WO2017119755A1 - Device for determining single nucleotide polymorphism by using two types of magnetic particles - Google Patents

Abstract

The present invention relates to a method and a kit for determining single nucleotide polymorphism (SNP). The method for determining SNP of the present invention uses two types of magnetic particles, and thus can perform SNP determination with high accuracy and can simplify and automatize a process.

Description

Single base polymorphism discrimination automation device using 2 kinds of magnetic particles

The present invention provides a single base polymorphism determination automation device and a single base polymorphism determination automation method using two kinds of magnetic particles; And it relates to a single base polymorphism determination kit.

There are differences between individuals in the entire nucleotide sequence of the genome, and the differences between individuals at the base level (more precisely, appearing at a frequency of more than 1% of the population) are called Single Nucleotide Polymorphisms (SNPs).

It is thought that SNPs exist in human DNA at a frequency of about 1000 bases. The majority of SNPs exist in the non-translated region and do not have a change in genetic characteristics, but SNPs in genes or control regions are likely to cause differences between genetic individuals. With the improvement of gene therapy and diagnostic technology, a single gene can be used for drug susceptibility (resistance and presence of side effects), resistance to infectious diseases, environmental factors of lifestyle diseases, and the list of various diseases such as cancer, Alzheimer's disease, and animal genetic diseases. Many explanatory examples of base polymorphism have been found, and the possibility of individual Taylor-made or predictive medicine using SNP interpretation is widely expected.

In order to realize tailor-made medical care and predictive medicine, it is urgent to establish a method and a system for tying a single base polymorphism that can be realized quickly and simply and at low cost in the medical field. The market for genetic analysis is expanding, and many techniques for gene array analysis are being developed. Among these, it is divided into using the recognition efficiency of a specific sequence by an enzyme which performs extension, ligation, and cleavage of nucleic acid by using hybridization of polynucleotides. Recently, the research on the typing method of SNP is being actively conducted.

As the SNP determination method, a method of detecting SNPs by fluorescence intensity emitted from a probe hybridized to template single-stranded DNA, a method of detecting SNPs using a restriction enzyme that recognizes and cleaves specific sequences, and the like are known. For example, Korean Patent Laid-Open Publication No. 10-2013-0065258 discloses a SNP determination method using DNA melting analysis including a dilution step of a PCR amplicon, and Korean Patent Publication No. 10-2012-0046018 Disclosed are methods and kits for polymorphism detection during real time PCR.

As described above, various methods for SNP determination have been reported, but each equipment is required for each step of SNP determination, and there are problems such as lowering precision when several processes are combined.

On the other hand, the gene polymorphism of osteoporosis-related TGF-β1 may be due to mutation of the coden 10 portion of exon1 of the TGF-β1 gene. The mutation in Codon 10 causes Leu to turn Pro. The normal nucleotide sequence of this region shows CTG, but many osteoporosis patients have CCG, and this polymorphism is distributed at high frequency in patients with osteoporosis. In addition, the gene of TGF-β1 has a high GC content, which makes it difficult to raise a high efficiency PCR effect and does not have a restriction enzyme site useful for SNP detection by RFLP analysis.

Known methods of interpreting the gene polymorphism of TGF-β1 first extract DNA from blood or extract mRNA to prepare cDNA by reverse transcription. Subsequently, PCR is performed using a primer set so as to amplify a portion containing the desired polymorphism by using the prepared DNA as a template, and then the base sequence of the obtained PCR product is analyzed. In addition, the base sequence analysis of the PCR product does not necessarily determine the complete base sequence can be interpreted as a restriction fragment length polymorphism.

Detection of the presence of osteoporosis risk factors based on genetic analysis results can also be used to determine whether the identified genotype is already the same or different genotype that proves to be a osteoporosis risk factor by examining the presence or absence of osteoporosis. For example, if the TGF-β1 gene polymorph is mutated from CTG of Codon 10 of exon 1 of the human TGF-β1 gene to CCG, the genotype of CTG is a risk factor.

An object of the present invention is to provide a hybridization reaction unit that hybridizes with a SNP probe with high efficiency and high precision within a short time, and automates the entire process from DNA extraction to monobasic polymorphism detection.

In addition, it is an object of the present invention to provide an automated apparatus that can perform reaction temperature management so as to obtain accurate detection results, but does not require a wide stage.

A first aspect of the present invention provides a single nucleotide polymorphism (SNP) determination automated device, comprising: a first reaction vessel, a reaction vessel support, and a means for applying magnetic force to the bottom of the reaction vessel in an ON / OFF manner; A first reaction unit performing a DNA separation process using a magnetic particle having a (+) charge and a PCR process using a PCR primer modified with a first functional group; Means for moving the first functional group modified polynucleotide containing PCR reaction product from the first reaction portion to the second reaction portion; And a second reaction vessel, a reaction vessel support, and a means for applying magnetic force to the lower portion of the reaction vessel in an ON / OFF manner, wherein the magnetic particles are modified with a second functional group that binds the first functional group-modified polynucleotide to the first functional group. Single base polymorphism determination automatic device characterized in that it comprises a second reaction unit for performing a process of immobilizing to, a single stranded (single stranded) process of the first functional group-modified polynucleotide and hybridization (Sbridization) with the SNP discrimination probe To provide.

The second aspect of the present invention is a method for determining single nucleotide polymorphism (SNP), wherein the first reaction vessel uses a positively charged magnetic particle to determine the SNP from a sample of an individual. Adsorbing the prepared DNA, applying magnetic force to collect magnetic particles having the DNA adsorbed under the first reaction vessel, and removing the supernatant; A second step of injecting a PCR reagent containing a primer modified with a first functional group into a first reaction vessel and performing PCR using DNA adsorbed on magnetic particles as a template to obtain a first functional modified polynucleotide-containing PCR reaction product; A third step of transferring the PCR reaction product in the supernatant of the first reaction vessel to the second reaction vessel; Fourth to obtain the magnetic particles bound to the first functional group modified polynucleotide through the combination of the first functional group and the second functional group by using the magnetic particles modified with the second functional group that binds to the first functional group in the second reaction vessel. step; A fifth step of treating the magnetic particles to which the first functional group-modified polynucleotide is bound to give an alkaline solution to obtain a single stranded first functional-modified polynucleotide; Hybridizing a single-stranded first functional group-modified polynucleotide to a label-modified SNP determination probe; A seventh step of applying magnetic force to collect magnetic particles adsorbed to the SNP determination probe and the polynucleotide adsorbed under the second reaction vessel, and to remove the supernatant containing the SNP determination probe which has not been hybridized; And an eighth step of measuring the level of the labeling substance of the SNP determination probe which is not removed from the second reaction vessel.

A third aspect of the invention provides a magnetic particle having a (+) charge, a PCR primer modified with a first functional group, a magnetic particle modified with a second functional group bound to the first functional group, and optionally a probe modification for SNP determination. It provides a kit for determining the single-base polymorphism comprising a label.

Since the SNP determination method using two kinds of magnetic particles according to the present invention in different reaction vessels can be automated through magnetic force control, it is possible to perform single base polymorphism determination with high determination accuracy that can be easily carried out in a medical field and various diseases. It can be usefully used for the prevention and treatment of.

1 is a conceptual diagram showing the internal configuration of a single-base polymorphism detection apparatus according to an embodiment of the present invention.

2 is a conceptual diagram illustrating a nucleic acid extraction process by magnetic particles.

Figure 3 is a flow chart according to one embodiment for detecting SNP TGF-β1 gene.

Figure 4 is a diagram showing the result of determining the SNP of TGF-β1 in 784 blood samples by the SNP determination method of the present invention.

5 is a conceptual diagram showing each step of the single-base polymorphism determination method according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

Single nucleotide polymorphism (SNP) refers to the case where two or more alleles exist in one locus, and that only a single base varies among humans among polymorphic sites.

An allele refers to several types of genes that exist at the same locus of homologous chromosomes. Alleles are also used to indicate polymorphism, for example, SNPs have two kinds of bialleles.

In the present invention, "individual" may refer to all animals, especially humans, to determine the presence and genotype of the SNP using the SNP determination apparatus or method of the present invention. In the present invention, a "sample of an individual" is separated from the individual, and may be urine, blood, various body fluids, saliva, and the like, and is included without limitation as long as a genetic material can be obtained for the purposes of the present invention.

In the present invention, DNA is not limited to native DNA and cDNA, as well as a polynucleotide as long as it can be amplified by PCR as well as a negative charge and can be hybridized with an SNP probe.

SNPs are currently being studied for the prevention and treatment of various diseases. For example, it is known to cause various human diseases including sickle cell anemia, beta-thalassemia, cystic fibrosis, and the apolipoprotein E (APOE) gene is associated with a high risk of Alzheimer's disease. Known. In addition, TGF-β1 is a marker of osteoporosis, and it is known that a mutation from T ²⁹ (Leu ¹⁰ ) to C ²⁹ (Pro ¹⁰ ) of the TGF-β1 gene is associated with bone loss.

Single base polymorphism (SNP) determination method may include a series of DNA collection process, PCR process, hybridization (hybridization) to the SNP determination probe and SNP detection process (eg, fluorescence measurement process).

 The present inventors have diligently tried to develop a SNP discrimination method that simplifies the process while showing high precision, and as a result, (i) positively charged magnetic particles capable of binding to nucleic acids, and (ii) In the case of using magnetic beads that can be combined with a PCR product, the present invention was found to be able to automate the entire process of SNP determination while having high precision for SNP.

The present invention is characterized by the use of two or more types of magnetic particles in the separation (B) / glass (F) separation for automation. That is, the present invention uses magnetic particles modified with a second functional group that binds to the first functional group to immobilize the positively charged magnetic particles for DNA extraction and the PCR product of the first functional group modified primer. to be.

The present invention is based on the discovery that the yield of PCR does not drop even when the negatively charged DNA is electrostatically adsorbed to the positively charged magnetic particles, and thus, nucleic acid extraction, PCR, Another feature is that hybridization with SNP probes and optionally SNP detection are both designed to be automated in one system unit.

The single-base polymorphism determination device of the present invention is a second binding to the first functional group to immobilize the magnetic particles having a (+) charge in the first reaction vessel, the immobilized PCR product of the first functional group modified primer in the second reaction vessel By using the magnetic particles modified with functional groups in sequence and separating the bound (B) / free (F) by an externally generated magnetic force, that is, the polynucleotide bound to the magnetic particles is magnetic By separating the supernatant from the substance that is not bound to the particles and removing the supernatant, the polynucleotide bound to the magnetic particles can be easily separated without contamination, and the SNP can be discriminated with high precision. Automation of the process can also be achieved by controlling the magnetic particles.

The present invention collects the magnetic particles adsorbed nucleic acid extracted to a specific site in the container by the magnetic force control device, the magnetic particles adsorbed single-stranded polynucleotide, or the polynucleotide hybridized with the probe is adsorbed. Magnetic particles can be collected.

The present invention can easily discard the supernatant by pipetting after performing various reactions including washing with respect to the polynucleotide fixed to the reaction vessel through the magnetic particles. In addition, the present invention can distribute a variety of liquids, including the cleaning liquid to the reaction vessel by pipetting, it is possible to induce agitation by pipetting.

In the present invention, the magnetic particles are at least one metal selected from the group consisting of iron (Fe), nickel (Ni), cobalt (Co), manganese (Mn), bismuth (Bi) and zinc (Zn). It may be an alloy of or a metal oxide or alloy oxide selected from them.

In the present invention, since the magnetic particles having a positive charge have a positive charge on the surface, the negatively charged nucleic acid is adsorbed on the surface of the magnetic particles, so that the nucleic acid can be extracted from the sample, and the PCR is carried out while the extracted DNA is adsorbed. Can be used. In one embodiment of the present invention, the magnetic particles having a (+) charge may be amino group-modified magnetic beads modified with an amino group on the surface of the magnetic beads.

The positively charged magnetic particles may lose positive charges or exhibit negative charges as pH increases, so that the positive surface charges may change negatively.

The positively charged magnetic particles may be modified by, for example, organosilane functionalization, where the organosilane molecules are bonded to the surface, and the surface may be modified with a banded functional group by the bound organosilane molecules. .

The functional group is 3-aminopropyl group, aminomethyl group, 2-aminoethyl group, N-methyl-3-aminopropyl group, N- (2-aminoethyl) -3-aminopropyl group, N-aminoethyl-3-amino It may be a propyl group or an N, N-di (2-aminoethyl) -3-aminopropyl group, but is not limited thereto as long as it can carry a (+) charge.

Positive charges include, but are not limited to, organosilane precursors that provide a functional group, such as 3-aminoproryltriethoxysilane (APTES), aminomethyltriethoxysilane, 2-aminoethyltriethoxysilane, N-methyl-3-aminoproryltriethoxysilane, N- (2-aminoethyl) -3- aminoproryltriethoxysilane, N-aminoethyl-3-aminoproryltriethoxysilane, N, N-di (2-aminoethyl) -3-aminoproryltriethoxysilane or mixtures thereof.

A polynucleotide modified with a first functional group prepared from a PCR reaction product obtained by amplifying a DNA separated by the (+)-charged magnetic particle with a primer modified with a first functional group was added to a magnetic particle modified with a second functional group. Non-limiting examples of the bond between the immobilizing first and second functional groups include coordinate bonds, covalent bonds, metallic bonds, hydrogen bonds, and ionic bonds. bonds, antigen-antibody binding and ligand-receptor binding. Specific binding is preferable for the bond between the first functional group and the second functional group.

Ligand receptor binding between biotin and avidin may be formed between the bionucleotide-modified polynucleotide and the avidin-modified magnetic particle or vice versa. Alternatively, since the avidin has a plurality of biotin binding sites, the biotinylated polynucleotide and the magnetic bead may bind to avidin. The avidin includes, without limitation, avidin, streptavidin, deglycosylated avidin (NeutrAvidin).

In one embodiment of the present invention, the magnetic particles modified with the second functional group that binds to the first functional group may be streptavidin-modified magnetic beads modified with streptavidin on the surface of the magnetic beads. Therefore, biotin-modified polynucleotides can be obtained through a biotin-streptavidin reaction from a PCR reaction product obtained by amplifying the DNA extracted by the amino group-modified magnetic beads with a biotin-modified primer.

In the present invention, single stranded refers to a process of making a double-stranded polynucleotide into a single-stranded polynucleotide for hybridization of a probe, and may be performed by treatment with an alkaline solution. Specifically, the alkaline solution may be a NaOH solution, but is not limited thereto.

In the present invention, a probe is a polynucleotide capable of hybridizing to a single-stranded DNA fragment, and is an oligonucleotide capable of sequence-specific binding to the complementary strand of a nucleic acid. Probes of the present invention are allele specific probes that can hybridize to only one of the alleles and cause hybridization differences between different allelic forms. In addition, depending on the genotype can be modified with different labeling substances. Since the probe to be bound depends on the genotype of the sample, a difference in the signal generated from the labeled material modified in the probe occurs. Thus, the hybridization difference can be detected by a label modified in the probe, the genotype of the SNP can be determined by the ratio of the measured label.

In the present invention, the labeling substance is a substance capable of generating a detectable signal. The labeling material applicable to the present invention may be a quantum dot, magnetic bead nanoparticles, gold nanoparticles, fluorescent dyes (eg, Cy3 or Cy5), fluorescent proteins, nanophosphors or silicon nanoparticles, but for the purposes of the present invention It is not limited as long as it is a substance capable of generating a detectable signal due to hybridization. The label may be detected by fluorescence microscopy, SEM, TEM, CT, MRI and the like.

The SNP determination probe modified with the labeling substance may be a probe modified with different labeling substances according to the genotype of the SNP. The genotype can be determined by adding a label-modified probe bound to a specific genotype to a sample and measuring the signal of the label released from the bound probe.

Single base polymorphism determination automated apparatus according to the present invention,

A first reaction vessel, a reaction vessel support, and a means for applying magnetic force to the lower portion of the reaction vessel in an ON / OFF manner, and a DNA separation process using a magnetic particle having a (+) charge and modified with a first functional group A first reaction unit performing a PCR process using a PCR primer;

Means for moving the first functional group modified polynucleotide containing PCR reaction product from the first reaction portion to the second reaction portion; And

A second reaction vessel, a reaction vessel support, and a means for applying magnetic force to the lower portion of the reaction vessel in an ON / OFF manner, the magnetic particles modified with a second functional group that binds the first functional group-modified polynucleotide to the first functional group. And a second reaction unit performing a step of immobilizing, a single stranded step of modifying a first functional group-modified polynucleotide, and a step of hybridizing with a probe for determining an SNP.

Therefore, the single nucleotide polymorphism detection apparatus according to the present invention, (1) DNA separation step, (2) PCR step, (3) immobilization step, (4) single stranded step, (5) SNP discrimination Hybridization with the probe (hybridization) and (6) labeling step detection can be performed automatically.

Specifically, the following steps can be performed using an automated monobasic polymorphism determination device according to the invention:

In the first reaction vessel, magnetic particles having a positive charge are used to adsorb the DNA prepared from the sample of the individual to be determined for the SNP, and magnetic particles having the DNA adsorbed are collected under the first reaction vessel by applying magnetic force. A first step of removing the supernatant;

A second step of injecting a PCR reagent containing a primer modified with a first functional group into a first reaction vessel and performing PCR using DNA adsorbed on magnetic particles as a template to obtain a first functional modified polynucleotide-containing PCR reaction product;

A third step of transferring the PCR reaction product in the supernatant of the first reaction vessel to the second reaction vessel;

Fourth to obtain the magnetic particles bound to the first functional group modified polynucleotide through the combination of the first functional group and the second functional group by using the magnetic particles modified with the second functional group that binds to the first functional group in the second reaction vessel. step;

A fifth step of treating the magnetic particles to which the first functional group-modified polynucleotide is bound to give an alkaline solution to obtain a single stranded first functional-modified polynucleotide;

Hybridizing a single-stranded first functional group-modified polynucleotide to a label-modified SNP determination probe;

A seventh step of applying magnetic force to collect magnetic particles adsorbed to the SNP determination probe and the polynucleotide adsorbed under the second reaction vessel, and to remove the supernatant containing the SNP determination probe which has not been hybridized; And

An eighth step of measuring the level of the labeling substance of the SNP determination probe that was not removed from the second reaction vessel.

1 is a conceptual diagram showing the internal configuration of a single-base polymorphism detection apparatus according to an embodiment of the present invention.

In the first reaction unit 1, a DNA separation process using magnetic particles having a (+) charge and a PCR process using a primer modified with a first functional group are performed. To this end, the first reaction unit 1 includes a first reaction vessel, a reaction vessel support, and a means for applying magnetic force to the lower portion of the reaction vessel in an ON / OFF manner.

The single-base polymorphism discrimination apparatus is provided with respect to the first reaction part (1), from the sample contained in the first reaction container in the first reaction container equipped with the magnetic particles having a (+) charge. After binding the charged nucleic acid to the magnetic particles and applying a magnetic force to separate the nucleic acid by the binding (B) / free (F) separation (see Figure 2), (ii) the first reaction vessel in the first Using a PCR primer modified with a functional group, PCR is performed to amplify the DNA portion defined by a pair of primers by conducting PCR in a state where the negatively charged DNA is attached to the magnetically charged magnetic particles. have. At this time, one strand of the amplified DNA which is a PCR product contains a primer having a first functional group. Subsequently, the single nucleotide polymorphism discrimination apparatus is programmed to (iii) move the DNA amplified by PCR from the first reaction section to the second reaction section.

In the second reaction unit (2), the DNA amplified by PCR is immobilized to a magnetic particle modified with a second functional group that binds to the first functional group, a single stranded DNA process, and hybridized with an SNP probe. (See FIG. 3). To this end, the second reaction unit 2 includes a second reaction vessel, a reaction vessel support, and a means for applying magnetic force to the lower portion of the reaction vessel in an ON / OFF manner.

A single base polymorphism discrimination apparatus is a PCR modified with a first functional group using magnetic particles modified with a second functional group that binds to a first functional group in a second reaction vessel in relation to the second reaction unit (2). The amplified DNA was immobilized on the magnetic particles using a primer, (v) single stranded DNA in the second reaction vessel, and (vi) one or more SNP probes and magnetic particles in the second reaction vessel. It is programmed to hybridize with a single strand of DNA immobilized on. In this case, since the DNA strand including the primer modified with the first functional group in the PCR product is immobilized on the magnetic particle modified with the second functional group, the SNP probe is complementary to the DNA strand including the primer modified with the first functional group. It is preferable.

The first reaction vessel and the second reaction vessel may comprise one or more distinct wells capable of receiving a sample and carrying out various reactions. For example, it may be a 24-96 well maicroplate capable of simultaneously testing a plurality of samples. The reaction vessel may be made of a resin having high versatility, but is not particularly limited in shape and material. The reaction vessel 8 may be detachably replaced or fixed to the reaction vessel support.

The first reaction unit 1 and the second reaction unit 2 include a support 9 for holding and holding the reaction vessel, a means for applying magnetic force to the lower portion of the reaction vessel in an ON / OFF manner (insertion diagram), and optionally The magnetic force control device 14 further includes.

The reaction vessel support 9 is preferably made of metal having a shape in close contact with the surface of the reaction vessel.

The magnetic force control device generates magnetic force at an appropriate time for the purpose of the present invention to enable the magnetic particles to be collected at specific portions of the container.

The means for applying the magnetic force in an ON / OFF manner and / or the magnetic force control device 14 generates a magnetic force corresponding to each of the one or more compartments of the reaction vessel, thereby immobilizing the magnetic particles under each compartment of the reaction vessel. Can be. Therefore, through the magnetic force control, it is possible to apply a magnetic force to the magnetic particles used for separation in the container type (B) / glass (F). If a means for applying magnetic force in an ON / OFF manner is provided below each section of the reaction vessel, preferably directly below, the magnetic force in the lower portion of the reaction vessel can be collected in a narrow area under the container by turning on and off. For example, by approaching the source of magnetism under the vessel, the magnetic force acting on the magnetic particles in the reaction vessel can be increased, and the magnetic force acting by isolating the source of magnetism downward or laterally from the vessel can be reduced. As the source of magnetic force approaches the bottom of the container, the magnetic source may extend up and down in accordance with the magnetic flux to collect the magnetic particles at a specific part of the container, and the area where the magnetic particles collect may be the bottom of the container, but is not limited thereto. no.

Therefore, in the first reaction vessel using magnetic particles having positive charges, the magnetic force is applied to collect the positively charged magnetic particles bound to the negatively charged nucleic acid to the reaction vessel. Nucleic acid can be separated by bound (B) / free (F) separation. In addition, in the second reaction vessel that hybridizes the DNA strand immobilized on the SNP discrimination probe and the magnetic particles, the SNP discrimination probe which is not hybridized to the DNA strand can be removed through the supernatant by applying magnetic force. In addition, by applying a magnetic force under the conditions for separating the SNP probe hybridized to the DNA strand and collecting the magnetic particles in the reaction vessel, the probe for determining the SNP from the magnetic particles to detect the SNP determination probe hybridized in the next step Can be separated.

On the other hand, a reaction vessel made of a resin capable of adsorbing a large amount of magnetic particles at an interface inside the container is inappropriate.

Non-limiting examples of magnetic sources include permanent magnets, electromagnets, and the like. Permanent magnets are preferred in terms of magnetic flux density and volume. Permanent magnets are arranged side by side on the array so as to correspond to each well of the reaction vessel having two or more compartments, and alternating N and S poles next to each other can prevent confusion of magnetic flux acting on the reaction vessel. Magnetic particles can be aggregated efficiently.

In addition, the first reaction unit and the second reaction unit may further include a heating cooling device (10, 11) and / or temperature sensor 12 for adjusting the temperature of the support and / or the reaction vessel.

The warm cooling device may include a heater 10 for warming the reaction vessel support 9 and a cooler 11 for cooling. In addition, a temperature sensor 12 for measuring the temperature of the support may be connected to the temperature control device 13 to control the operation of the heater or cooler.

Accordingly, the warm cooling devices 10 and 11 and / or the temperature sensor 12 for controlling the temperature of the support and / or the reaction vessel in the first reaction section and the second reaction section are hybridized during PCR or with SNP probes. It helps to precisely control the temperature required.

Specifically, in the first reaction vessel, a PCR process may be performed through temperature control, and in the second reaction vessel, denature, annealing, and the like for hybridization with an SNP probe through temperature control. Combined (B) / glass (F) separation can be performed (see FIG. 3). In the second reaction vessel, a process of hybridizing single-stranded DNA and SNP probe in a PCR product is performed under stringent temperature conditions (FIG. 3).

Conventional DNA probe measuring devices, including DNA samples, reagent units, reagent dispensing units, reaction vessel transport units, and hybridization units, are known for the critical temperature and B / F of the reaction in the hybridization process. Since the temperature control at the time of separation was not carried out, an error of the detection result occurred, such as an increase in non-specific adsorption.

The temperature control device 13 may control the inside of the reaction vessel at a DNA denature temperature (eg, 96 ° C.) and at an annealing temperature (eg, 40 to 70 ° C.). In addition, the temperature control device can also control the reaction time by the timer function.

Measurement of sample nucleic acid through hybridization using SNP discrimination probe requires simple repetition because of the large number of samples, and requires a large installation area because equipment used in each process is independent. It takes a long time to control the reaction temperature and progress the reaction, and it is necessary to automate the hybridization process in order to obtain a high precision for a small amount of sample.

In one embodiment of the present invention, by attaching a heating cooling device and a magnetic force control unit to a reaction section that performs hybridization, the hybridization process can be automated even with a simple operation. It is possible.

On the other hand, the single base polymorphism determination automation apparatus of the present invention may be provided with a movable head portion 7 equipped with one or more pipettes. The head portion 7 may include an arm unit moving in the X-Z axis direction.

Each arm unit comprises means for moving a tip nozzle 8 and a head portion; Means for attaching and detaching a tip to each tip nozzle; And means for sucking and releasing the treatment liquid (waste liquid, washing liquid) from the mounted tip.

Pipettes may inhale and dispense liquid by generating a partial vacuum in the liquid receiving chamber and optionally vacuuming a vaccum release. In the present specification, a pipette may be used interchangeably as a tip rack and a pipettor. The tip of the pipette can be removed or fixed from the rack.

Single base polymorphism determination automated device comprises a reagent storage container; Waste liquid portion; The cleaning liquid storage container and the cleaning liquid with a cooling device may further include.

The moving means mounted on the head part can move the head part to the first reaction part, the second reaction part, the reagent storage container, the waste liquid part and the cleaning liquid part by a program.

Accordingly, the pipette of the head portion may carry reagents in the first reaction vessel or the second reaction vessel or waste liquid out of the first reaction vessel or the second reaction vessel according to a program. In addition, the pipette of the head portion may clean the pipette tip in the cleaning liquid portion according to the program.

The device according to the invention may comprise a reagent tray 6 with all the reagent holding vessels required from nucleic acid extraction to SNP level detection. Reagent trays can contain labeled reagents or enzyme chromogenic substrates, for example, when labeling is required to detect hybridized nucleic acids. When using a sample labeled with a nucleic acid in advance, it is not necessary to have the reagent tray.

Reagents that can be used in the single-base polymorphism determination automated device include blood hemolytic agents, mixed solution for PCR (2X PCR buffer, dNTP, MgCl ₂ , BSA, Taq polymerase), and one or more types of SNPs identified as markers. There is a probe.

The apparatus according to the present invention includes a waste liquid portion (3) for discarding the sample liquid in the tip, a tip rack (4) for holding a dispensing tip for dispensing the cleaning liquid and reagents, and the waste liquid sucked from the reaction portion during magnetic separation / cleaning. A waste container for discharge and storage is placed underneath. Space can be reduced by placing the tip rack and the waste container perpendicular to the moving plane of the head. Tip rack (4) has a hole in the taper section of the disposer tip so that the disposer tip (5) is prepared before starting the measurement. Install the tip rack filled with tip after sterilization to the tip rack fixing part (4).

In addition, the head portion 7 may further include a buffer dispenser.

On the other hand, the single base polymorphism determination automated device may include a movable label detector. The detector may be mounted to the head portion or may be provided separately from the head portion.

The detector may include one or more chips capable of sensing a label such as a phosphor; A chip nozzle which can be equipped with the chip and sucks and injects the treatment liquid from the second reaction vessel; Means for attaching and detaching chips to each chip nozzle; Movement in the X-Z axis direction.

The chip may, for example, comprise means capable of detecting one or more fluorescent signals.

The present invention includes a magnetic particle having a (+) charge, a PCR primer modified with a first functional group, a magnetic particle modified with a second functional group that binds to the first functional group, and optionally a label for modifying a probe for SNP determination. It provides a single base polymorphism determination kit.

The kit may be made of a number of separate packaging or compartments containing the above components. Instructions describing the optimum reaction performance conditions may be further included. Instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide may include information disclosed or provided through an electronic medium such as the Internet.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

In the following example, a single base polymorphism detection of osteoporosis marker TGF-β1 was performed with an automated device according to the present invention with blood of 784 suspected osteoporosis patients.

<Material preparation>

(1) Preparation of amino-modified magnetic beads and streptavidin-modified magnetic beads

In the automated device according to the present invention, Invitrogen's Dynabeads M-270 Amine was used, and as Streptavidin-modified magnetic beads, MyOne Streptavidin C1 from Invitrogen was used.

(2) primer pairs for PCR

sense 5 '-CGCCTCCCCCATTCCGCCCT-3' SEQ ID NO: 1 Antisense 5 '-ACCAGTAGCCACAGCAGCGGTAGCAG-3' SEQ ID NO: 2

Sense primer 5 'was modified with biotin.

(3) Synthesis of SNP Detection Probe

A probe designed to detect a wild type having T ²⁹ (Leu ¹⁰ ) of TGF-β1 of osteoporosis and a detection probe DNA to determine a variant having C ²⁹ (Pro ¹⁰ ). Synthetic oligo DNA was fluorescently labeled at the 5 'end of DNA.

T allele detection probe Cy3-5'-TGCTGCTGCTGCTG-3 ' SEQ ID NO: 3 C allele detection probe Cy5-5'-TGCTGCCGCTGCT-3 ' SEQ ID NO: 4

(4) Preparation of Sample DNA

Ten microliters of whole blood of 784 suspected osteoporosis patients were dispensed per well of a 96 microplate.

(5) reagent preparation

(i) 15 μl of blood hemolysin per well of 96 microplates, mixed solution for performing PCR (2X PCR buffer, dNTP, MgCl ₂ , BSA, Taq polymerase), Cy3 modified probe, Cy5 modified probe for reagent tray (6) It prepared in advance and stored 4 degreeC.

(ii) PBS buffer solution, alkaline solution, neutral solution, cleaning solution and detection solution are prepared in an external container and the first reaction part (1) and the second reaction part through a buffer dispenser (9) attached to the arm unit. It is supplied to (2).

(iii) Install the tip rack filled with the tip to the tip rack fixing part (4) after sterilization.

Example 1. Extraction of Nucleic Acids

10 μl of blood was dispensed into each well of a 96 well microplate containing amino group-modified magnetic beads, and the head portion 7 equipped with a tip took 15 μl of the hemolytic buffer from the reagent tray 6, and the first reaction portion ( After dispensing into a 96-well microplate mounted in 1), hemolysis of blood was performed by repeating suction and discharge (piping) about 10 times. After incubation at a constant temperature for 20 minutes (amino acid modified magnetic beads were attached to the nucleic acid extracted from the blood.

Then, magnetic beads were recovered by applying a magnetic field to the 96 well microplate by operating the magnetic force control device. The head 7 with the new tip was then taken out of the supernatant from each well of the 96 well microplate and discarded in the waste 3.

Then, using the buffer dispenser attached to the head portion 7, the cleaning liquid is injected into each well of the 96-well microplate using a buffer dispenser, and the pipette is turned on after the magnetic force controller is turned off. After collecting the magnetic particles in the container, the supernatant was discarded.

Example 2. PCR

PCR reagents necessary for PCR and primers modified with biotin at 5 'were mixed and prepared in the reagent tray (6) in advance. The head part equipped with a tip in each well of the 96 well microplate of the first reaction part 1 after nucleic acid extraction was taken from the reagent tray 6 and injected with a PCR reagent. Thereafter, PCR was performed by operating the temperature control function of the warm cooling device of the 96-well microplate.

Example 3. Immobilization

After injecting streptavidin-modified magnetic beads from the reagent tray 6 into each well of the 96 well microplate mounted on the second reaction section 2, the PCR product of the first reaction section 1 was subjected to the second reaction. Each well of the 96-well microplate of the part (2) was injected using a head and incubated for 15 minutes while pipetting. At this time, the temperature control function of the warm cooling device of the second reaction unit 2 was operated to set the 96-well microplate to 25 ° C. After maintaining for 5 minutes, the biotin-modified nucleic acid of the sample in the 96-well microplate was immobilized to streptavidin-modified magnetic beads.

Example 4 Single Stranded

After the completion of the immobilization, the alkaline solution was injected into each of 96 wells using a head buffer buffer dispenser from an external container, followed by pipetting. Then, the magnetic force control device was operated on the 96 well microplate to generate magnetic attraction force to collect magnetic particles. After the magnetic force control device was operated, it was left as it was. Then, the head portion 7 equipped with the new tip took the supernatant from the reaction vessel 2 and discarded it in the waste liquid portion 3. Next, the neutral solution was injected into each well of a 96 micro plate using a buffer dispenser from an external container, pipetting was performed, and magnetic attraction was generated by collecting magnetic particles. After the magnetic force control device was operated, it was left as it was. The head 7 with the new tip was then taken out of the supernatant from each well of the 96 well microplate and discarded into the waste liquid 3. This operation was repeated three times.

Example 5. Hybridization

In the 96-well microplate of the second reaction part 2, a streptavidin-biotin-modified single-stranded DNA sample is present. Cyc, Cy5 fluorescently modified probes were injected into 96-well microplates using a head, and then stirred by pipetting sufficiently, followed by denature. At this time, the temperature control function of the warm cooling device of the second reaction unit 2 was operated to set the 96-well microplate to 70 ° C. Thereafter, pipetting was followed by annealing. At this time, the temperature control function of the heating / cooling device of the second reaction unit 2 was operated to control the temperature so that the 96-well microplate dropped to 25 ° C. After completion of the annealing, the magnetic force control device was operated to generate magnetic attraction force to collect the magnetic beads in the container. After the magnetic force control device was operated, it was left as it was. Afterwards, move the arm unit moved to the waste part downward, mount the dispos tip on the tip nozzle, move the arm unit to the second reaction part, lower it as it is, stop it at a suitable position, and then use a 96-well microplate using the tip nozzle. The supernatant in water was aspirated and discharged by moving to the waste liquid portion. This operation was repeated three times. This allowed the unhybridized probe to be removed from the sample.

Example 6. Fluorescence detection

The head portion equipped with the fluorescence detector was moved to the second reaction portion 2 to measure the change in fluorescence light generated in each well of the 96 well microplate.

Specifically, heat treatment was performed to release Cy3 or Cy5 fluorescently labeled probes from the bound target DNA. The magnetic force control device was operated to separate the magnetic beads from the emitted probes, and cooled to 4 ° C. to detect the intensity of the fluorescence emitted from the fluorescent material.

Analysis of SNP Discrimination Results

SNP detection of the gene marker TGF-ß1 from DNA extraction and osteoporosis using the SNP detection automatic device of the present invention with 784 blood provided from a hospital. Shown in

The dotted line indicates the position where the ratio of the signals of Cy3 and Cy5 becomes the ratio of 2 or 0.5. The measured data were automatically genotyped by calculating the fluorescence intensity ratio using T allele and C allel. This threshold is 2 or 0.5, which is T-type homo when greater than 2, C-type homo when less than 0.5, and otherwise hetero. This result was compared with the result judged by Sequence. The discrimination result was found to be consistent with the discrimination result by the DNA sequence sequencing method. As a result, it was confirmed that this detection system is valid.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (18)

In the automatic device for discriminating single nucleotide polymorphism (SNP), A first reaction vessel, a reaction vessel support, and a means for applying magnetic force to the lower portion of the reaction vessel in an ON / OFF manner, and a DNA separation process using a magnetic particle having a (+) charge and modified with a first functional group A first reaction unit performing a PCR process using a PCR primer; Means for moving the first functional group modified polynucleotide containing PCR reaction product from the first reaction portion to the second reaction portion; And A second reaction vessel, a reaction vessel support, and a means for applying magnetic force to the lower portion of the reaction vessel in an ON / OFF manner, the magnetic particles modified with a second functional group that binds the first functional group-modified polynucleotide to the first functional group. A second reaction part performing a process of immobilization, a single stranded process of the first functional group modified polynucleotide, and a process of hybridizing with a probe for determining an SNP Single base polymorphism determination automation device characterized in that provided with. According to claim 1, wherein the first reaction vessel is characterized in that the magnetic particles having a (+) charge is accommodated, the second reaction vessel is characterized in that the magnetic particles modified with a second functional group that is bonded to the first functional group. Single base polymorphism discrimination apparatus. The method of claim 1, (i) In a first reaction vessel having magnetic particles with (+) charge, the magnetic force is induced after combining the negatively charged nucleic acid from the sample contained in the first reaction vessel with the magnetic particles. Nucleic acid is separated by binding (B) / free (F) separation, (ii) PCR was carried out using a PCR primer modified with a first functional group in a first reaction vessel in a state in which (-) charged DNA is attached to a magnetic particle carrying (+) charge, and a pair of primers are used. Amplify the DNA portion defined by (iii) transfer the DNA amplified by PCR from the first reaction vessel to the second reaction vessel, (iv) immobilizing the amplified DNA to the magnetic particles using a PCR primer modified with a first functional group using a magnetic particle modified with a second functional group that binds to the first functional group in a second reaction vessel, (v) single stranded DNA in a second reaction vessel, (vi) A single nucleotide polymorphism determination automation device, characterized in that it is programmed to hybridize with a DNA single strand immobilized on at least one SNP discrimination probe and magnetic particles in a second reaction vessel. The method of claim 1, wherein the magnetic force is charged to the first reaction vessel to collect the (+) charged magnetic particles bound to the negatively charged nucleic acid in a specific portion of the reaction vessel, Single base polymorphism discrimination characterized in that the magnetic force is applied to the second reaction vessel under conditions for separating the SNP probe hybridized to a single strand of DNA to separate the SNP discrimination probe from the magnetic particles while collecting the magnetic particles in a specific portion of the reaction vessel. Automation device. The apparatus of claim 1, further comprising a heating cooling device for adjusting the temperature of the support or the reaction vessel, and optionally a temperature sensor. The apparatus of claim 1, wherein the SNP determination probe is modified with a labeling substance. The apparatus of claim 1, further comprising a movable head portion equipped with one or more pipettes. The method according to claim 7, wherein the pipette (pipette) is to transport a reagent selected from the group consisting of blood hemolytic agent, PCR mixture, at least one SNP probe modified with a label to the first reaction vessel or the second reaction vessel Single base polymorphism discrimination automatic device. 8. The apparatus of claim 7, wherein the reaction is carried out by pipetting to promote the reaction in the reaction vessel. 8. The apparatus of claim 7, wherein the SNP probe or the labeling substance detector is mounted on the head.  In the method of determining the single nucleotide polymorphism (SNP), In the first reaction vessel, magnetic particles having a positive charge are used to adsorb the DNA prepared from the sample of the individual to be determined for the SNP, and magnetic particles having the DNA adsorbed are collected under the first reaction vessel by applying magnetic force. A first step of removing the supernatant; A second step of injecting a PCR reagent containing a primer modified with a first functional group into a first reaction vessel and performing PCR using DNA adsorbed on magnetic particles as a template to obtain a first functional modified polynucleotide-containing PCR reaction product; A third step of transferring the PCR reaction product in the supernatant of the first reaction vessel to the second reaction vessel; Fourth to obtain the magnetic particles bound to the first functional group modified polynucleotide through the combination of the first functional group and the second functional group by using the magnetic particles modified with the second functional group that binds to the first functional group in the second reaction vessel. step; A fifth step of treating the magnetic particles to which the first functional group-modified polynucleotide is bound to give an alkaline solution to obtain a single stranded first functional-modified polynucleotide; Hybridizing a single-stranded first functional group-modified polynucleotide to a label-modified SNP determination probe; A seventh step of applying magnetic force to collect magnetic particles adsorbed to the SNP determination probe and the polynucleotide adsorbed under the second reaction vessel, and to remove the supernatant containing the SNP determination probe which has not been hybridized; And And an eighth step of measuring a level of a label of the SNP determination probe that is not removed from the second reaction vessel. 12. The method of claim 11, wherein each step is automated by a program. 12. The method of claim 11, wherein the method is performed using the single base polymorphism detection device according to any one of claims 1 to 10. 12. The method of claim 11, wherein the labeling substance modified SNP determination probe is characterized in that different labeling substances are modified according to the genotype of the SNP. The method of claim 11, wherein the labeling material is a quantum dot, magnetic bead nanoparticles, gold nanoparticles, fluorescent dyes, fluorescent proteins, nanophosphors or silicon nanoparticles. The method of claim 11, wherein the drug sensitivity, resistance to infectious diseases, environmental factors of lifestyle diseases, cancer, Alzheimer's disease, and genetic diseases of animals are used to identify via SNP. 12. The method of claim 11, wherein the method is used for individual Taylor-made medical care or preventive medicine through SNP. A single base comprising a positively charged magnetic particle, a PCR primer modified with a first functional group, a magnetic particle modified with a second functional group that binds to the first functional group, and optionally a label for modifying the probe for SNP determination Polymorphism kit.

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