efficiency For 10 to 60 seconds at 50 to < RTI ID = 0.0 > 70 C. < / RTI >

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

[Example 1]

Columnar Hydrogel  Containing porous microparticles PCR Reaction chip  Produce

As one embodiment of the present invention, a PCR reaction chip in which a plurality of hydrogel porous microparticles were loaded in different positions was prepared.

First, a mixture of PEG 600 (poly (ethylene glycol)) (Mn 600) and DNase-free water (Irgacure 1173) at a ratio of 20: 40: 35: The polymer solution was mixed with 100 μM of CYP2C9 mutant-specific ADP in a volume ratio of 9: 1 to prepare an ADP-prepolymer mixed solution.

In another embodiment, an ADP-prepolymer mixed solution was prepared in the same manner as described above, except that ADP was used as CYP2C9 wild-specific ADP, VKORC1 mutant-specific ADP and VKORC1 wild-specific ADP, respectively.

At this time, the double-labeled nucleotide contained in each ADP was purchased from IDT (Integrated DNA Technologies). Iowa Boack (TM) FQ was used as a fluorophore at the 5 'end of each double-labeled nucleotide and i6 FAMK at the 3' end, and an A-spacer having 50 A bases as a linkage. Includes Acrydite as government.

Then, the ADP-prepolymer mixed solution in which the four different ADPs were mixed was loaded into the PCR reaction chip at different positions. The PCR reaction chip was photocrosslinked by irradiating UV (365 nm, 728 mW / cm ² ) for 100 ms through a mask. Then, ADP in the solution state without photo-crosslinking was washed by injecting DNase-free water to prepare a PCR reaction chip including four post-hydrogel porous microparticles having four kinds of ADPs Respectively.

Columnar Hydrogel  Porous microparticle-based single nucleotide polymorphism Simultaneous multiplexing  real time Polymerase chain reaction

To the prepared PCR reaction chip, 1 μL of DNA solution, 0.8 μL of 20 μM CYP2C9 specific forward primer, 0.8 μL of 20 μM CYP2C9 specific reverse primer, 0.8 μL of 20 μM VKORC1 specific omni-directional primer, 20 μM 0.8 μL of VKORC1specific reverse primer in μM concentration, 3.8 μL of DNase-free water, and 8 μL of TaqMan master mix. At this time, the DNA contained in the solution was gDNA extracted from a common human, and each primer was purchased from IDT (Integrated DNA Technologies).

PCR conditions were repeated 40 cycles after the first denaturation (95 ° C., 8 sec), followed by denaturation (95 ° C., 3 sec), annealing and elongation (60 ° C., 15 sec) Respectively.

[Example 2]

In an embodiment of the present invention, a plurality of hydrogel porous microparticles are prepared so as to have different colors, and a PCR reaction chip in which the porous microparticles are loaded is prepared.

Disc-shaped Hydrogel  Preparation of porous fine particle prepolymer

First, all four ADP solutions, which were the same as ADP used in [Example 1], were 100 μM CYP2C9 mutant-specific ADP, CYP2C9 wild-specific ADP, VKORC1 mutant-specific ADP and VKORC1 wild- Prepared.

Next, prepolymer mixed solutions having four different colors, each having a volume ratio of 1: 9 with each of the ADP solutions, were prepared. The proportions of the components to be mixed were as follows.

Specifically, a prepolymer mixed solution for fine particles of colorless cord was prepared by mixing PEG 700DA: PEG 600: DNase-free water: Irgacure 1173 in a volume ratio of 20: 40: 35: 5. In another embodiment, the prepolymer mixed solution for fine particles of red and green cords has a volume ratio of PEG 600: acrylated-quantum dot: DNase-free water: Irgacure 1173 = 40: 20: Mixed. The prepolymer mixed solution for the blue cord microparticles was mixed in a volume ratio of PEG 600: acrylated-quantum dot: DNase-free water: Irgacure 1173: blue foodcoloring = 40: 20: Prepared.

Stop flow lithography (SFL) Disc-shaped Hydrogel  Preparation of Porous Fine Particles

A 7-way PDMS mold was plasma-bonded by O ₂ plasma bonding to a polydimethylsilane (PDMS) -coated glass substrate, and the first to sixth 7-way PDMS inlets were filled with the colorless, red, , And blue cord prepolymer solution, respectively. The final seventh inlet connects the tube containing one of the four ADP solutions and the outlet of the PDMS channel connects the collection tube containing the 1X TET (Tris-EDTA-Tween) solution. One of the six injection ports to which the color code polymer solution was connected and the injection port to which the ADP solution was connected was moved at a pressure of about 1: 3. In the pressure actuator, the flow time and the stop time were set to about 500 ms. After setting the mask size in accordance with the width of the PDMS channel, the exposure time was set to 100 ms and the hold time was set to 200 ms.

Subsequently, disk-shaped hydrogel porous fine particles having the above four colors were prepared by SFL (stop flow lithography), respectively, and then stored at 4 ° C. In this case, the CYP2C9 mutant-specific ADP is colorless, the CYP2C9 wild-specific ADP is the red code, the VKORC1 mutant-specific ADP is the green code, and the VKORC1 wild-specific ADP is the blue code.

Disc-shaped Hydrogel  Porous microparticle-based single nucleotide polymorphism Simultaneous multiplexing  real time Polymerase chain reaction

1 μL of DNA in solution, 0.8 μL of 20 μM CYP2C9 specific upstream primer, 0.8 μL of 20 μM CYP2C9 specific downstream primer, 0.8 μL of 20 μM concentration of VKORC1 specific upstream primer, 20 μM , 0.8 μL of VKORC1specific downstream primer, 3.8 μL of DNase-free water, and 8 μL of TaqMan master mix. At this time, the DNA and the sequence information of each primer contained in the solution are the same as those described in [Example 1] above.

PCR conditions were repeated 40 cycles after the first denaturation (95 ° C., 8 sec), followed by denaturation (95 ° C., 3 sec), annealing and elongation (60 ° C., 15 sec) Respectively.

[Test Example 1]

As one embodiment of the present invention, the analysis efficiency of single nucleotide polymorphism (SNP) genotypes according to the length of the connecting portion included in each ADP, i.e., T-spacer, in the hydrogel porous microparticles of the single base polymorphism (SNP) Respectively.

First, real-time PCR was performed using the PCR reaction chip prepared in the same manner as in [Example 1]. The PCR reaction chip contained two kinds of hydrogel porous microparticles, and the two microparticles contained ADP containing a double label nucleotide having the following base sequence.

-CYP2C9 allele 2-mutant-specific ADP (SEQ ID NO: 1): 5'-GAGCATTGAGGACCGTGTTCAAGAGGA-3 '

Each of the ADPs included 12 or 30 T-bases (T-spacer length: 12T, 30T).

The primer sequence information of the PCR solution included in the real-time PCR was as follows.

- omni-directional primer (SEQ ID NO: 2): 5'-GGA ATT GTT TTC AGC AAT GGA AAG-3 '

- Reverse primer (SEQ ID NO: 3): 5'-GGT CAG TGA TAT GGA GTA GGG TCA-3 '

FIG. 5 shows the results of a real-time PCR reaction of hydrogel porous microparticles having a T-spacer length of 12T (ADP sequence: 5Acryd / 12T / i6 FAMK / probe (27nt) / 3BHQ_1). As can be seen from FIG. 5, when the target gene of the ADP was added to the PCR solution of the real-time PCR (SNP target (+)), the fluorescence was developed in the hydrogel porous microparticles, (SNP target (-)), fluorescence was not developed in the hydrogel porous microparticles.

FIG. 6 shows a real-time PCR reaction of hydrogel porous microparticles having a T-spacer length of 30T (ADP sequence: 5Acryd / 30T / i6 FAMK / probe (27nt) / 3IABkFQ). As can be seen from FIG. 6, when the target gene of ADP was added to the PCR solution of the real-time PCR, fluorescence was observed in the hydrogel microparticles.

FIG. 7 is a graph comparing fluorescence intensities according to the length of the T-spacer shown in FIGS. 5 and 6. It can be seen that fluorescence coloration is more remarkable when the length is 30T, and the analysis efficiency is excellent.

[Test Example 2]

As an embodiment of the present invention, the analysis efficiency of single nucleotide polymorphism (SNP) genotypes according to the pore size of the hydrogel porous microparticles of the single base polymorphism (SNP) genetic analysis apparatus and the length of the junctions included in ADP .

8A is a schematic diagram of a prepolymer mixed solution prepared by mixing PEG 700DA, PEG 600, DNase-free water and Irgacure 1173 in a volume ratio of 20: 40: 35: 5 (v / v) 50A, 50T, and 72T, the PCR reaction chip prepared in the same manner as in [Test Example 1] above was used to perform real-time PCR. In this case, 30T means a connection portion composed of 30 T bases, 50A means a connection portion composed of 50 A bases, 50T means a connection portion composed of 50 T bases, and 72T means a connection portion composed of 72 T bases.

8B is a graph showing the results obtained by using a prepolymer mixed solution prepared by mixing PEG 700DA: PEG4000 (50% w / v): DNase-free water: Irgacure 1173 in a volume ratio of 20: 65: 10: 5 (v / v) The results of real-time PCR using the PCR reaction chip prepared in the same manner as in [Test Example 1] except that the junctions were 30T, 50A, 50T, and 72T, respectively.

8C is a graph showing the results of using a prepolymer mixed solution prepared by mixing PEG 700DA: PEG6000 (50% w / v): DNase-free water: Irgacure 1173 in a volume ratio of 18: 58: 19: 5 (v / v) The results of real-time PCR using the PCR reaction chip prepared in the same manner as in [Test Example 1] except that the junctions were 30T, 50A, 50T, and 72T, respectively.

As the molecular weight of the porogen inducible polymer increased, the fluorescence intensity of the hydrogel porous microparticles containing mutant-specific ADP increased and the detection efficiency of single nucleotide polymorphism (SNP) increased. In addition, as the length of the linkage included in ADP was increased, the degree of freedom of the photocrosslinked primer was increased and the detection efficiency of SNP was increased. When the number of T base was more than a certain number, the difference in efficiency was not significant.

[Test Example 3]

As one embodiment of the present invention, PCR amplification efficiencies according to changes in PCR conditions in the analysis of single base polymorphism (SNP) genetic traits were compared.

FIG. 9 shows that the annealing and elongation conditions are 60 ° C. and 15 sec (Condition # 1), 60 ° C. and 25 sec (Condition # 2), 55 ° C. and 15 sec 4), a real-time PCR reaction was carried out in the same manner as in [Test Example 1] above. In FIG. 9, post refers to the case where hydrogel porous microparticles are used, and "sol" refers to the result of the experiment using the conventional qPCR in a solution state under the annealing and elongation conditions as a comparative example.

As a result, it can be confirmed that the PCR amplification efficiency is most excellent when annealing and elongation conditions are 60 ° C and 15 sec (Condition # 1).

<110> Korea Institute of Science and Technology <120> METHOD FOR HYDROGEL-BASED GENOTYPING OF SINGLE NUCLEOTIDE          POLYMORPHISM AND THE APPARATUS THEREFOR <130> 17P113IND <150> KR 10-2016-0038413 <151> 2016-03-30 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9 allele 2-mutant-specific ADP sequence <400> 1 gagcattgag gaccgtgttc aagagga 27 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of CYP2C9 allele 2-mutant-specific ADP sequence <400> 2 ggaattgttt tcagcaatgg aaag 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of CYP2C9 allele 2-mutant-specific ADP sequence <400> 3 ggtcagtgat atggagtagg gtca 24

Claims (11)

A polymerase chain reaction (PCR) chip comprising a plurality of hydrogel porous microparticles,
In one or more of the hydrogel porous microparticles, a first ADP (Anchored Double-labled Probe) comprising a double-labeled nucleotide consisting of a sequence complementary to a single nucleotide polymorphism (SNP) nucleotide sequence of a gene to be analyzed And a second ADP comprising a fluorescent labeling agent, a quencher and a double-labeled nucleotide sequence complementary to the wild gene to be analyzed is immobilized on the one or more other hydrogel porous microparticles,
Wherein a fluorophore is attached to the 5 'end of the double label nucleotide and a quencher is attached to the 3' end of each double labeled nucleotide. The method of claim 1, wherein the first ADP and the second ADP comprise a connection comprising 1 to 72 T bases or 1 to 50 A bases and the connection is fixed to the hydrogel porous microparticles. Basic polymorphism (SNP) genetic characterization device. The method of claim 1, wherein the first ADP and the second ADP are chemically immobilized on the hydrogel porous microparticles by an acrylate function, an Avidin-Biotin bond or a Thiol-Ene bond. (SNP) genetic trait analyzing apparatus. The method of claim 1, wherein the fluorescent colorant is selected from the group consisting of 6-carboxyfluorescein, tetrachlorofluorescein, and 6-carboxy-2,4,4,5,7,7-hexachlorofluorescein succinimidyl ester. (SNP) &lt; / RTI &gt; genetic trait analyzing device. 2. The apparatus according to claim 1, wherein the quencher is selected from the group consisting of tetramethylrhodamine (TAMRA), Iowa Black (TM) FQ, and BHQ (TM). 2. The method according to claim 1, wherein, when the gene to be analyzed contains a SNP base sequence, it fluoresce completely by complementary binding with the double label nucleotide of the first ADP, and incomplete with the double label nucleotide of the second ADP (SNP) genetic trait analyzing apparatus according to claim 1 or 2, wherein fluorescence is not developed. The method according to claim 1, wherein if the gene to be analyzed is a wild gene that does not contain a single nucleotide polymorphism (SNP) base sequence, the fluorescence is not developed due to incomplete complementary binding with the double label nucleotide of the first ADP, (SNP) genetic trait analyzing device, wherein fluorescence is generated by completely complementary binding with double label nucleotides. A method for analyzing a SNP genotype, comprising the steps of: injecting a PCR solution containing a gene to be analyzed, an omnidirectional primer and a reverse primer into a PCR reaction chip of the SNP genetic analysis apparatus of any one of claims 1 to 7;
A PCR step of amplifying the gene to be analyzed by an omni-directional primer and a reverse primer included in the PCR solution to generate a gene amplification product;
The gene amplification diffuses into the hydrogel porous microparticle and binds to the first ADP or the second ADP; And
Confirming the fluorescence of the hydrogel porous fine particles contained in the PCR reaction chip after completion of the PCR;
(SNP) genetic trait analysis method. 9. The method of claim 8, wherein the gene amplification is diffused into the hydrogel porous microparticles and binding to the first ADP or the second ADP
Wherein the gene to be analyzed contains a single nucleotide polymorphism (SNP) base sequence, the second labeled nucleotide completely complementary binds to the double labeled nucleotide of the first ADP and incompletely complementarily binds to the double labeled nucleotide of the second ADP, Polymorphism (SNP) genetic trait analysis method. 9. The method of claim 8, wherein the gene amplification is diffused into the hydrogel porous microparticles and binding to the first ADP or the second ADP
If the gene to be analyzed is a wild gene that does not contain a single nucleotide polymorphism (SNP) base sequence, it is incompletely complementary to the double label nucleotide of the first ADP and completely complementary to the double label nucleotide of the second ADP (SNP) genetic trait analysis method. 9. The method of claim 8,
When the first ADP-immobilized hydrogel porous microparticle shows fluorescence color, the gene to be analyzed contains a single nucleotide polymorphism (SNP) nucleotide sequence, and the second ADP-immobilized hydrogel porous microparticles have fluorescence And determining that the gene to be analyzed is a wild gene that does not include a single nucleotide polymorphism (SNP) nucleotide sequence.

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