KR101488436B1 - Method for Detecting Single Mismatches in DNA Hybridization Events Using Gold Nanoparticles - Google Patents

Abstract

The present invention relates to a method for detecting a single mismatch in a DNA hybridization reaction using gold nanoparticles, and more particularly, to a method for detecting a mismatch between a DNA probe and a detection probe capable of complementarily binding with a target nucleic acid, The present invention relates to a method for detecting a single base mutation of a target nucleic acid by controlling the reaction, quantitatively analyzing the difference between the absorbance of a complete hybridization of the target nucleic acid and the incompletely hybridized single base mutation.
The method for detecting a single base mutation of a target nucleic acid of the present invention quantitatively analyzes a difference in absorbance of a target nucleic acid and a single base mutation through a DNA-DNA hybridization reaction and a salt concentration control process, It is possible to detect a single base mutation and to use early prediction of the risk of a disease such as breast cancer.

Description

Technical Field [0001] The present invention relates to a method for detecting a single mismatch in DNA hybridization using gold nanoparticles,

The present invention relates to a method for detecting a single mismatch in a DNA hybridization reaction using gold nanoparticles, and more particularly, to a method for detecting a mismatch between a DNA probe and a detection probe capable of complementarily binding with a target nucleic acid, The present invention relates to a method for detecting a single base mutation of a target nucleic acid by controlling the reaction, quantitatively analyzing the difference between the absorbance of a complete hybridization of the target nucleic acid and the incompletely hybridized single base mutation.

Single nucleotide polymorphism (SNP) is a genetic mutation in which a single nucleotide sequence is substituted for a DNA sequence, which causes a person-to-individual difference, such as a cause of disease or a response to a therapeutic agent. Detection and confirmation of single nucleotide sequence mutations are attracting attention as they lead to the development of new drugs as well as custom drugs.

For rapid detection of single nucleotide sequence variation, various detection methods employing real-time PCR technology have been used. Representative methods include analysis using a DNA intercalating fluorescent substance, a method using a DNA probe, or a method using a PNA probe. However, it has limitations in using DNA intercalating fluorophores and has the disadvantage of using a program to analyze the melting curve (Kirk M. Ririe meat al ., Analytical Biochemistry 245: 154,1997; U. Hladnik meat al ., Clin Exp Med. , 2: 105, 2002)

Surface plasmon resonance (SPR) methods have been used extensively in many studies on the interaction between biomolecules immobilized on the surface, since they can quantitatively detect the reaction without complex pre-purification and labeling processes. The SPR method can be used to detect the adsorption of biomolecules on thin metal films due to the sensitive and fast response to changes in refractive index at the sensor surface. For the past several decades, the use of various SPR-based biomolecules to measure biomaterial concentration, thickness, and binding kinetics data for specific biochemical analytes including antigen / antibody, ligand / receptor, protein / protein reaction, Interaction analysis has been performed.

On the other hand, gold nanoparticles become red due to the collective vibration of free electrons of particles known as surface plasmon resonance and the interaction of incident light, and the probe using gold nanoparticles adsorbs and binds to the target material, may provide certain information, there is a high sensitivity and specificity, these DNA hybridization based on gold nanoparticle probes (probe) has been extensively developed in order to detect the target nucleic acid (Storhof et. al., Biosensors and Bioelectronics , 19: 875, 2004; CA Mirkin et . al ., Nature, 382: 607,1996). In addition, double-stranded DNA that is fully hybridized with a single base pair that makes mismatches appears to have a different propensity for adsorption to gold nanoparticles due to different electrostatic interactions (H. Li and L. Rothberg, PNAS , 101: 14036, 2004), and has been studied extensively in single nucleotide polymorphism (SNP) analysis using gold nanoparticles (SJ Park et al . , ≪ / RTI > Science , 295: 1503,2002; YWC Cao et al. , Science , 297: 1536, 2002)

In addition, DNA sensing using a complementary hybridization process between single stranded DNA (ssDNA) samples has recently been shown to be more sensitive, since small changes in the gene can affect biological predisposition, such as cancer or congenital disease Many studies have been conducted and the separation and signal detection of target nucleic acids using mainly liquid nanoparticles have been studied (Y. Weizmann et al . , J Am Chem Soc. , 133: 3238, 2011; S. Cai et al. Analyst , 135: 615, 2010; JT Petty et al . , Anal Chem. , 84: 356, 2012).

However, since mutant DNA detection using liquid nanoparticles in an unstable state is affected by the hybridization efficiency by controlling temperature and pH, the optical characteristics of the nanoparticles vary depending on the hydrodynamic effects, pH, and mixed factors, , And sensors using liquid nanoparticles suffer from the problems of low reaction efficiency and low reproducibility since they are subjected to various complicated steps (AG Kanaras et al., Angew Chem Int Edit , 42: 191, 2003).

Therefore, the present inventors have made intensive efforts to effectively detect a single base mutation of a target nucleic acid. As a result, the present inventors have found that a capture probe in which a capture DNA capable of binding complementarily to a target nucleic acid is bound to a gold nanoparticle, A hybridization reaction of the prepared capture probe and the detection probe with the target nucleic acid was carried out by controlling the salt concentration, As a result, it was confirmed that the difference in absorbance of a single base mutation that underwent incomplete hybridization with a target nucleic acid having a complete hybridization can be quantitatively analyzed, and the present invention was completed.

It is an object of the present invention to provide a method for multiplexing a single base mutation of a target nucleic acid in a DNA hybridization reaction using gold nanoparticles.

(A) fixing gold nanoparticles on a transparent support, and then binding the capture DNA capable of binding complementarily to one side of the target nucleic acid to the gold nanoparticles, thereby forming a capture probe capture probe; (b) performing hybridization of the capture probe-target nucleic acid by adding a single strand of the target nucleic acid to the prepared capture probe; (c) a hybridization reaction of the capture probe-target nucleic acid-detecting probe with a detection probe to which the detection DNA capable of binding complementarily to the other side of the target nucleic acid is added to the gold nanoparticle is added to the hybridization reaction product ; (d) deriving the hybridization difference between the target nucleic acid having a single base mutation and the target nucleic acid having no single base mutation through the control of the salt concentration, and then measuring the absorbance; And (e) analyzing the number of gold nanoparticles in the detection probe hybridized from the measured absorbance to detect the presence or absence of single base mutation in the target nucleic acid or DNA base hybridization reaction using gold nanoparticles A method for detecting a single base mutation of a target nucleic acid.

The method for detecting a single base mutation of a target nucleic acid of the present invention quantitatively analyzes a difference in absorbance of a target nucleic acid and a single base mutation through a DNA-DNA hybridization reaction and a salt concentration control process, It is possible to detect a single base mutation and to use early prediction of the risk of a disease such as breast cancer.

1 is a schematic diagram illustrating the overall process of the present invention. (A) shows a method for producing a capture probe on an amino-coated glass substrate, (B) a method for producing a detection probe, (C) And shows the overall process of detecting a single mismatch.
Fig. 2 is a schematic diagram showing electrostatic bonding according to the DNA-DNA binding between the matched DNA and the mismatched DNA.
FIG. 3 is a graph showing the difference (A) between the gold nanoparticles used for the production of a capture probe and a detection probe and the difference in absorbance according to the size of gold nanoparticles (B).
FIG. 4 is a graph (A) showing the shape of a capture probe fixed on a glass substrate and a change in absorbance due to hybridization of DNA between a capture probe and a detection probe (B).
FIG. 5 is a graph showing a process of electrophoresis in order to isolate a detection probe uniformly bound with DNA and a change in absorbance of a detection probe according to a change in salt concentration (B)
FIG. 6 shows AFM analysis results of the manufactured sensor (red: capture probe, yellow peak: detection probe combined).
7 is a graph (A) showing the height of the sensor surface prepared by AFM analysis and a graph (B) showing the change in absorbance according to the hybridization of the capture probe-target nucleic acid-detection probe.
8 is a graph showing the DNA hybridization efficiency of the matched DNA and the mismatched DNA through the number of detection probes according to the salt concentration.
9 is a graph showing the density of gold nanoparticles different from the target nucleic acid concentration change by (A) colorimetric method, (B) is a graph showing the relationship between the concentration of gold nanoparticles in the buffer, 1/5 diluted serum, And detecting the DNA of the target nucleic acid.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention provides a gold nanoparticle comprising (a) a gold nanoparticle immobilized on a transparent support, and then binding the capture DNA capable of binding complementarily to one side of the target nucleic acid to the gold nanoparticle to form a capture probe, ; (b) performing hybridization of the capture probe-target nucleic acid by adding a single strand of the target nucleic acid to the prepared capture probe; (c) a hybridization reaction of the capture probe-target nucleic acid-detecting probe with a detection probe to which the detection DNA capable of binding complementarily to the other side of the target nucleic acid is added to the gold nanoparticle is added to the hybridization reaction product ; (d) deriving the hybridization difference between the target nucleic acid having a single base mutation and the target nucleic acid having no single base mutation through the control of the salt concentration, and then measuring the absorbance; And (e) analyzing the number of gold nanoparticles in the detection probe hybridized from the measured absorbance to detect the presence or absence of single base mutation in the target nucleic acid or DNA base hybridization reaction using gold nanoparticles To a method for detecting a single base mutation of a target nucleic acid.

The 'target nucleic acid' of the present invention means a nucleic acid sequence to be detected, and is annealed or hybridized with the probe under hybridization conditions. The 'target nucleic acid' is not different from the 'target nucleic acid', 'target nucleic acid sequence' or 'target sequence', and is used in this specification.

The 'capture DNA' of the present invention refers to a base sequence complementary to one side (= one side) in a target nucleic acid sequence, and 'one side' refers to one end (5 'or 3') of a target nucleic acid, And a portion far from one end. The term "other DNA" refers to a nucleotide sequence capable of complementarily binding to the other side (= other side) in the target nucleic acid sequence, and the term "other side" means the other end (5 'or 3' , A portion close to the other end, and a portion apart from the other end. That is, the portion where the capture DNA hybridizes with the detection DNA in the target nucleic acid is a different portion. In the present invention, when both the capture DNA and the detection DNA hybridize to the target nucleic acid, And the presence or absence of a single base mutation of the target nucleic acid was detected.

"Hybridization" of the present invention means that complementary single-stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur when perfect complementarity between two nucleic acid strands is achieved (perfect match) or some mismatching base is present. The degree of complementarity required for hybridization can be varied depending on the hybridization conditions, and is adjusted using the salt concentration change in the present invention.

In the DNA hybridization reaction using the gold nanoparticles of the present invention, a method for detecting a single base mutation of a target nucleic acid is a method of detecting a DNA having perfect hybridization and one or more mismatches ) Of the target nucleic acid through the difference in the DNA constituting the target nucleic acid. DNA that forms a complete hybridization is strong and stable, whereas a DNA having one or more base mutations is not well formed due to hydrogen bonding, resulting in a lower binding force and a greater influence depending on temperature and pH. In addition, as shown in Fig. 2, DNA having one or more base mutations is characterized in that an unstable portion occurs in the DNA-DNA bond and the electrostatic binding force weakens. In the present invention, the single base mutation of the target nucleic acid was detected using the difference in the electrostatic binding force through the control of the salt concentration.

In the present invention, the gold nanoparticles of the capture probe and the detection probe are prepared by reducing HAuCl _{4 with} citrate and have an average size of 10 to 20 nm. The size of the gold nanoparticles is determined by the amount of the reducing agent and the reaction time. In one embodiment of the present invention, gold nanoparticles having an average diameter of about 10 nm (9.992 ± 1.14 nm) and 20 nm (20.09 ± 2.22 nm) (Fig. 3A). 10 nm gold nanoparticles are used to generate optical signals in sensitive SPRs due to their low reflectivity, and 20 nm gold nanoparticles provide the space needed to recognize a particular sequence, making them suitable for the manufacture of capture probes.

In the present invention, the gold nanoparticle surface in step (a) is an organic adsorbent _{HS (CH 2) 11 (OCH} 2 CH 2) 3 OH (EG 3 -OH) , and _{HS (CH 2) 11 (OCH} 2 CH ₂ ) ₆ OCH ₂ COOH (EG ₆ -COOH).

In the present invention, the transparent support of step (a) may be a glass coated with an amine group. However, it is not limited thereto, and a support capable of transmitting light may be used. In order to facilitate bonding with the nanoparticles, an amine group can be coated on a transparent support.

As shown in FIG. 1A for forming self-assembled monolayers (SAMs) so that the surface of the gold nanoparticles prepared above has an oligomer (ethylene glycol) (oligo (ethyleneglycol); OEG) The adsorbent HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₃ OH (EG ₃ -OH) and HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₆ OCH ₂ COOH (EG ₆ -COOH) The surface of the gold nanoparticles was modified by reacting with the particles. EG ₆ -COOH is a linker that assists in binding capture DNA and gold nanoparticles with amine groups coated glass substrates or amine groups, while EG ₃ -OH is a spacer that provides the space required to recognize a particular base sequence

The gold nanoparticles having diameters of 10 nm and 20 nm were modified with oligo (ethylene glycol) and measured with a UV / VIS spectrophotometer (FIG. 3B). The 20 nm gold nanoparticles were observed at 10 nm The gold nanoparticles having a size of 20 nm were used for the fabrication of the capture probe and the gold nanoparticles having a size of 10 nm Gold nanoparticles were used in the production of detection probes.

In one embodiment of the present invention, N-hydroxysuccinimide and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide are added to gold nanoparticles having a size of 20 nm modified with oligo (ethylene glycol) The mixed solution is treated with an amine-coated glass to fix the gold nanoparticles on a glass plate, and then the complementary metal complex is added to the target nucleic acid to prepare a gold capture nanoprobe. (3'-amine-functionalized ssDNAs) with binding to the gold nanoparticles prepared above were added to a fixed glass plate to prepare a capture probe.

As a result of observing the capture probe fabricated by the above method, it was confirmed that the gold nanoparticles were uniformly fixed on the glass plate at a ratio of 93.9% (FIG. 4A). After the gold nanoparticle surface was modified and fixed on the glass plate, As shown in FIG. 4B, when a gold nanoparticle and a capture DNA were bound (step 2) and a nanoparticle and a capture DNA (FIG. 4B) were observed, (Step 3), the red shifts of 4 nm and 3 nm were observed, respectively, and it was confirmed that the capture probe was well-made.

Gold nanoparticles prepared by reducing sodium citrate with HAuCl ₄ are surrounded by negative charge layers around the nanoparticles by citrate ions having a negative charge in the gold colloid state, The layer is compressed or expanded depending on the total ion concentration of the surrounding solution. When the gold nanoparticles are reacted in such a colloid state without fixing them on the glass plate, the gold nanocolloid is exposed to the electrolyte solution due to the electrostatic effect of the charge layer, thereby decreasing the stability and reactivity, and the sensitive plasmon band of the nanoparticles Ionic strength and change in mixing parameters. In addition, in a complicated reaction system such as the method of detecting a single base mutation of a target nucleic acid of the present invention, there is a disadvantage that noise is generated due to interaction between a target nucleic acid and a non-target nucleic acid and Brownian motion of nanoparticles.

However, when the gold nanoparticles are fixed on a glass substrate to produce a capture probe, it is possible to (1) uniformly arrange the gold nanoparticles on the glass substrate to optimize the overall reaction, and (2) (3) the reaction solution can be simplified for each step to reduce the noise, (4) the binding site with the detection probe can be prevented, and (5) It is possible to maintain the reactivity of the capture probe in order to improve the reproducibility, and it is possible to overcome the limitation of the gold nanocolloid and to detect the super-sensitive DNA .

The surface of the gold nanoparticle modified with self-assembled monolayers (SAMs) such that the oligo (ethylene glycol) (OEG) of the present invention is at the terminal end not only keeps the stability of the particles, Since it can protect the surface from adsorption, it plays an important role in the construction of capture nanoprobe.

Previous studies have shown that the three-dimensional structure of nanoparticles bound to high-density DNA oligomers has limited ability to hybridize probes due to electrostatic repulsion and steric hindrance (C. Cao and SJ Sim, J Nanosci Nanotechno , 7, : 3754, 2007). Capture probes prepared by the method of the present invention were improved in the hybridization efficiency by modifying the surface to have both functionalized groups and spacers through oligo (ethylene glycol) , Linker systems using EG ₆ -COOH not only provide a large number of binding sites for increased sensitivity, but also have the effect of reducing background intensities due to non-specific binding during the hybridization reaction.

In the present invention, the detection probe of step (c) may be prepared by (a) treating gold nanoparticles with 3-mercaptopropionic acid (MPA) and dATP and reacting them to form melopentopropionic acid and dATP Coupling; And (b) detecting DNA and a salt capable of binding complementarily to the target nucleic acid to the gold nanoparticles to which the melphopropopropionic acid and dATP are bound, and the detection probe is prepared by exchanging the detection DNA with dATP.

In one embodiment of the present invention, 10 nm sized gold nanoparticles conjugated with melapentopropionic acid (MPA) and dATP using a competitive binding method are incubated with detection DNA (thiol -DNA, detection probe) was prepared and the detection DNA was bound to the gold nanoparticles through the ligand exchange of the detection DNA (thiol-DNA, detection probe) and dATP. Since the number of detection DNAs bound to each gold nanoparticle differs between the prepared detection probes, electrophoresis was performed in order to separate a uniform detection probe. As shown in FIG. 5A, the number of DNAs bound to gold nanoparticles The moving speeds are different from each other, so that a uniform detection probe can be selected.

As shown in FIG. 1B, when mercapto propionic acid (MPA) and dATP are reacted at the surface of gold nanoparticles at room temperature, self-assembled monolayers are rapidly formed and dATP-bound gold nanoparticles have a high concentration (WT Zhao et < RTI ID = 0.0 > al . , Langmuir , 23: 7143, 2007). By changing the temperature from 45 to 55 ° C, dATP is separated from the gold nanoparticles, and the ligand exchange between the detection DNA in which the dATP and the thiol group are bound is accomplished within 2 hours by adjusting the salt concentration. In addition, the dATP residue of gold nanoparticles helps reduce non-specific DNA binding to nanoparticles due to nitrogen-containing bases, and the reduction of nonspecific DNA binding by dATP Helps in the proper orientation of single stranded DNA in the binding process.

In the present invention, even when gold nanoparticles are treated at a high temperature in order to remove dATP, the mercapto propionic acid (MPA) remains in a state of being continuously bonded to the gold nanoparticle surface by salt-induced aggregation, (Fig. 5B) that the detection probe was stably maintained even after the treatment with the salt of the concentration of the enzyme.

The carboxyl group of the mercapto propionic acid (MPA) bound to the gold nanoparticles increases the electron density of the gold nanoparticles through the negative charge induction at a high pH to increase the plasmon resonance phenomenon and improve the sensitivity But it has the effect of improving the stability of nanoparticles through electrostatic repulsion. In addition, in the gel electrophoresis, a positive charge for transferring the gold nanoparticles is provided to the bipolar electrode, thereby increasing the separation efficiency of the detection probe.

The capture probe and the detection probe prepared in the present invention form a sandwich form having a capture probe-target nucleic acid-detection probe configuration after hybridization with a target nucleic acid. As shown in FIG. 6, the capture probe and the detection probe are subjected to atomic force microscopy AFM). As a result, it was confirmed that the capture probe (red) and the detection probe (yellow peak) were stably bonded onto the glass substrate. In addition, when hybridization with a capture probe-target nucleic acid-detecting probe was performed, the height of the gold nanoparticle surface of the capture probe was analyzed by an atomic force microscope because the height was increased (16.8 nm). As a result, It was confirmed that the probe and the detection probe were combined.

In order to confirm whether the system using the capture probe and the detection probe of the present invention specifically reacted with the target nucleic acid, the absorbance of the capture probe (line 1 in FIG. 7B), the case where the detection probe was inserted into the capture probe treated with the non- (FIG. 7B line 2) and the absorbance when the detection probe was put in the capture probe treated with the target nucleic acid (line 3 in FIG. 7B) was measured. When the detection probe was inserted into the capture probe to which the non- The capture probe-non-target nucleic acid-detection probe was not coupled and the difference between the absorbance of the capture probe and that of the capture probe hardly occurred. However, when the detection probe was inserted into the capture probe to which the target nucleic acid was bound, It is confirmed that the detection probe is coupled and the absorption spectrum moves. That is, it was confirmed that the system using the detection probe and the detection probe of the present invention specifically binds to the target nucleic acid.

In the present invention, the gold nanoparticle number of the detection probe in the step (e) may be analyzed by the following equation.

[Equation 1]

(N = number of gold nanoparticles measured per unit volume (number of gold nanoparticles in the detection probe), Abs ₄₅₀ = absorbance at ₄₅₀ nm, R ' = vibration amount of free electrons)

(N) of a detection probe hybridizing with a target nucleic acid can be measured by substituting the absorbance of a single base mutation that incompletely hybridizes with the target nucleic acid of the present invention, which is the complete hybridization of the present invention, into Equation (1) Quot; means the number of target nucleic acids bound to the probe.

In order to investigate the influence of the salt concentration on the hybridization efficiency of the nanoparticles and DNA in the system using the capture probe and the detection probe of the present invention, the kinetic behavior of DNA hybridization was evaluated as a function of sodium ion concentration . As a result of measuring the number of detection probes coupled to the capture probe using the equation, it was confirmed that the amount of the detection probe coupled to the capture probe increases as the salt concentration increases as shown in FIG. 8, and the incomplete hybridization It was confirmed that the single base mutation made was significantly lower than that of the target nucleic acid which made a complete hybridization, thus reducing the number of detection probes bound to the capture probe.

Since the sodium ion of the salt to be added is concentrated in the DNA grooves of the target nucleic acid hybridized with the capture probe and the detection probe by the electrostatic interaction, the hybridization efficiency and the stability line of the target nucleic acid are improved when the salt concentration is increased, The degree of hybridization between the probe and the target nucleic acid of the detection probe is dependent on the electrostatic interaction controlled by the salt concentration.

In the case of a single base mutation, the electrostatic binding force is weakened in the DNA hybridization reaction, so that there is a difference in the degree of binding with the detection probe when compared with the target nucleic acid which forms a complete hybridization. Therefore, it is possible to optimize the salt concentration to increase the difference in the number of the detection probes bound to the target nucleic acid (PMT) and the single base mutation (MMT), which achieve complete hybridization by controlling the salt concentration. As shown in FIG. 8, it was confirmed that the number of detection probes bound to the target nucleic acid (PMT) and the single base mutation (MMT) at a salt concentration of 0.2 M showed a difference of about 5.5 × 10 ¹⁰ at the maximum.

In one embodiment of the present invention, a target nucleic acid having a concentration of 0.1 fM to 10 nM is treated to measure the degree of DNA hybridization according to the target nucleic acid concentration to measure the number of gold nanoparticles hybridized with the target nucleic acid (detection probe) As a result, it was confirmed that the method of the present invention was able to detect a target nucleic acid at a maximum of 10 fM (Fig. 9A), and the detection method of the present invention can detect not only a target nucleic acid amount but also a single target nucleic acid The degree of nucleotide variation can be grasped.

In order to confirm whether the assay method of the present invention can be applied to a human serum sample, analysis was performed using human serum containing a target nucleic acid and a non-target nucleic acid. As a result, as shown in FIG. 9B, It was confirmed that the target nucleic acid contained in the serum and the buffer diluted 1/5 was detected. This means that the method of the present invention can not only detect a specific gene contained in a serum immediately without complicated processes such as PCR, but also detect a mutation (base mutation) of a specific gene.

In the present invention, the target nucleic acid may be BRCA- 1 (breast cancer type 1), which is a breast cancer-related gene, and the detection method using the capture probe and the detection probe of the present invention is not limited thereto. Can be detected. To ensure that that can be effectively as the detection method of the present invention detects a BRCA gene BRCA -1 -1 gene and a single nucleotide mutation occurred, naemeurosseo identify a mutation of a single base mutation, it is possible to predict the risk of breast cancer for early .

In the DNA hybridization reaction using the gold nanoparticles of the present invention, a single base change detection method of a target nucleic acid can be performed by adjusting the difference in electrostatic binding force between the DNA that undergoes complete hybridization and the DNA that undergoes hybridization to a salt concentration, The method of measuring the number of detection probes combined with the target nucleic acid using Equation 1 is used so that not only the detection of a single base mutation but also the detection of a nucleotide mutation caused by a mutation of a target nucleic acid Mismatch can be detected. In addition, since the number of detection probes bound to the target nucleic acid can be measured and the target nucleic acid can be detected and quantified, it is possible to diagnose a disease caused by a gene or gene mutation that induces a specific disease.

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 examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Gold nanoparticles DNA of Hybridization  Single base mutation detection kit of target nucleic acid using reaction

1-1: Preparation of gold nanoparticles

In the present invention, gold nanoparticles for producing a single base mutation detection kit using a hybridization reaction between gold nanoparticles and DNA are prepared by a known method (C. Cao and SJ Sim, J Nanosci Nanotechno , 7: 3754, 2007) was prepared by reducing sodium chloroaurate (HAuCl ₄ ) with sodium citrate.

First, a 3: 1 mixture of hydrochloric acid (HCl) and nitric acid (HNO ₃ ) was added to a large beaker to prevent nucleation from being uneven due to contaminants during gold nanoparticle formation. Regia was prepared, and all the flasks and magnetic stirrer used to make the gold nanoparticles were immersed for at least 15 minutes to remove contaminants and then washed with ultrapure water.

20 ml of a 0.25 mM solution of HAuCl ₄ was added to the flask prepared above and stirred on a hot plate. When the solution started to boil, 0.2 ml of a 0.5 mM sodium citrate solution was rapidly added. Then, the color of the solution changed from light yellow to dark red, and after boiling for 10 minutes, the solution was stirred at room temperature for about 15 minutes. After the solution was cooled to room temperature, the evaporated water was corrected using ultrapure water (18.2 mΩ / cm). To remove aggregated gold nanoparticles or other impurities during the making process, The membrane was filtered with a membrane filter, and the resulting gold nanoparticles were stored at 4 ° C in a refrigerator.

1-2: Of the capture probe  Produce

To form self-assembled monolayers (SAMs) such that oligo (ethylene glycol) (OEG) is at the terminal on the surface of the gold nanoparticles prepared above, C. Cao and SJ Sim, J Nanosci Nanotechno , 7: 3754, 2007; C. Cao and SJ Sim, Biosensors & bioelectronics , 22: 1874, 2007).

As shown in FIG. 1A, the organic absorbent is _{HS (CH 2) 11 (OCH} 2 CH 2) 3 OH (EG 3 -OH) , and _{HS (CH 2) 11 (OCH} 2 CH 2) 6 OCH 2 COOH (EG 6 - COOH) were mixed and reacted with gold nanoparticles to modify the gold nanoparticle surface. 9 ml of gold nanoparticle solution was added to 1 ml of absolute ethanol mixed with EG ₃ -OH and EG ₆ -COOH in a ratio of 1: 9 so that the final concentration of gold nanoparticles was 0.5 mM. The reaction was allowed to proceed overnight at room temperature while gently shaking to form self-assembled monolayers (SAMs) on the surface of gold nanoparticles. Then, the supernatant was discarded by centrifugation at 14000 rpm for 30 minutes at 4 ° C, and the remaining precipitate (gold nanoparticles) was washed with PBS (0.01M, pH 6) The above washing procedure was repeated three or more times to remove unbound OEG (ethylene glycol).

Finally, the carboxyl-stabilized AuNP solution was concentrated to 5 mL, and 100 μL of 0.1 M N-hydroxysuccinimide (NHS, Sigma aldrich) was added immediately Then, 100 μl of 0.1 M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDS, Sigma aldrich) was added thereto and the reaction was carried out for 10 minutes . Subsequently, the mixed solution was treated with amine-coated glass, reacted at room temperature overnight, and carefully washed with deionized water.

As a result of observing the capture probe fabricated by the above method, it was confirmed that the gold nanoparticles were uniformly fixed on the glass plate at a ratio of 93.9% (FIG. 4A). After the gold nanoparticle surface was modified and fixed on the glass plate, As shown in FIG. 4B, when a gold nanoparticle and a capture DNA were bound (step 2) and a nanoparticle and a capture DNA (FIG. 4B) were observed, (Step 3), the red shifts of 4 nm and 3 nm were observed, respectively, and it was confirmed that the capture probe was well-made.

To prepare a gold capture nanoprobe, the gold nanoparticles prepared above were coated on a fixed glass substrate with 3'-amine-functionalized ssDNAs (SEQ ID NO: 1) was added and captured DNA was bound to the gold nanoparticles while gently shaking at room temperature for 8 hours, and then washed three times to remove single-stranded capture DNA not bound to gold nanoparticles.

Capture DNA

[SEQ ID NO: 1] 5'-GAAACCCTATGTATGCTCTTTTTTTTTT-Amine-3 '

Gold nanoparticles having an average diameter of about 10 nm (9.992 占 1.14 nm) and 20 nm (20.09 占 2.22 nm) were fabricated by the method of Example 1-1 (FIG. 3A), and gold nanoparticles having 10 nm and 20 nm sizes (Ethylene glycol) and measured with a UV / VIS spectrophotometer (Fig. 3B). The 20 nm gold nanoparticles were significantly broader and longer wavelength than the 10 nm gold nanoparticles due to the larger optical cross sections It was confirmed that the peak was shifted to the side. Therefore, in the present invention, gold nanoparticles having a size of 20 nm were used to fabricate a capture probe, and gold nanoparticles having a size of 10 nm were used in the production of a detection probe.

1-3: Detection Probe ( detection probe ) Produce

3-mercaptopropionic acid (MPA, sigma aldrich) and dATP (300 μM; bioneer) were added to 10 nm-sized gold nanoparticles prepared by the method of Example 1-1 at pH 10 to 30 Min, and centrifugation and resuspension were repeated three or more times to remove the remaining solution. 10 mM PBS buffer (pH 8) and 0.1 M sodium chloride (NaCl) solution were treated with gold nanoparticles pretreated with MPA and dATP as described above. Then, 500 μM of thiol-bound thiol-DNA And reacted at 50 ° C for 2 hours with gentle shaking (FIG. 1B).

Detection DNA (Detection DNA)

[SEQ ID NO: 2] 5'-Thiol-TTTGTATGAATTATAATCAAA-3 '

Since the number of DNAs bound to each gold nanoparticle differs in the detection probe prepared above, electrophoresis was performed in order to separate a uniform detection probe. The same amount of 10% glycerol as the solution containing the detection probe was added to 2% agarose gels. After electrophoresis in 0.5X Tris-Borate-EDTA buffer at 5 V / cm, As shown in FIG. 5A, since the detection probes are separated and the moving speed varies according to the number of DNAs bound to gold nanoparticles, a uniform detection probe can be selected. The separated detection probes were stored at 4 ° C using PBS buffer.

Also, in the present invention, even when the gold nanoparticles are treated at a high temperature to remove dATP, the mercapto propionic acid (MPA) is still bound to the gold nanoparticle surface by salt-induced aggregation , And it was confirmed by absorbance measurement that the detection probe was stably maintained even after treatment with a high concentration of salt (Fig. 5B).

Manufactured Capture Probe and  detection Of the probe On hybridization  Identify features

2-1: Capture Probe - Target nucleic acid - detection Of the probe Hybridization  Confirm

Experiments were conducted to analyze the degree of hybridization with the target nucleic acid using the capture probe and the detection probe prepared in Example 1 of the present invention.

First, a single-stranded target nucleic acid (SEQ ID NO: 3) was treated with a capture probe on which gold nanoparticles were immobilized on a glass substrate, reacted for 1 hour while gently shaking at 37 DEG C, Lt; / RTI > Then, 100 μl of the detection probe was added and incubated for 1 hour to react with the capture probe and the hybridized target nucleic acid, and then washed 3 times to remove the remaining detection probes.

As a result of the DNA hybridization reaction, a sandwich structure of a capture probe (20 nm gold nanoparticle) - a target nucleic acid-detection probe (10 nm gold nanoparticle) was formed and analyzed using atomic force microscopy (AFM) It was confirmed that the capture probe (red) and the detection probe (yellow peak) were stably bonded onto the glass substrate (Fig. 6). In addition, when hybridization with a capture probe-target nucleic acid-detecting probe was performed, the height of the gold nanoparticle surface of the capture probe was analyzed by an atomic force microscope because the height was increased (16.8 nm). As a result, It was confirmed that the probe and the detection probe were combined.

2-2: Target nucleic acid detection

DNA hybridization was performed using a target nucleic acid and a non-target nucleic acid to confirm whether the system using the capture probe and the detection probe of the present invention specifically reacted with the target nucleic acid.

The target nucleic acid and the non-target nucleic acid base sequence Sequences (5'-3 ') SEQ ID NO: Target nucleic acid
(target DNA) 5'-GAGCATACATAGGGTTTCTCTTGGTTTCTTTGATTATAATTCATAC-3 ' SEQ ID NO: 3 Non-target nucleic acid
(nontarget DNA) 5'-ACACGCTTGGTAGACTTTTTTTTTTAGCATCGATAACGTTAAAAAA-3 ' SEQ ID NO: 4

After the non-target nucleic acid and the target nucleic acid were treated and reacted with the capture probe, the detection probe was added to induce DNA hybridization, and the absorbance was measured using a UV / Vis spectrophotometer. The absorbance of the capture probe Were measured. As a result, when the detection probe was inserted into the non-target nucleic acid-bound capture probe (line 2 in FIG. 7B), the capture probe-non-target nucleic acid-detection probe was not bonded and the absorbance of only the capture probe 1). However, when a detection probe was inserted into the capture probe to which the target nucleic acid was bound (line 3 in FIG. 7B), the capture probe-target nucleic acid-detection probe was bound to confirm that the absorption spectrum was shifted there was. That is, it was confirmed that the system using the detection probe and the detection probe of the present invention specifically binds to the target nucleic acid.

2-3: detection bound to the target nucleic acid Probe  Check number

In order to quantitatively analyze the difference in absorbance of a single base mutation that incompletely hybridizes with a target nucleic acid that forms a complete hybridization in the present invention, the number of gold nanoparticles of a detection probe bound to a target nucleic acid and a single base mutation Were measured.

[Equation 1]

Here, N means the number of gold nanoparticles (number of gold nanoparticles in the detection probe) measured per unit volume, Abs _{450 indicates} the absorbance at ₄₅₀ nm, and R ' indicates the amount of free electrons.

Equation 1 is a modification of the commonly used Mie equation (Equation 2) and the Riccati-Besel equation (Equation 3). When the absorbance value is substituted, the number of gold nanoparticles (N) of the detection probe is finally measured .

&Quot; (2) "

Here, σ _ext = extinction cross section of the gold nanoparticles, ω = radiation frequencies (the radiation frequency), c = the speed (light speed) of light, ε _m = dielectric constant, V = the volume of gold nanoparticles, R ' = Radius of free electron ( R '= R-r ), R = radius of gold nanoparticle, r = radius of electron cloud around gold nanoparticle, .

&Quot; (3) "

Here, Q _ext = σ _ext / πR ² means the extinction efficiency, and l ₀ = the length of the path used to measure the absorbance.

That is, the absorbance of a single base mutation that incompletely hybridizes with the target nucleic acid of the present invention which forms the complete hybridization can be substituted into Equation (1) to measure the number of gold nanoparticles (N) of the detection probe hybridizing with the target nucleic acid, This can be taken to mean the number of target nucleic acids bound to the capture probe.

Single base mutation detection of target nucleic acid by salt concentration

In order to investigate the hybridization efficiency of nanoparticles and DNA according to the increase of the salt concentration and the effect of detecting the single mutation base of the target nucleic acid (SEQ ID NO: 5) in the system using the capture probe and the detection probe of the present invention, Kinetic behavior was measured using a function of sodium ion concentration.

Target nucleic acid and single mutation base sequence Sequences (5'-3 ') SEQ ID NO: Target nucleic acid
(target DNA) 5'-GAGCATACATAGGGTTTCTCTTGGTTTCTTTGATTATAATTCATAC-3 ' SEQ ID NO: 3 Single base mutation
(Single mismatches) 5'-GAGCATACATAGGGTTTCTCTTGGTTTCTTTGATTAT C ATTCATAC-3 ' SEQ ID NO: 5

The target nucleic acid and the single base mutation were each treated at 0.2 M and the experiment was performed by increasing the salt concentration from 0.1 M to 0.5 M while keeping the concentration of the detection probe (gold nanoparticle) and the target nucleic acid constant.

After hybridization of the capture probe-target nucleic acid-detection probe, the absorbance was measured and the number of detection probes bound to the capture probe was measured using Equation (1). As a result, as shown in FIG. 6, it was confirmed that the amount of the detection probe hybridized with the target nucleic acid was increased with an increase in the salt concentration, and the single mutation base had a lower electrostatic binding force than the target hybridizing nucleic acid Therefore, it was confirmed that the number of detection probes coupled to the capture probe was reduced (FIG. 8).

Also, it was confirmed that the number of detection probes bound to the target nucleic acid (PMT) and the single base mutation (MMT) at a salt concentration of 0.2M showed a maximum difference of about 5.5 × 10 ¹⁰ .

Gold nanoparticles DNA Hybridization  Detection and quantification of target nucleic acid using reaction

Quantitative analysis was performed to measure the degree of DNA hybridization according to the target nucleic acid concentration according to the present invention.

100 μl of the target nucleic acid having a concentration of 0.1 fM to 10 nM was reacted with the capture probe for 1 hour and then reacted with the detection probe to induce hybridization of the capture probe-target nucleic acid-detecting probe to measure the absorbance of the gold nanoparticle . The number of gold nanoparticles hybridized to the target nucleic acid (detection probe) was measured using the measured absorbance value and the formula (1), and it was confirmed that the method of the present invention was able to detect a target nucleic acid at a maximum of 10 fM 9A).

In order to confirm whether the assay method of the present invention can be applied to human serum samples, analysis was performed using human serum containing target nucleic acid and non-target nucleic acid.

Serum containing 50 nM of target nucleic acid and non-target nucleic acid, 1/5 diluted serum and 100 쨉 l of buffer were added to a capture probe immobilized with gold nanoparticles on a glass substrate, reacted for 1 hour while gently shaking at 37 캜 , And washed three times with a buffer to remove unhybridized DNA. Then, 100 μl of the detection probe was added and incubated for 1 hour to react with the capture probe and the hybridized target nucleic acid, and then washed 3 times to remove the remaining detection probes.

In order to measure the amount of target nucleic acid bound to the capture probe, the absorbance was measured and the number of gold nanoparticles was calculated by the following equation (1). As a result, as shown in FIG. 9B, The target nucleic acid contained in the serum and the buffer was detected, and it was confirmed that the detection probe of the present invention binds only to the target nucleic acid and shows a signal.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Korea University Research and Business Foundation <120> Method for Detecting Single Mismatches in DNA Hybridization          Events Using Gold Nanoparticles <130> P13-B015 <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> capture DNA <400> 1 gaaaccctat gtatgctctt tttttttt 28 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> detection DNA <400> 2 tttgtatgaa ttataatcaa a 21 <210> 3 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> target DNA <400> 3 gagcatacat agggtttctc ttggtttctt tgattataat tcatac 46 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> nontarget DNA <400> 4 acacgcttgg tagacttttt tttttagcat cgataacgtt aaaaaa 46 <210> 5 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> single mismatches <400> 5 gagcatacat agggtttctc ttggtttctt tgattatcat tcatac 46

Claims (8)

Detection of a single base mutation of a target nucleic acid in a DNA hybridization reaction using gold nanoparticles comprising the steps of:
(a) The surface of the transparent support is coated with an organic adsorbent such as HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₃ OH (EG ₃ -OH) or HS (CH ₂ ) ₁₁ (OCH ₂ CH ₂ ) ₆ OCH ₂ COOH ₆ -COOH), and then preparing a capture probe by binding a capture DNA capable of binding complementarily to one side of the target nucleic acid to the gold nanoparticle, thereby preparing a capture probe ;
(b) performing hybridization of the capture probe-target nucleic acid by adding a single strand of the target nucleic acid to the prepared capture probe;
(c) a hybridization reaction of the capture probe-target nucleic acid-detecting probe with a detection probe to which the detection DNA capable of binding complementarily to the other side of the target nucleic acid is added to the gold nanoparticle is added to the hybridization reaction product Steps to Perform
Here, the detection probe may be prepared by (i) treating gold nanoparticles with 3-mercaptopropionic acid (MPA) and dATP and reacting them to bind melphantopropionic acid and dATP to the surface of gold nanoparticles; And (ii) a detection DNA and a salt capable of binding complementarily to the target nucleic acid to the gold nanoparticles to which the melphopropopropionic acid and dATP are bound are added, and the detection DNA and the salt, which are bound to the gold nanoparticle surface at 45 to 55 ° C preparing a detection probe by exchanging the detection DNA with dATP;
(d) adjusting the salt concentration to a concentration of 0.1 to 0.5 M to induce a hybridization difference between the target nucleic acid having a single base mutation and the target nucleic acid having no single base mutation, and then measuring the absorbance; And
(e) analyzing the gold nanoparticle number of the hybridized detection probe from the measured absorbance to detect the presence or absence of a single base mutation in the target nucleic acid or the number of single base mutations.
The method of claim 1, wherein the gold nanoparticles of the capture probe and the detection probe are prepared by reducing HAuCl ₄ to citrate and have an average size of 10-20 nm. Mutation detection method.
The method according to claim 1, wherein the transparent support of step (a) is a glass coated with an amino group.
delete delete The method according to claim 1, wherein the salt of step (ii) is added with 0.1 to 0.5 M sodium chloride.
The method according to claim 1, wherein the salt of step (d) is sodium chloride.
The method according to claim 1, wherein the number of gold nanoparticles in the detection probe in step (e) is analyzed by the following equation.
[Mathematical Expression]

here,
N = number of gold nanoparticles measured per unit volume (number of gold nanoparticles in the detection probe)
Abs ₄₅₀ = Absorbance at ₄₅₀ nm
R ' = vibration amount of free electrons.

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