KR101809587B1 - PCR Composition Containing Graphene Oxide Coated by Au Nanoparticles and Method for PCR Using The Same - Google Patents

Abstract

The present invention relates to a PCR composition containing graphene oxide (Au @ GO) coated with gold nanoparticles, and more particularly to a PCR composition containing graphene oxide (Au @ GO) coated with gold nanoparticles, nucleic acid polymerase, And a method for amplifying a nucleic acid using the PRC composition.
The PCR composition comprising graphene oxide coated with gold nanoparticles according to the present invention has an effect of significantly improving the PCR efficiency due to interaction with the Taq DNA polymerase and directional interaction (π-π stacking).

Description

PCR Composition Containing Graphene Oxide Coated by Au Nanoparticles and Method for PCR Using The Same}

The present invention relates to a PCR composition containing graphene oxide (Au@GO) coated with gold nanoparticles, and in more detail, graphene oxide (Au@GO) coated with gold nanoparticles, nucleic acid polymerase and target nucleic acid It relates to a PCR composition comprising a primer pair capable of amplifying and amplifying a nucleic acid using the PCR composition.

Polymerase Chain Reaction (PCR) is a molecular biological method that can exponentially amplify the number of copies of a desired part of DNA. Any part of the DNA can be amplified through PCR if the sequence is known. PCR was designed by K. Mullis in the mid-1980s and is still widely used in many biology-related studies, including molecular genetics, to study and analyze genes. PCR is a method using the characteristics of DNA replication by DNA polymerase. DNA polymerase can synthesize complementary DNA using single-stranded DNA as a template. Such single-stranded DNA can be obtained simply by boiling double-stranded DNA. have. This process is called DNA denaturation. For DNA polymerase to start replicating DNA, the starting site must be in the form of a double-stranded DNA. Therefore, in PCR, a small piece of DNA that can complementarily bind to the template DNA must be put together at both ends of the DNA sequence to be amplified, and this must be annealed to both ends of a specific DNA sequence to form a two-stranded DNA. Only in this case can DNA replication by DNA polymerase be initiated. A small piece of DNA that can complementarily bind to the end of the template DNA is called an oligonucleotide primer or a primer for short. Once the primer is bound to the end of the template DNA, DNA synthesis proceeds to the other end by the action of DNA polymerase. Because the PCR reaction is based on this series of mechanisms, the PCR cycle is a denaturation step that converts the two-stranded template DNA into single-stranded DNA, a binding step in which the primer binds to the end of the template DNA, and DNA synthesis is accomplished by the action of DNA polymerase. It consists of three stages: the elongation stage that reaches the other end. After the first PCR cycle is over, in the subsequent PCR cycle, both the original template DNA and the newly synthesized DNA by PCR become the DNA template. In this way, as the cycle of PCR continues to be repeated, the number of DNA templates continues to increase.

The PCR methods developed until recently include rapid PCR, which reduces the time required for amplification, direct PCR to omit the sample purification process, and RNA as a template by combining reverse transcription and PCR. Reverse transcriptase PCR (RT-PCR), a hot-start PCR reaction that improves the specificity of the PCR reaction by dramatically reducing non-specific amplification occurring at room temperature. There are real-time PCR (real-time PCR) that enables real-time monitoring of the device. In addition, many other methods and related technologies have been developed.

In addition, there have been attempts to improve the efficiency of the PCR reaction itself. Attempts to increase the efficiency of the PCR reaction have been made in various ways, such as improvement of the PCR machine, improvement of the protein engineering of the existing DNA polymerase, high purity of PCR reaction components, and PCR reaction. There were methods such as the application of a PCR reaction enhancer that can enhance the reaction, and the application of a stabilizer that can increase the stability of essential components of the PCR reaction such as DNA polymerase.

In particular, graphene has been used to increase PCR efficiency. In addition, gold nanoparticles (AuNP) are non-toxic and function to absorb proteins including small molecules and polymerases (Min Li, et al., Nucleic Acids Research, 33(21), e183, 2005).

However, in the case of graphene or gold nanoparticles, there is a limit to the interaction with the nucleic acid polymerase and the improvement of thermal conductivity.

Accordingly, as a result of the present inventors making diligent efforts to improve the efficiency of the PCR reaction, it was confirmed that when the graphene oxide coated with gold nanoparticles was added to the PCR reaction solution, the PCR efficiency could be significantly improved compared to the graphene oxide. And completed the present invention.

An object of the present invention is to provide a PCR composition containing graphene oxide coated with gold nanoparticles having an excellent effect in improving PCR efficiency.

Another object of the present invention is to provide a method for amplifying a target nucleic acid using a PCR composition containing graphene oxide coated with gold nanoparticles.

Another object of the present invention is to provide a kit for detecting a nucleic acid containing a PCR composition containing graphene oxide coated with gold nanoparticles.

In order to achieve the above object, the present invention provides a PCR composition comprising a gold nanoparticle-coated graphene oxide (Au@GO), a nucleic acid polymerase, and a primer pair capable of amplifying a target nucleic acid.

The present invention also provides a method of amplifying a target nucleic acid, characterized in that the PCR composition is used.

The present invention also provides a kit for detecting a nucleic acid comprising a PCR composition comprising graphene oxide (Au@GO) coated with the gold nanoparticles.

The PCR composition comprising graphene oxide coated with gold nanoparticles according to the present invention has an effect of remarkably improving PCR efficiency by interaction with Taq DNA polymerase and directional interaction (π-π stacking).

1 is an image showing an aromatic-aromatic interaction (π-π stacking) between graphene oxide (GO) and DNA of a nucleobase.
2 is an image showing a process of synthesizing graphene oxide coated with gold nanoparticles in Example 1. FIG.
3 is a graph showing the UV spectrum of graphene oxide coated with gold nanoparticles.
4 is a TEM image of Au@GO prepared in Example 1. The scale ruler represents 0.1 μm and 10 nm.
Figure 5 is a schematic diagram showing the PCR protocol of the embodiment.
6 shows the PCR yield according to the concentration of gold nanoparticles (AuNPs) of Example 3-1 (M: DNA marker, Con: positive control without gold nanoparticles; (1): AuNPs 1X10 ^-3 μg/µl , (2): AuNPs 3X10 ^-3 µg/µl, (3): AuNPs 5X10 ^-3 µg/µl, (4): AuNPs 7X10 ^-3 µg/µl, (5): AuNPs 9X10 ^-3 µg/µl, ( 6): AuNPs 1X10 ^-2 µg/µl, (7): AuNPs 3X10 ^-2 µg/µl, (8): AuNPs 5X10 ^-2 µg/µl, (9): AuNPs 7X10 ^-2 µg/µl, (10) : AuNPs 9X10 ^-2 µg/µl).
7 shows the PCR yield according to the graphene oxide (GO) concentration of Example 3-1 (M: DNA marker, Con: positive control without graphene oxide; (1): GO 1X10 ^-3 μg/µl , (2): GO 3X10 ^-3 µg/µl, (3): GO 5X10 ^-3 µg/µl, (4): GO 7X10 ^-3 µg/µl, (5): GO 9X10 ^-3 µg/µl, ( 6): GO 1X10 ^-2 µg/µl, (7): GO 3X10 ^-2 µg/µl, (8): GO 5X10 ^-2 µg/µl, (9): GO 7X10 ^-2 µg/µl, (10) : GO 9X10 ^-2 µg/µl).
8 shows the PCR yield according to the concentration of graphene oxide (Au@GO) coated with gold nanoparticles of Example 3-1 (M: DNA marker, Con: no graphene oxide coated with gold nanoparticles Positive control; (1): Au@GO 1X10 ^-3 µg/µl, (2): Au@GO 3X10 ^-3 µg/µl, (3): Au@GO 5X10 ^-3 µg/µl, (4): Au @GO 7X10 ^-3 µg/µl, (5): Au@GO 9X10 ^-3 µg/µl, (6): Au@GO 1X10 ^-2 µg/µl, (7): Au@GO 3X10 ^-2 µg/µl , (8): Au@GO 5X10 ^-2 µg/µl, (9): Au@GO 7X10 ^-2 µg/µl, (10): Au@GO 9X10 ^-2 µg/µl).
9 shows the PCR yield according to the optimum concentration of gold nanoparticles (AuNPs), graphene oxide (GO) and graphene oxide (Au@GO) coated with gold nanoparticles of Example 3-2 (M: DNA marker, Con: positive control without nanoparticles; (1): AuNPs 7X10 ^-2 µg/µl, (2): AuNPs 9X10 ^-2 µg/µl (3): Go 1X10 ^-3 µg/µl, (4) : Go 3X10 ^-3 µg/µl, (5): Au@GO 1X10 ^-2 µg/µl, (6): Au@GO 3X10 ^-2 µg/µl).
FIG. 10 shows gold nanoparticles (AuNPs), graphene oxide (GO), and gold nanoparticles for each DNA sample of E. coli BL21 DNA (EGFP) into which fish DNA, Listeria monosite DNA (LM), and green fluorescent protein are inserted. It is a graph showing the quantitative PCR (qPCR) yield when the coated graphene oxide (Au@GO) is added.
FIG. 11 is a graph showing a cycle (Cq) in which replication starts for quantitative PCR (qPCR) for each DNA sample of E. coli BL21 DNA (EGFP) into which fish DNA, Listeria monosite DNA (LM), and green fluorescent protein are inserted. to be.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by an expert skilled in the art to which the present invention belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.

In the present invention, "GO" represents graphene oxide, "AuNP" represents gold nanoparticles, and "Au@GO" represents graphene oxide coated with gold nanoparticles.

In the present invention, in the quantitative PCR (qPCR), Cq (quantitative cycle) is the number of cycles of the fluorescence signal indicating the threshold, which is the point at which all templates start to react and amplification starts actively, that is, the number of cycles at which DNA replication starts. It represents a number, and the earlier the cycle appears, the more templates are replicated.

In the present invention, a PCR composition containing graphene oxide (Au@GO) coated with gold nanoparticles was prepared, and a PCR method in which it was added was performed. As a result, compared with the positive control containing nothing and gold nanoparticles (AuNPs) and graphene oxide (GO), the PCR composition containing graphene oxide (Au@GO) coated with gold nanoparticles was PCR efficiency. It was confirmed that it significantly improves.

Graphene oxide (Au@GO) containing gold nanoparticles used in the present invention is 1) interaction between Au@GO and Taq DNA polymerase (Au@GO- Taq complex having a positive charge has a negative charge) DNA and primer attraction), 2) DNA affinity (aromatic-aromatic interaction; π-π stacking) between graphene oxide (GO) and DNA of a nucleobase (Fig. 1), 3 ) Significantly improves PCR efficiency, including chemical interaction between gold nanoparticles and DNA and excellent thermal conductivity.

Accordingly, in one aspect, the present invention relates to a PCR composition comprising a gold nanoparticle-coated graphene oxide (Au@GO), a nucleic acid polymerase, and a primer pair capable of amplifying a target nucleic acid.

In the present invention, it may be characterized in that it further comprises a polymerase, a fluorescent substance capable of amplifying by binding to a specific nucleic acid site, a primer pair, and a buffer.

In the present invention, the primer pair may be selected from the group consisting of SEQ ID NOs: 1 to 2, SEQ ID NOs: 3 to 4, and SEQ ID NOs: 5 to 6, but is not limited thereto.

In the present invention, the polymerase may be characterized in that it is selected from the group consisting of Taq polymerase, VENT polymerase, DEEPVENT polymerase, PWO polymerase, and Pfu polymerase, but is limited thereto. no.

In the present invention, the buffer may be characterized in that it contains a gel loading buffer, but is not limited thereto.

In the present invention, a fluorescent substance that emits fluorescence by binding to a target nucleic acid may be further included, and the fluorescent substance is selected from the group consisting of SYBR GREEN, Cy5.5, Cy5, Cy3.5, and Cy3. can do.

In the present invention, the concentration of the graphene oxide (Au@GO) coated with the gold nanoparticles may be 1 X 10 ^-2 μg/μl to 3 X 10 ^-2 μg/μl, but is limited thereto. It does not become.

In another aspect, the present invention relates to a method of amplifying a target nucleic acid, characterized in that the PCR composition is used.

In the present invention, "target nucleic acid" refers to all nucleic acids that can be amplified by an amplification reaction such as PCR, and may be, for example, a nucleic acid such as DNA or RNA containing one or a plurality of base mutation sites.

In one embodiment of the present invention, a PCR composition using mackerel DNA as a template, graphene oxide (Au@GO) coated with gold nanoparticles, and graphene oxide (GO) and gold nanoparticles (AuNP) as a control, is used. Thus, as a result of detecting higher genes, gold nanoparticles (AuNP), graphene oxide (GO), and gold nanoparticles coated graphene oxide (GO@Au) were used for PCR compared to the control group (Con). The specificity and yield were high, and in particular, in the case of graphene oxide (Au@GO) coated with gold nanoparticles, it was confirmed that a stronger band appeared than gold nanoparticles (AuNP) and graphene oxide (GO). (Lanes 5 and 6 in Fig. 9).

In another aspect of the present invention, as a result of qPCR using EGFP DNA, which is a green fluorescent protein (GFP), and Listeria monocytogenes (ATCC 19115) DNA, which is a bacteria bacteria, graphene oxide coated with gold nanoparticles regardless of the type of DNA. It was confirmed that the final amplification amount of (Au@GO) was the largest.

In another aspect, the present invention relates to a kit for detecting a nucleic acid comprising the PCR composition.

In one aspect of the kit of the present invention, in addition to the above-described DNA polymerase, a composition comprising various components necessary for DNA synthesis by PCR method, such as dNTP, magnesium chloride, a buffer component for maintaining the reaction solution at an appropriate pH, etc. Can be mentioned. Moreover, this composition may contain the said acidic substance. Such a composition can be prepared so that a reaction solution can be prepared by adding a primer for amplifying a target DNA fragment and a template DNA, and then adding water or a buffer as necessary. In addition, when a DNA fragment to be amplified by the kit is determined, this composition may contain a primer suitable for amplification of this fragment.

[Example]

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

Preparation of samples and equipment

_{Phosphorus pentoxide (P 2} O ₅ ) used in the synthesis of graphene oxide, potassium persulfate (K ₂ S ₂ O ₈ ), sulfuric acid (H ₂ SO ₄ ,95~98%), potassium permanganism ( KMnO ₄ ) was purchased from Sigma-Aldrich (USA), and graphite powder was purchased from ANTO (China). Hydrochloric acid (HCl, 35.0%-37.0%) was purchased from SAMCHUN.

Gold(III) Chloride trihydrate used for the synthesis of graphene oxide (Au@GO) coated with gold nanoparticles was purchased from Sigma-Aldrich (USA), and sodium citrate was purchased from Bio Basic (CANADA). .

0.1 μm filter paper was purchased from Whatman (Germany), and tertiary distilled water (Milli-Q grade water 18.2 MΩ cm, Millipore) was used in all experiments.

Example 1: Preparation of GO@Au

1-1. Preparation of graphene oxide

Graphene oxide was synthesized using the Hummer and Offeman method (J. Am. Chem. Soc. 80 (1958) 1339), which is a representative method of synthesizing graphene oxide. 27 mL 50 wt% sulfuric acid (H ₂ SO ₄ ) was added to a 100 mL beaker and the temperature was raised to 84˚C. Add 2g of potassium persulfate (K ₂ S ₂ O ₈ ) and 2 g of phosphorus pentoxide (P ₂ O ₅ ) and stir until dissolved, while maintaining the temperature at 84°C, add graphite powder and stir for 5 hours I did. The temperature was lowered to room temperature, 500 mL of distilled water was added and stirred for 15 hours. Filter was performed using 0.1 μm filter paper and dried at room temperature. 50 wt% sulfuric acid (H ₂ SO ₄ ) 270 mL was made cold, and dried graphite powder was added. And it stirred while slowly adding 10 g of potassium permanganism (KMnO _{4 ).} Thereafter, the solution was stirred for 1 hour while maintaining the cold state, stirred at room temperature for 24 hours, and then 500 mL of distilled water and 30 wt% hydrogen peroxide (H ₂ O ₂ ) were added and stirred for 5 minutes. Then, the filter was performed using 0.1 μm filter paper and washed with 10 wt% hydrochloric acid (HCl) and distilled water.

1-2. Graphene oxide coated with gold nanoparticles

75 mg of graphene oxide was added to 275 mL of distilled water, and ultrasonic waves were applied for 1 hour. _{0.51 mM gold chloride (HAuCl 4} ) was prepared in 250 mL of distilled water. _{250 mL of gold chloride (HAuCl 4} ) solution was added to the graphene oxide solution to which ultrasonic waves were applied, and stirred for 30 minutes. The temperature was raised to 80˚C _{, 10 mL of 0.88 M sodium citrate (C 6} H ₅ Na ₃ O ₇ ·2H ₂ O) was added and stirred for 1 hour, and then the temperature was lowered to room temperature and stirred for 15 hours. Filtered with 0.1 μm filter paper and dried in an oven at 60°C.

Then, a uniformly diluted solution to which the prepared nanoparticles were added was prepared. Nanoparticles (1 mg) were individually suspended in autoclaved distilled water (1 ml). Since the nanoparticles are hydrophobic and do not mix with water, ultrasonic treatment (frequency 40 kHz, ultrasonic 100 W) was performed for 30 minutes using a water bath ultrasonic grinder (Rainbow, Korea) (FIG. 2). Then, the concentration of the nanoparticles was diluted using vortex and distilled water. In order to properly mix and prevent aggregation between nanoparticles in distilled water, ultrasonic treatment was performed immediately before the dilution step. It was confirmed that gold nanoparticles were coated on the graphene oxide using an absorption spectrum (UV/visible spectroscopy) and a transmission electron microscope (TEM) (FIGS. 3 and 4).

Example 2: PCR method using GO@Au

2-1. PCR procedure

First, PCR for amplifying DNA was performed using two specific primers (VF2_t1_M13 SEQ ID NO: 1; FishR2_t1_M13 SEQ ID NO: 2) (Table 1). Optimization and amplification of DNA was performed using the MAXIME PCR preMIX Kit (INtRON, Korea). As a DNA template, ^{DNA extraction extracted from mackerel using G-DEX TM} IIc For Cell/Tissue Genomic DNA Extraction Kit (INtRON, Korea) was used. The PCR reaction mixture contained the following final concentrations: i-Taq ^TM DNA polymerase 2.5 U/µl in reaction buffer, dNTP 2.5 mM, reaction buffer 1X, gel loading buffer 1X. In this reaction buffer mixture, 1 µl of template DNA, 1 µl of forward primer (10 pmol/µl), and 1 µl of reverse primer (10 pmol/µl) while making the concentration of nanoparticles the same or different until the total volume is 20 µl. ) 1 μl and distilled water were added.

After PCR using a BIO-RAD T100 ^TM Thermalcycler, electrophoresis was performed using 0.8% wt/v agarose gel (Seakem ^ㄾ LE Agarose, LANZA) to visualize the PCR product. ^{Mupid ㄾ} -2plus (ADVANCE) was used as an electrophoresis device. In addition, Marker 1 kb plus (ACCUGEN) was used as a size marker.

Forward primer
VF2_t1_M13(#1) Reverse primer
FishR2_t1_M13(#2) Band (amplicon)
size order 5'-CAACCAACCACAAAGACATTGGCAC-3' 5'-ACTTCAGGGTGACC
GAAGAATCAGAA-3'

800bp Tm (℃) 63 62 GC content (%) 48 33.3

2-2. PCR protocol

First, the temperature was continuously increased to 95°C in the initial denaturation step (95°C, 5 minutes). Non-specific annealing was observed according to the interaction between the nanoparticles and the primers. The primer was attached to the surface of the nanoparticles, forming an Au@GO- Taq complex. As a result, it was possible to minimize the phenomenon that primers are incorrectly attached to the template DNA. Eventually, it was found that PCR efficiency can be improved.

In the denaturation step (step 1; 95°C, 30 seconds), the hydrogen bonds in the double-stranded DNA are broken.

The Au@GO-Taq complex combined with a specific primer in the annealing step (step 2; 55° C., 30 seconds) has a strong affinity and pulls DNA through a π-π stacking interaction (FIG. 1). In addition, the hydrophobic interaction has the effect of attracting the target DNA. The reaction mixture was cooled to 55°C so that the primers bind to the single-stranded DNA attached to the Au@GO surface.

In the extension step (step 3; 72° C., 50 seconds), the synthesis of the complementary chain of the target DNA occurs (FIG. 5). Au@GO has high thermal conductivity. Thus, the PCR product added to the nanoparticles could quickly reach thermal equilibrium during the heating and cooling steps. Finally, rapid thermal equilibration brings high efficiency to the PCR system. The amplification of the above 3 steps was performed 30 times in the same manner. Finally, 4°C was maintained.

After PCR, it was analyzed in 0.8% wt/v agarose gel, and visualized using Gel-Doc. For electrophoresis, since the gel loading buffer was contained in the PCR reaction buffer, gel loading could be performed without any treatment.

Example 3: PCR result

3-1: PCR yield according to the concentration of nanoparticles

In the PCR system, a fish (mackerel) DNA extraction 800 bp fragment was used as a template. First, in order to select the optimal concentration of each graphene oxide (GO) and graphene oxide (Au@GO) coated with gold nanoparticles, GO and Au@GO, respectively, ^{from 1X10 -3} ㎍/µl to 9X10 ^-2 ㎍ Experiments were conducted at ten different concentrations between /µl. The total volume of the nanoparticles was 10 μl. When an extreme concentration (>1 μg/µl) is added to the PCR mixture, the PCR reaction is completely inhibited. This is judged by aggregation between the nanoparticles. It was found that both GO, AuNP, and Au@GO improve the amplification efficiency compared to the positive control group without nanoparticles (FIGS. 6, 7 and 8). When the concentration of GO is 1 X 10 ^-3 ~3X10 ^-3 µg/µl (lane 1 and 2), 7 X 10 ^-3 ~9X10 ^-3 µg/µl (lane 4 and 5), Au@GO When the concentration was 1 X 10 ^-2 µg/µl and 3 X 10 ^-2 µg/µl (lanes 6 and 7), a strong band was shown compared to the positive control without nanoparticles. As a result, it was confirmed that the PCR yield was highly dependent on the concentration of the nanoparticles.

3-2: GO@Au's PCR yield improvement effect

In the case of FIG. 9, when PCR was performed at the optimum concentration of each nanoparticle, gold nanoparticles (AuNP), graphene oxide (GO), and graphene oxide (Au@GO) coated with gold nanoparticles were used. A result was obtained that showed higher specificity and yield for PCR than the control group (Con). In particular, in the case of graphene oxide (Au@GO) coated with gold nanoparticles, it was confirmed that a stronger band appeared compared to gold nanoparticles (AuNP) and graphene oxide (GO) (lanes 5 and 6 of FIG. 9). .

Example 4: qPCR method using GO@Au

4-1: qPCR procedure

PCR for amplification of DNA was performed using six different primers (VF2_t1_M13 SEQ ID NO: 1; FishR2_t1_M13 SEQ ID NO: 2; EGFP_F_Nd SEQ ID NO: 3; EGFP_R_H3 SEQ ID NO: 4; LM0_F SEQ ID NO: 5; LM0_R SEQ ID NO: 6;). Tables 1 to 3). DNA extracted from fish DNA as a DNA template using G-DEX ^TM IIc For Cell/Tissue Genomic DNA Extraction Kit (INtRON, Korea) and green fluorescence that emits bright green fluorescence when exposed to light in the ultraviolet range from blue visible light EGFP DNA, which is a protein (GFP), and Listeria monocytogenes (ATCC 19115), which are bacteria were used. The qPCR reaction mixture included: Taq DNA polymerase, SYBR Green qPCR buffer, SYBR Green I, ROX Passive reference dye. In this reaction buffer mixture, 1 µl of template DNA, 1 µl of forward primer (10 pmol/µl), and 1 µl of reverse primer (10 pmol/µl) while making the concentration of nanoparticles the same or different until the total volume is 20 µl. ) 1 μl and distilled water were added.

After qPCR using the BIO-RAD CFX96 Touch ^TM Real-Time PCR Detection System, BIO-RAD CFX Manager 3.1 was used to visualize the PCR product.

Forward primer
EGFP_F_Nd(# 3) Reverse primer
EGFP_R_H3(# 4) Band (amplicon)
size order 5'-AAAATTCCATAT
GGTGAGCAAGGGCGA-3' 5'-TTCTAAGCTTTTA
CTTGTACAGCTCGTC-3'

800bp Tm (℃) 64.9 55.4 GC content (%) 44.4 39.3

Forward primer
LM0_F(# 5) Reverse primer
LM0-R(#6) Band (amplicon)
size order 5'-GCGCCACTACGGAC
GTTTAACCAAG-3' 5'-ACAATCGCATCCGCAA
GCACTGTAG-3'

200bp Tm (℃) 55.7 47.3 GC content (%) 56 52

4-2: qPCR protocol

First, the temperature was continuously increased to 95°C in the initial denaturation step (95°C, 5 minutes). In the denaturation step (step 1; 95°C, 30 seconds), the hydrogen bonds in the double-stranded DNA are broken. In the annealing step (step 2; 55°C, 30 seconds), a specific primer and a target DNA are bound. Features shown here are the same as in Example 2-3.

The amplification of step 2 was performed 50 times in the same manner.

In the extension step (step 3), the synthesis of the complementary chain of the target DNA occurs, attaches to a large groove of the DNA, absorbs light energy (500 nm) of a specific wavelength, and then emits light (530 nm) at a specific wavelength. The phosphorus SYBR Green I was combined together.

After qPCR, it was analyzed and visualized in BIO-RAD CFX Manager 3.1. The amount of amplified DNA could be quantified by measuring the degree of fluorescence of SYBR Green I attached to the DNA. Visualized using Gel-Doc.

Example 5: qPCR results

5-1: Au@GO's qPCR yield improvement effect

In the case of FIG. 10, as a result of repeatedly performing qPCR according to each DNA type, it was confirmed that the final amplification amount of graphene oxide (Au@GO) coated with gold nanoparticles was the largest regardless of any DNA type. As a result, it was confirmed that a noticeable difference appeared between qPCR yields.

5-1: Changes in the number of qPCR replication cycles of Au@GO

In qPCR, Cq (Quantification cycle) refers to the number of cycles at which replication begins. The smaller the number of cycles of Cq, the greater the expression with the target DNA.

In the case of FIG. 11, three types of DNA having different sizes of each DNA reaction product were used, and the Cq values of Au@GO were compared. It was confirmed that the Cq value of graphene oxide (Au@GO) coated with gold nanoparticles was the smallest compared to the control group.

As described above, specific parts of the present invention have been described in detail, and it is obvious that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> CHUNG ANG University industry Academic Cooperation Foundation <120> PCR Composition Containing Graphene Oxide Coated by Au Nanoparticles and Method for PCR Using The Same <130> P14-B337 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 caaccaacca caaagacatt ggcac 25 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acttcagggt gaccgaagaa tcagaa 26 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aaaattccat atggtgagca agggcga 27 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ttctaagctt ttacttgtac agctcgtc 28 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcgccactac ggacgtttaa ccaag 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acaatcgcat ccgcaagcac tgtag 25

Claims (12)

Graphene oxide (Au @ GO) coated with gold nanoparticles at a concentration of 1 × 10 ^-3 μg / μl to 1 × 10 ^-2 μg / μl, a nucleic acid polymerase and a primer pair capable of amplifying the objective nucleic acid PCR composition.
2. The PCR composition according to claim 1, wherein the concentration is 1 X 10 &lt; ^-3 &gt; to 5 X 10 &lt;&quot; ³ &gt;
2. The PCR composition of claim 1, wherein said nucleic acid polymerase is selected from the group consisting of Taq polymerase, VENT polymerase, DEEPVENT polymerase, PWO polymerase and Pfu polymerase.
The PCR composition according to claim 1, further comprising a fluorescent substance which fluoresces in combination with the target nucleic acid.
5. The PCR composition of claim 4, wherein the fluorescent material is selected from the group consisting of SYBR GREEN, Cy5.5, Cy5, Cy3.5, and Cy3.
The PCR composition of claim 1, further comprising a buffer solution.
7. The PCR composition of claim 6, wherein the buffer solution comprises a gel loading buffer.
2. The PCR composition according to claim 1, wherein said primer pair is selected from the group consisting of SEQ ID NOS: 1 to 2, SEQ ID NOS: 3 to 4, and SEQ ID NOS: 5 to 6.
The PCR composition according to claim 1, which is a composition for qPCR.
delete A method for amplifying a target nucleic acid, which comprises using the PCR composition of any one of claims 1 to 9.
A kit for detecting nucleic acid comprising the PCR composition of any one of claims 1 to 9.

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