KR20180095357A - Matrix comprising cDNA or amplicons and a carrier thereof - Google Patents

Abstract

The present invention provides a carrier carrying cDNA or amplicon. According to the present invention, it is possible to capture, replicate or amplify a target polynucleotide from a sample with high sensitivity in a hydrogel microparticle, and to safely store or carry the replicated or amplified target polynucleotide in a fixed state in the hydrogel microparticle. In addition, the stored target polynucleotide or the carrier containing the same can be reused, if necessary, for secondary PCR, sequencing, and the like.

Description

cDNA or amplicons and a carrier thereof. < RTI ID = 0.0 > [0002] <

New applications of hydrogel microparticles are disclosed herein.

Polymerase chain reaction (PCR) is the most widely used method for detecting specific genetic biomarkers to diagnose various cancers and infectious diseases. In addition, gene sequencing is a major contributor to the association of disease, gene mutation, and expression by reading gene information. When PCR or sequencing is used, the complexity of the patient sample, such as the quality of the target gene, the purity, and the amount of the sample in the clinical detection, serves as a factor for lowering the target gene detection efficiency.

Korean Patent Publication No. 10-2015-0048964 (May 2015)

In one aspect, the present invention relates to a method for replicating or amplifying a target gene so as to accurately and efficiently detect the target gene, separating the resultant cDNA (complementary DNA) or amplicon from the external environment safely and easily, And to provide a structure.

In order to solve the above-mentioned object, one aspect of the present invention relates to a cDNA or an amplicon, which is a duplicate of a target polynucleotide; And a carrier carrying the cDNA or the amplicon, wherein the carrier comprises at least one of a forward primer and a reverse primer of the target polynucleotide, wherein the at least one primer is immobilized on the hydrogel fine particle And wherein said cDNA or amplicon is associated with said at least one primer.

In one aspect of the present invention, there is provided a method for producing the structure, comprising the steps of: placing an RNA or DNA sample of a target polynucleotide into a hydrogel microparticle in which at least one primer of the forward primer and the reverse primer of the target polynucleotide is immobilized, incubating the primer with a primer and an RNA or DNA gene as a target polynucleotide; And reverse transcription of the incubated primer and the RNA or DNA gene to synthesize a first strand cDNA or DNA template (first strand cDNA / DNA template).

In one aspect of the present invention, there is provided a method for producing the structure, comprising the steps of: placing an RNA or DNA sample of a target polynucleotide into a hydrogel microparticle in which at least one primer of the forward primer and the reverse primer of the target polynucleotide is immobilized, incubating the primer with a primer and an RNA or DNA gene as a target polynucleotide; And synthesizing a first strand cDNA or a DNA template by reverse transcription of the incubated primer and the RNA or DNA gene, Further comprising the steps of placing the hydrogel microparticles in a PCR solution and amplifying the target polynucleotide by PCR (polymerase chain reaction).

The present invention provides a construct comprising cDNA or a carrier carrying an amplicon. According to the present invention, it is possible not only to capture and amplify the target polynucleotide from the sample with high sensitivity in the hydrogel fine particle, but also to immobilize the replicated or amplified target polynucleotide in the hydrogel fine particle Can be safely stored or transported. In addition, the stored target polynucleotide or a carrier containing the same can be reused as needed for secondary PCR, sequencing, and the like.

FIG. 1 illustrates a process of synthesizing a first-strand cDNA or DNA template using hydrogel fine particles fixed with a reverse primer according to an embodiment of the present invention.
FIG. 2 is a diagram illustrating separation of an amplicon, which is a target gene amplified after qPCR, with a restriction enzyme, according to an embodiment of the present invention.
FIG. 3 is a graph comparing the results of PCR according to the presence or absence of washing after the whole RNA incubation of the hydrogel microparticles according to one embodiment of the present invention.
FIG. 4 is a graph showing fluorescence images according to the progress of a PCR cycle when hydrogel fine particles are washed after synthesis of a first-stranded DNA template according to an embodiment of the present invention.
FIG. 5 is a chart comparing the conventional solution qRT-PCR, which is a comparative example, with the PCR results of hydrogel fine particle (PIN) qRT-PCR, which is an embodiment of the present invention.
FIG. 6 is a chart comparing NTR (no-template control) results of conventional solution qRT-PCR, which is a comparative example, and hydrogel fine particle (PIN) qRT-PCR, which is one embodiment of the present invention.
Figure 7 compares the amplicons of PCR with solution qRT-PCR and hydrogel fine particle (PIN) qRT-PCR in agarose gel.
FIG. 8 is a graph showing the result of performing qRT-PCR with hydrogel fine particles according to an embodiment of the present invention, followed by sequencing only with restriction enzyme treatment.

Hereinafter, preferred embodiments of the present invention will be described in detail in order to facilitate the present invention by those skilled in the art.

The term " amplicon " used herein has the same meaning as " gene amplification " and means an amplification product such as polynucleotide or oligonucleotide such as DNA or RNA. The amplicon is not limited to the number of times of gene amplification but may be the maximum including all the amplification products of the gene. For example, the amplicon may include a PCR result of the target polynucleotide.

As used herein, the term "target polynucleotide" is used interchangeably with "target polynucleotide", "target gene", "target gene" and includes all DNA, RNA, It means the best. Such analysis or action includes treatment such as copying, amplification, sequencing, etc. of the polynucleotide.

One embodiment of the present invention provides a construct comprising an amplicon, which is a cDNA or an amplicon, which is a copy of a target polynucleotide, and a carrier that carries the cDNA or the amplicon.

One embodiment of the carrier is a structure wherein at least one of a forward primer and a reverse primer of the target polynucleotide is immobilized on a hydrogel microparticle, It may be combined with a primer.

Hereinafter, a process for producing a structure including the hydrogel fine particles according to an embodiment of the present invention as a carrier for cDNA or amplicon will be described with reference to FIG. FIG. 1 is a diagram showing a quantitative reverse transcription polymerase chain reaction (qRT-PCR) process of a target gene RNA or DNA using hydrogel fine particles fixed with a reverse primer according to an embodiment of the present invention. As shown in FIG. 1, the reverse primers immobilized within the particles can selectively bind the target gene during the incubation of the total RNA or DNA with the hydrogel fine particles. When reverse transcription is performed, a first strand cDNA template is synthesized (cDNA synthesis) or a first strand template synthesis is performed by complementary binding with the target RNA, and the synthesized genes are fixed in the hydrogel fine particle (Captured) and stay. Then, when the surrounding solution is replaced with a PCR solution containing forward primers for amplification of the target gene and PCR is performed, the amplified gene remains fixed in the hydrogel fine particle. Therefore, the hydrogel microparticles of the present invention can be stored in a state where specific RNA and / or DNA is immobilized on the particles. In addition, the amplified gene can be separated and purified (Restriction enzyme digestion), and reused if necessary.

In one embodiment, the forward primer or the reverse primer may be a polynucleotide consisting of 10 to 50 contiguous DNA sequences. However, if the primer is selectively bound to the target polynucleotide, the sequence type and the sequence length of the primer are not limited to the target polynucleotide Modifications are possible without limitation.

In one embodiment, the reverse primer is immobilized to the hydrogel microparticles in the case of PCR, and the forward primer can be injected into the PCR solution at the time of PCR, prior to the PCR step for amplification of the target gene. That is, the forward and reverse primers may be for annealing a target gene.

Also, in one embodiment, the carrier may comprise a primer for each of the one or more target polynucleotides, and one or more cDNAs or amplicons associated therewith. That is, the carrier contains primers for various kinds of target polynucleotides, and as a result, various kinds of gene copies (cDNA) or amplifications (amplicons) can be stored or carried.

In one embodiment of the present invention, the primer may be linked to an ester linkage with hydrogel fine particles. Alternatively, the primer may be immobilized within the pores of the hydrogel microparticles by incorporating hydrogel microparticle immobilization functional groups at the 5 'end or the 3' end of the base sequence. In one embodiment, the functional group may include a functional group capable of being covalently bonded to the hydrogel monomer, and examples thereof include acrydite. In another embodiment, the functional group may include a functional group that can be linked to the hydrogel through a peptide bond, and examples thereof include an amine group and a carboxyl group.

In addition, as an example, the 5 'end or the 3' end of the nucleotide sequence of the primer may further include a restriction enzyme to separate the cDNA or the amplicon of the target gene from the hydrogel fine particle. Alternatively, as an example, a basic catalyst may be used at the 5 'end or the 3' end of the nucleotide sequence of the primer to separate the cDNA or the amplicon of the target gene from the hydrogel fine particles. The basic catalyst may provide a hydroxyl group (-OH) capable of separating an amplicon of a target gene synthesized by PCR from the hydrogel microparticles when the primer is immobilized by ester bonding with the hydrogel microparticles Any of them may be used without limitation. In one embodiment, the basic catalyst is exemplified by ammonium hydroxide.

[Chemical Formula 1]

The above formula (1) shows a mechanism for separating oligonucleotides from hydrogel fine particles by a basic catalyst (corresponding to Base of Chemical Formula 1) when the primer is fixed by ester bonding with hydrogel fine particles. In the counterclockwise direction from the upper left corner of the formula (1), when the primer fixed to the hydrogel fine particle by the ester bond becomes an oligonucleotide by PCR, the ester bond is broken by the basic catalyst and the oligonucleotide is hydrogel fine particle . After separation, the basic catalyst can be restored to originally hydroxyl groups due to the surrounding water molecules, and the recovered basic catalyst attacks another ester bond of the hydrogel fine particles, and the other oligonucleotides immobilized on the particles are immersed in the hydrogel fine Can be separated from the particles.

The hydrogel microparticles according to an embodiment of the present invention may have a particle size of, for example, an average particle size of 10 μm to 500 μm, and more specifically an average particle size of 100 μm to 300 μm. The shape is not limited as long as it is a three-dimensional structure, and more specifically, a spherical shape is exemplified. The material of the hydrogel fine particles can be used without limitation as long as it is a pre-polymer that can be solidified. Specifically, polyethylene glycol-diacrylate (PEG-DA) or polyacrylamide (PAAm) May be used.

In one embodiment, the hydrogel microparticles may be a porous structure comprising a void. In one embodiment, the primer may be immobilized within the pores of the hydrogel fine particles.

 In one embodiment, when the hydrogel microparticles comprise voids, the porosity of the microparticles may be between 10% and 80% by volume, more specifically between 20% and 70% by volume relative to the total volume of microparticles . Outside of this range, the porosity may be deteriorated or the stability of the structure may become unstable.

Specifically, the hydrogel fine particles can be prepared using a photo-crosslinkable hydrogel solution containing specific primers. In the hydrogel fine particle according to an embodiment of the present invention, the primer is immobilized inside the particle rather than on the surface of the fine particle, so that capturing, reverse transcription and amplification of the target gene can be sequentially and easily performed in PCR, Of the cDNA or amplicon can be safely stored and transported. In addition, the problem of nonspecific signal generation of conventional solution-based PCR can be solved.

The construct according to an embodiment of the present invention can selectively transcribe and amplify a target gene from a small amount of total RNA mixture with excellent sensitivity. Only the amplified target gene is captured in the particle while the unwanted genes are dispersed and removed, so the false positive signal is reduced. In one embodiment, the amplified gene, that is, the amplicons, can be isolated and detected by electrophoresis after restriction enzyme digestion. The isolated gene can be used for subsequent analysis such as gene sequencing. Therefore, the present invention can be used for various applications for detection and quantification of target genes.

The present invention can provide a method of manufacturing any one or more of the structures described above as another embodiment.

In one embodiment, the method comprises introducing RNA or DNA samples of the target polynucleotide into hydrogel microparticles in which at least one primer of an forward primer and a reverse primer of the target polynucleotide is immobilized therein Incubating the RNA or DNA gene, which is a target polynucleotide, with the primer; And synthesizing a first strand cDNA or DNA template (first strand cDNA / DNA template) by reverse transcription of the incubated primer and the RNA or DNA gene.

In one embodiment, the method further comprises amplifying the target polynucleotide by PCR (polymerase chain reaction) by adding the hydrogel fine particles containing the synthesized first chain cDNA or DNA template into a PCR solution .

In one embodiment, the method may further comprise a washing step to remove nonspecifically bound RNA or DNA in advance of reversing the incubated hydrogel microparticles. In the present invention, only the target polynucleotide is captured in the hydrogel microparticles, and the non-target genes can be washed out through the washing step, thereby preventing unnecessary reverse transcription of the non-target gene causing nonspecific amplification in the PCR process.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 schematically shows an example of a method for producing a construct comprising the cDNA or the carrier carrying the amplicon of the present invention. 1, the method of the present invention will be described. In one embodiment, the total RNA (total RNA) or DNA sample is added to the hydrogel fine particle immobilized inside the particle with a reverse primer for trapping the target gene, To 70 < 0 > C for 1 minute to 100 minutes. In one embodiment, the washing may be performed between the incubation step and a reverse transcription step such as the first chain template synthesis. Specifically, the hydrogel fine particles may be introduced into a buffer And washing to remove nonspecifically bound RNA or DNA (Nonspecifically-bound RNA / DNA). In one embodiment, when the used sample is RNA, a first strand cDNA template is synthesized by a reverse transcription reaction using a reverse transcriptase, and when the sample is DNA, a gene extension method using a gene polymerase 1 strand DNA template (First strand DNA template). In one embodiment, the hydrogel microparticles can be incubated with each total RNA for 5 minutes at about 65 ° C, and are reverse transcribed and amplified.

In addition, the method according to an embodiment of the present invention may further include a step of separating the cDNA of the target nucleotide synthesized after the reverse transcription or the step of separating the amplified target polynucleotide after the PCR. For example, in the case where the reverse primer immobilized on the hydrogel microparticles further comprises a restriction enzyme, the step of separating the amplified target gene comprises the step of separating the target amplified using the restriction enzyme And isolating the polynucleotide. Alternatively, in one embodiment, separating the amplified target polynucleotide may comprise separating the amplified target polynucleotide from the hydrogel microparticle using a basic catalyst, wherein the basic catalyst is not limited Ammonium hydroxide, for example, may be mentioned.

In one embodiment, the step of amplifying the target polynucleotide may comprise the steps of synthesizing the first chain cDNA or DNA template, replacing the surrounding solution with a PCR solution containing the forward primer, May be injected into the PCR to amplify the first chain cDNA or DNA template in a form fixed to the hydrogel microparticles. At this time, the nucleic acid fluorescent dye SYBR® green I can be used to measure the amplified gene in real time.

In one embodiment, the amplified target polynucleotide may be separated from the microparticles and used for gel electrophoresis or sequencing.

In one embodiment, the RNA or DNA sample of the target polynucleotide may comprise a cell lysate. Since only the target polynucleotide is captured in the hydrogel microparticles, even if a cell lysate containing various proteins and other substances is used as the sample, only the target gene can be selectively captured into the particle.

In one embodiment of the present invention, the RNA or DNA sample of the target polynucleotide comprises RNA or DNA samples for one or more target polynucleotides, and the hydrogel microparticles are one or two or more of the above- Or a primer for each of the above target polynucleotides. For example, since the present invention can include one or more primers for one or more target genes, it is possible to selectively capture non-coding RNA in addition to protein coding RNA . In addition, two or more kinds of various genes can be captured at the same time by using hydrogel fine particles fixed with different gene primers, and the ampicrons can be stored or transported.

In one embodiment, the amplicons may be separated from the hydrogel fine particles through restriction enzyme treatment by further including a restriction enzyme in front of the 5 'end of the primer.

According to one embodiment of the present invention, during the PCR, only the target amplicon can be captured in the particle through the primer immobilized within the hydrogel microparticle. The byproducts that are not immobilized to the other particles are dispersed in the surrounding solution, so that they do not affect the fluorescence that is increasing due to the amplification of the target gene in the hydrogel microparticles. Additionally, fluorescent accumulation of intercalating dyes gives a high signal to the noise ratio.

Also, according to one embodiment of the present invention, the RT and PCR are performed in the same hydrogel microparticle, thus reducing the possibility of contamination of samples that may occur by separate experimental sets . Further, since the reverse transcription and the PCR are performed in several nonoliter volumes, RT-PCR using the hydrogel microparticles of the present invention can be performed using a small amount of sample. In fact, the single small particle can be the chamber for the RT-PCR reaction of the target one, so there is an advantage in that multiple PCR applications can be performed by fixing different primers to each particle. If PCR is combined with the reverse transcription process in the hydrogel microparticles of the present invention, it can be used as a multiple diagnosis system for mRNA pattern analysis in the clinical field.

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.

[Manufacturing Example]

Hydrogel fine particles having a primer fixed in a particle according to an embodiment of the present invention were prepared according to the following method.

Cell culture and RNA extraction

Human chronic myelogenous leukemia cell line K562 was used as a positive control for Bcr-Abl and human acute promyelocytic leukemia cell line HL60 was used as a negative control for Bcr-Abl. The two cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37 ° C and 5% CO ₂ . Total RNA was extracted from K562 and HL60 using AccuZol ( ^TM ). The total RNA extraction solution (Bioneer, Korea) was prepared according to the manufacturer's instructions.

Primer design

Abl omni-directional primers for the capture and amplification of forward and reverse primers and Abl genes were designed as follows to detect the Bcr-Abl gene as a target gene.

- omni-directional primer: 5'-TGG GTT TCT GAA TGT CAT CGT CCA CTC A-3 '(SEQ ID NO: 1)

- reverse primer: 5'-AGT TCC AAC GAG CGG CTT CAC TCA-3 '(SEQ ID NO: 2)

For separation of the hydrogel microparticles and the target gene, acrydite and restriction enzyme EcoRI site (GAATTC: SEQ ID NO: 3) were added to the 5 'end of the reverse primer.

The forward primer of Abl was designed as 5'-AAG CTG GTG GGC TGC AAA TCC AAG-3 '(SEQ ID NO: 4).

The omni-directional primer of Abl is a standard omni-directional primer. The positive control, K562, has a Bcr-Abl gene fused to Bcr rather than normal Abl, and a negative control, HL60, has a common Abl. When real-time PCR is performed by mixing cells, it is possible to compare the Ct values which are obtained by decreasing the number of positive control cells by having a standard of Ct value. Therefore, since the number of HL60 cells as the negative control group was the same for each experiment, the following experiment was carried out based on the expression level (Ct) of the Abl gene which appears only in HL60, the forward primer used in this case is the primer of SEQ ID NO: 4, SEQ ID NO: 2.

Preparation of hydrogel fine particles

A prepolymer solution for the preparation of hydrogel fine particles (PIN particles) was prepared by mixing 20% v / v of UV-photopolymerizable poly (ethylene glycol) diacrylate (PEGDA, Sigma- Aldrich, MW700), poly (ethylene glycol) (Sigma-Aldrich, Sigma-Aldrich, Sigma-Aldrich), 5% v / v of photoinitiator 2-hydroxy-2-methylpropiophenone v / v and 0.15% Tween-20 (Sigma-Aldrich). The PEG 600 was used to increase the porosity of the hydrogel microparticles for ease of use in the reverse transcription and PCR steps. 200 [mu] M of the acrylate binding-reverse primer of the Bcr-Abl prepared above was added to make the final concentration of the pre-polymer solution 20 [mu] M. The hydrogel microparticles were exposed to UV light (360 nm wavelength, 35 mJ / cm ² ) for 1 minute after jetting system (Arrayer 2000, Advanced Technology Inc., Korea) Spotting < / RTI > The photo-crosslinked hydrogel microparticles were released from the PDMS surface by mild agitation and were washed by TE buffer containing 0.05% Tween-20 to remove unbridged compounds such as reverse primer, PEGDA and PEG.

[Experimental Example 1]

As one embodiment of the present invention, the reverse primer for detection of the target gene in the above-mentioned production example was subjected to RT-PCR using the hydrogel fine particles immobilized inside the particle according to the following method.

cDNA synthesis

The reverse transcription step was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermo scientific, USA). First, the hydrogel microparticles of the above preparation example were incubated for 5 minutes at 65 DEG C with 1 mu g of total RNA extracted from K562 or HL60 cells in Example 1, respectively, and then ice-cooled.

Some of the particles were then washed with TE buffer (Tris-EDTA-buffer) containing 0.15% Tween-20 to remove non-specifically bound RNA.

After the washing step, the reaction buffer, dNTP and reverse transcriptase were added and incubated at 42 ° C for 60 minutes. The reaction was heated at 70 ° C for 5 minutes and then terminated. The hydrogel microparticles with the first synthesized cDNA were stored at -20 ° C for the next experiment.

As a comparative example, reverse transfer was performed without using hydrogel fine particles according to the prior art. Specifically, the total RNA was incubated at 42 ° C. for 60 minutes with a reverse primer (SEQ ID NO: 2), reaction buffer, dNTP and reverse transcriptase, and the reaction was heated at 70 ° C. for 5 minutes and then terminated. The synthesized cDNA was stored at -20 DEG C for the next experiment.

PCR

QRT-PCR using the above hydrogel microparticles was performed using an UltraFast LabChip Real-time PCR G2-3 System (Nanobiosys, Seoul, Korea). For qPCR using the hydrogel microparticles, deionized water containing 8 μL 2X SYBR Green master mix (Nanobiosys, Korea) and 0.8 μL of 10 μM omni-directional primer (SEQ ID NO: 1) was mixed to give a final volume of 16 μL .

As a comparative example, 0.8 μL of a 10 μM reverse primer and a cDNA template were further added to the solution in the conventional solution RT-PCR.

Single hydrogel fine particles were trapped in the PCR chamber, and the mixed solution was injected into the chamber. PCR conditions were 95 ° C for 8 seconds, pre-denaturation 1 cycle, denaturation at 95 ° C for 3 seconds, annealing at 60 ° C for 11 seconds, and elongation 40-50 cycles . Fluorescence images of each cycle were captured and the fluorescence intensity was calculated using software. The fluorescence intensity values were normalized by software and plotted as a linear graph (Figs. 3 and 4).

FIG. 3 is a graph showing experimental results of positive and negative control groups with and without washing steps performed after the entire RNA incubation. In the negative control (HL60), nonspecific amplification appears when there is no washing step, and no nonspecific signal appears when washing is performed. The positive control group had no significant influence on the presence or absence of the washing process.

FIG. 4 is a graph showing fluorescence images according to the progress of a PCR cycle when hydrogel fine particles are washed after synthesis of a first-stranded DNA template according to an embodiment of the present invention. It can be seen that the nonspecific reaction was suppressed by the washing step.

This is because a non-specific strand generated in the process of synthesis of the first chain template by reverse transcription nonspecifically binds to the non-specific RNA in the total RNA can be amplified in the PCR process, and the non- Lt; RTI ID = 0.0 > RNA < / RTI >

Further, although not described in the present specification, the present inventors have experimented with reverse primers at various concentrations (5 μM to 50 μM) since a sufficient amount of reverse primers are required for reverse transcription and PCR, Ct values were not changed. Thus, the reverse primer of 20 [mu] M was sufficient for reverse transcription or PCR.

[Experimental Example 2]

In order to prepare the construct carrying the target gene amplicon according to the present invention as an example of the present invention, qRT-PCR was performed using the hydrogel fine particles immobilized in the inside of the particle with the reverse primer in the above Experimental Example 1 and The same procedure was followed.

In order to compare the Ct value in the hydrogel microparticle-based qRT-PCR (PIN qRT-PCR) with the Ct value in the conventional solution qRT-PCR (solution qRT-PCR), the conventional solution qRT- Lt; RTI ID = 0.0 > qRT-PCR. ≪ / RTI > Since the volume of the hydrogel fine particles is 20 nL, the cDNA solution was also used in the same volume for the PCR process.

FIG. 5 is a view comparing respective PCR results of the conventional solution qRT-PCR, which is the comparative example, and the hydrogel fine particle (PIN) qRT-PCR, which is one embodiment of the present invention.

Conventional solution qRT-PCR Ct values of the positive samples (K562) is a 28.10 ± 0.55 (n = 4) , the hydrogel microparticles (PIN) qRT-PCR Ct value of 29.01 ± 0.80 (n = 4) , the solution qRT-PCR And the difference in Ct value between hydrogel fine particle (PIN) qRT-PCR was less than 1. No signal from nonspecific amplification was detected in qRT-PCR in hydrogel microparticles (PIN) qRT-PCR, whereas nonspecific signals were found in solution qRT-PCR (Ct value: 32.59 ± 0.37 ( n = 4)). This implies that non-specific gene amplification occurred in the conventional solution qRT-PCR, but non-specific gene amplification was suppressed in the hydrogel fine particle-based qRT-PCR. Therefore, the hydrogel fine particle (PIN) qRT-PCR shows excellent efficiency in reverse transcription and amplification of the target gene.

Another problem with conventional solution-based qRT-PCR is that signals generated from primer-dimers interfere with signal analysis. Fluorescence signals from NTC (no-template control) were measured in both solution qRT-PCR and hydrogel fine particle (PIN) qRT-PCR systems using forward and reverse primers of the target gene. Specifically, in the case of a hydrogel fine particle (PIN) qRT-PCR system, a reaction solution containing only a forward primer is injected into fine particles in which RNA or DNA is not synthesized while only the reverse primer is immobilized in the fine particle, Respectively.

As a result, as shown in Fig. 6, the fluorescence rapidly increased in the solution qRT-PCR, but not in the hydrogel fine particle (PIN) qRT-PCR. That is, no fluorescence signal appeared. This is because, in the case of primers immobilized in the hydrogel microparticles, the primer-dimer formation is reduced due to the advantage that the chance of random binding of the primer is less, and the nonspecific reaction is suppressed. This means that the hydrogel microparticles according to the present invention have an advantage that other unwanted amplicons can be dispersed in the surrounding solution to capture and carry the target gene with higher purity.

[Experimental Example 3]

High purity amplification is required for accurate analysis of genes. In order to confirm the purity of the hydrogel fine particle-based qRT-PCR according to an embodiment of the present invention, hydrogel fine particle-based qRT-PCR was performed in the same manner as in [Experimental Example 1] Was isolated by using restriction enzyme EcoRI (SEQ ID NO: 3; TaKaRa Bio Inc., Japan) to isolate a specific site in front of the target (Bcr-Abl) gene. In order to visualize the amplified target gene on the agarose gel, 50 hydrogel fine particles were used for amplification. After amplification of the target gene, the hydrogel microparticles were washed three times with TE buffer containing 0.15% Tween-20 to remove PCR reagents and non-conjugated primers. The buffer was then replaced with deionized water for the EcoRI treatment and enzyme digestion was performed at 37 ° C for 2 hours. The amplified gene was detected using 3% agarose gel electrophoresis.

After PCR, hydrogel microparticles were collected and the amplified Bcr-Abl genes were released from the hydrogel microparticles by EcoRI, and the released genes were analyzed by gel electrophoresis and gene sequencing.

As a comparative example, gel electrophoresis was also performed using the same solution-based qRT-PCR amplification as in [Experimental Example 2].

As a result, as shown in Fig. 7, non-specific amplifications from both positive (K562) and negative (HL60) samples as well as the target amplification appeared on the agarose gel in the case of solution qRT-PCR. On the other hand, in the case of hydrogel microparticle qRT-PCR, only the target amplicon (Bcr-Abl) from the positive sample was apparent, and no other nonspecific amplification product from the negative sample was observed. This result implies that the hydrogel microparticles capture and carry the amplified product of the target mRNA with high purity and can use it for accurate analysis.

[Experimental Example 4]

The hydrogel microparticle-based qRT-PCR was performed in the same manner as in [Experimental Example 1], and the EcoR1 restriction enzyme (SEQ ID NO: 3) and the reaction buffer were added to the particles after completion of the RT-PCR reaction. The reaction was carried out for a period of time. After the reaction was completed, the supernatant except the particles was extracted and sequenced using Sanger sequencing using the separated nucleic acid and omni-directional primer (SEQ ID NO: 1). As a result, it can be confirmed that the sequence of the sequencer data matches the sequence of the target amplicon as shown in FIG. This means that sequencing can be performed only by restriction enzyme treatment without the need for sequencing of the target gene by purifying and separating the target gene through electrophoresis after completion of the PCR reaction.

<110> Korea Institute of Science and Technology <120> Matrix comprising cDNA or amplicons and a carrier thereof <130> 16P109 / IND <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Abl Forward primer <400> 1 tgggtttctg aatgtcatcg tccactca 28 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Abl Reverse primer <400> 2 agttccaacg agcggcttca ctca 24 <210> 3 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> Restriction enzyme EcoRI site <400> 3 gaattc 6 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Abl Standard Forward primer <400> 4 aagctggtgg gctgcaaatc caag 24

Claims (12)

CDNA (complementary DNA), which is a copy of the target polynucleotide, or amplicon, which is an amplification product; And a carrier carrying the cDNA or the amplicon,
Wherein the carrier is a hydrogel microparticle to which at least one of a forward primer and a reverse primer of the target polynucleotide is immobilized and the cDNA or the amplicon is combined with the at least one primer. Structure. 2. The method of claim 1, wherein the primer further comprises a restriction enzyme,
Wherein the restriction enzyme is for separating the cDNA or the amplicon from the hydrogel microparticles. The construct of claim 1, wherein the primer further comprises a hydrogel fine particle immobilization functional group at the 5 'end of the nucleotide sequence. The structure according to claim 1, wherein the hydrogel fine particles are porous. 5. The construct of claim 4, wherein the primer is immobilized within the pores of the hydrogel microparticles. 6. A method for producing a structure according to any one of claims 1 to 5,
An RNA or DNA sample of the target polynucleotide is put into hydrogel fine particles having one or more of a forward primer and a reverse primer of a target polynucleotide immobilized thereon and incubated, Selectively binding an RNA or DNA gene that is a target polynucleotide; And
And reverse transcribing said RNA or DNA gene with said incubated primer to synthesize a first strand cDNA or DNA template (first strand cDNA / DNA template). 7. The method according to claim 6, further comprising the step of putting the hydrogel fine particles containing the synthesized first chain cDNA or DNA template into a PCR solution and amplifying the target polynucleotide through PCR (polymerase chain reaction). 7. The method of claim 6, wherein the method further comprises a washing step to remove nonspecifically bound RNA or DNA prior to reverse transcription of the incubated hydrogel microparticles. 7. The method of claim 6, wherein the method further comprises separating the amplified target polynucleotide after PCR. 7. The method of claim 6, wherein the method further comprises separating the cDNA of the synthesized target polynucleotide after reverse transcription. 10. The method of claim 9, wherein separating the amplified target polynucleotide comprises separating the amplified target polynucleotide from the hydrogel microparticle using a basic catalyst. 7. The method of claim 6, wherein the RNA or DNA sample of the target polynucleotide comprises a cell lysate.

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