KR20140094043A - Oligo device for rapidly detecting nucleic acid amplification and detection method using thereof - Google Patents
Oligo device for rapidly detecting nucleic acid amplification and detection method using thereof Download PDFInfo
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- KR20140094043A KR20140094043A KR1020130003758A KR20130003758A KR20140094043A KR 20140094043 A KR20140094043 A KR 20140094043A KR 1020130003758 A KR1020130003758 A KR 1020130003758A KR 20130003758 A KR20130003758 A KR 20130003758A KR 20140094043 A KR20140094043 A KR 20140094043A
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- acid amplification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
Abstract
The present invention can obtain a nucleic acid amplification product comprising a fluorescent label and biotin labeled with a pair of primers used in a nucleic acid amplification process using a DNA polymerase, wherein the nucleic acid amplification product includes both fluorescent labels and biotin labels. In addition, the migration of the nucleic acid amplification product by the chromatography method and the nucleic acid amplification product by biotin-avidin or biotin-streptavidin binding can be separated. When the present invention is used, fluorescence label contained in the separated nucleic acid amplification product can be measured to quickly and easily confirm the amplification of the nucleic acid, and the nucleic acid amplification product can be easily separated.
Description
More particularly, the present invention relates to a method for rapidly and accurately detecting the presence or absence of a nucleic acid through mixing of a nucleic acid-specific base sequence and a non-specific base sequence, and an apparatus using the same.
In general, nucleic acid amplification technology is very important technology for molecular genetic analysis, and it is very important technology for analysis of human genome research, detection of infectious diseases and genetic diseases. The PCR (Polymerase Chain Reaction) method, which is a typical technique of this nucleic acid amplification technology, is the most widely known technology, and is a technique capable of selectively amplifying a specific nucleic acid sequence in a large amount. To analyze nucleic acid amplification products amplified by PCR or the like, hybridization techniques such as electrophoresis, Sourthern Blot or Northern Blot are used. However, the above-described analysis method requires a separate apparatus. Since the analysis method is complex, it takes a long time to analyze and there is a concern that the analytical sample may be denatured during a process of complicated process. Therefore, there is a problem that the sensitivity is deteriorated and the result is fluctuated according to the skill level of the inspector. In particular, in the case of the hybridization reaction, the analysis results may be very different depending on the influence of the external conditions such as the composition of the oligos used in the hybridization and the composition of the reaction composition. In order to obtain the optimal analysis result, And the like.
Therefore, the present inventors have completed the present invention in order to improve the efficiency of the analysis result and to develop a method for easily separating the analyzed material.
An object of the present invention is to provide a primer set capable of rapidly analyzing and separating nucleic acid amplification products.
It is still another object of the present invention to provide a method for rapidly analyzing and separating nucleic acid amplification products using the primer set.
One aspect of the present invention is to provide a primer set for amplifying a target DNA comprising a fluorescent substance and biotin.
In the primer set for amplifying the target DNA, the primer set may include an oligonucleotide including a fluorescent substance and an oligonucleotide including biotin.
In the present invention, the term "primer" is an oligonucleotide, which is a necessary single short strand DNA for performing nucleic acid amplification.
The fluorescent material may be contained inside the oligonucleotide. Preferably at the 5 ' end of the oligonucleotide. In addition, the fluorescent substance can be directly bound to an oligonucleotide. The fluorescent material may include a linker and may bind to an oligonucleotide through a linker.
In the present invention, the term "fluorescent-labeled primer or oligonucleotide conjugated with a fluorescent substance" refers to a fluorescence substance capable of emitting fluorescence energy at the 5 'end of a single stranded base fragment composed of oligonucleotide Means a structure in which a substance is connected. This is a structure for measuring the result of nucleic acid amplification by measuring the amount of fluorescence energy emitted by the fluorescent material.
In the present invention, the term "fluorescent substance-primer" refers to a fluorescent substance that is connected to the 5 'terminal of the primer, such as Hydroxycoumarin, Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer yellow, NBD, R-Phycoerythrin (Cyanine 3), Cy3B (Cyanine 3B), Cy3.5 (Cyanine 3.5), Cy5 (Cyanine 2),
Biotin may be contained within the oligonucleotide. Preferably at the 5 ' end of the oligonucleotide. In addition, the biotin can be directly bound to an oligonucleotide. Further, the biotin may include a linker and may bind to an oligonucleotide through a linker.
In the present invention, "biotin-labled primer" refers to a structure in which biotin is linked to the 5 'end of a single stranded base fragment composed of an oligonucleotide, preferably a single strand base segment. This is a structure for selectively capturing only biotin-labeled nucleic acid in the resultant product upon specific amplification with avidin (or streptavidin) fixed on the nitrocellulose membrane.
The oligonucleotides including the fluorescent substance and biotin can be used in pairs. Usually, a forward primer and a reverse primer are used as a primer.
One embodiment can bind the fluorescent material to the forward primer and bind the biotin to the reverse primer. At this time, the fluorescent substance can be bound to the 5 'end of the forward primer. In addition, the fluorescent material can be bound to both double strands of the forward primer. In addition, biotin can be bound to the 5 'end of the reverse primer. In addition, biotin can be bound to both double strands of the reverse primer.
In another embodiment, the fluorescent material and biotin are coupled to the forward primer and the biotin and the fluorescent material are coupled to the reverse primer. At this time, the position where the fluorescent substance and biotin are bound to the primer can be appropriately designed so that the amplified product can simultaneously contain the fluorescent substance and the biotin.
Yet another aspect of the present invention provides a primer set for amplifying a target DNA comprising a fluorescent substance and biotin, and a kit for detecting nucleic acid coupled with avidin or streptavidin.
Avidin or streptavidin can be bound directly to a nucleic acid detection device. In addition, the device may comprise a membrane bound to avidin or streptavidin. In addition, the membrane is characterized in that nitrocellulose or an equivalent medium capable of performing a chromatography method is used. In addition, the avidin or streptavidin is located in a test line on the nucleic acid detection device.
The kit may also be provided in the form of a kit comprising a packaging unit with one or more reagents. The kit may also include one or more of the following items: buffer, instructions for use, and positive or negative controls. The kits may comprise containers of reagents mixed in suitable proportions to carry out the methods described herein. The reaction reagent vessels preferably contain a unit quantity of reaction reagent so as to omit the measuring step when performing the method. In another embodiment, the kit reagent further comprises reagents for extracting genomic DNA or RNA from the biological sample. In addition, the kit reagent may include reagents for application to reverse transcriptase-PCR analysis.
In another aspect of the present invention, a target DNA is amplified using a primer set for amplifying a target DNA, which comprises an oligonucleotide including a sample containing a target DNA and an oligonucleotide including a fluorescent substance and an oligonucleotide comprising biotin ; Introducing the amplification product into a device coupled with avidin or streptavidin; And detecting whether the amplification product is attached to the avidin or streptavidin-coupled membrane through the detection of the fluorescent substance.
The method will be described in detail as follows.
First, amplifying the target DNA using the sample containing the target DNA and the primer set of
In the present invention, the term "template" means a substance having a target gene part to be amplified in a PCR reaction, and is usually double-stranded DNA. This should be added to the nucleic acid amplification reaction preparation step.
A sample containing the target DNA may contain a plurality of DNA strands. The sample can be obtained by a method which can be obtained by those skilled in the art besides tissues, cells, blood or body fluids. In addition, the target DNA can be obtained from mRNA.
At this time, a primer set for amplifying a target DNA comprising an oligonucleotide including a fluorescent substance and an oligonucleotide including biotin is mixed and PCR is performed.
Amplification of the nucleic acid using a pair of primers labeled with a fluorescent label and biotin yields a nucleic acid amplification product in which biotin is labeled at one end and fluorescence is labeled at the other end.
In addition, the detection method comprises the step of putting the amplification product into a device to which avidin or streptavidin is bound. Avidin or streptavidin can be bound directly to a nucleic acid detection device. In addition, the device may comprise a membrane bound to avidin or streptavidin. In addition, the membrane is characterized in that nitrocellulose or an equivalent medium capable of performing a chromatography method is used. In addition, the avidin or streptavidin is located in a test line on the nucleic acid detection device.
Further, the detection method includes the step of detecting whether the amplified product is attached to the avidin or streptavidin-coupled membrane through the detection of the fluorescent substance. By applying the result of nucleic acid amplification to a nitrocellulose membrane, the nucleic acid amplification product is moved on the nitrocellulose membrane by the chromatographic principle. At this time, biotin labeled at one end of the amplified nucleic acid product interacts with labeled avidin (or streptavidin) on the nitrocellulose membrane, and the amplified nucleic acid product is no longer able to migrate the nitrocellulose membrane Eventually, only the amplified nucleic acid is located at the site of avidin (or streptavidin) binding. Therefore, the amplification product can be detected by measuring whether the nucleic acid amplification product binds to avidin.
This reaction is completed within 1 to 15 minutes, 5 to 10 minutes, preferably 6 to 8 minutes, and the finished product is analyzed using a scanner or a detector capable of analyzing the fluorescent label of the nucleic acid amplification product, It is a method to confirm whether nucleic acid amplification is possible.
In another aspect of the present invention, a target DNA is amplified using a primer set for amplifying a target DNA, which comprises an oligonucleotide including a sample containing a target DNA and an oligonucleotide including a fluorescent substance and an oligonucleotide comprising biotin ; Introducing the amplification product into a device coupled with avidin or streptavidin; And isolating the amplification product from avidin or streptavidin.
A method for isolating the amplification product comprises first amplifying a target DNA using a primer set for amplifying a target DNA, which comprises an oligonucleotide including a fluorescent substance and an oligonucleotide including biotin. The amplification product is then introduced into a device coupled with avidin or streptavidin. Since the fluorescent substance and avidin are bound to the amplification product at the same time, it is possible to determine whether the amplification product is bound to avidin or streptavidin. In addition, the amplification product can be separated by performing a washing step and then separating biotin from avidin or streptavidin.
Another aspect of the present invention is to provide a probe for detecting a target DNA comprising a fluorescent substance and biotin.
As used herein, the term "probe" refers to an oligonucleotide that specifically binds to a target sequence and includes, for example, a sequence-specific Specific < / RTI > portion designed to hybridize in a manner that is well known in the art. In one embodiment, the oligonucleotide probe is in the range of 15 to 60 nucleotides. Preferably, the oligonucleotide probe is in the range of 18 to 30 nucleotides.
The fluorescent substance and biotin are simultaneously bound in the probe. In addition, the fluorescent substance and the biotin can bind through the linker.
The present invention provides a method for analyzing nucleic acid amplification more easily by providing a method and apparatus for rapidly and easily analyzing nucleic acid amplification and analyzing the resultant nucleic acid. Specifically, a specific label is attached to each end of the primer used in the nucleic acid amplification process, and the nucleic acid amplification product is rapidly separated using this labeling and chromatography method, and the nucleic acid amplification product separated by fluorescence labeling is rapidly and accurately Can be detected. Since the amplification can be easily confirmed through such a quick and accurate detection method, it can be very usefully used industrially.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing the structures of fluorescent and biotin-labeled primers presented in the present invention. FIG. FIG. 1 (A) is a schematic diagram showing the structure of a biotin-labeled oligopeptide, and FIG. 1 (B) is a schematic diagram showing the structure of an oligopeptide labeled with fluorescence (
FIG. 2 (A) is a schematic diagram showing a structure of binding to template DNA when nucleic acid is amplified using the labeled primers shown in FIG. 1, and FIG. 2 (B) is a schematic diagram showing a nucleic acid amplification product generated through nucleic acid amplification It is a schematic diagram.
Fig. 3 (A) is a schematic diagram showing the structure of a chromatographic strip for nucleic acid analysis. A test line zone to which avidin (or streptavidin) is bound, and an absorbent pad portion that absorbs the remaining amount of the nucleic acid amplification reaction solution. FIG. 3B is a schematic diagram illustrating the reaction of the nucleic acid amplification product in the chromatographic strip. In the nucleic acid amplification product introduced into the sample pad, only the biotin-bound nucleic acid migrates on the nitrocellulose membrane while the avidin (or streptavidin ), And the rest are all absorbed by the absorbing pad through the membrane.
4 is a schematic view showing the principle and structure of an apparatus for analyzing nucleic acid amplification products separated using a nucleic acid analysis strip according to the present invention. When energy is received from the light source, fluorescence of a specific wavelength is emitted from the fluorescence Dye labeled at one end of the nucleic acid amplification product. At this time, the fluorescence light emitted is detected by a silicon diode detector through a fluorescence filter.
FIG. 5 is a graph showing the results of genomic amplification of rice genomic DNA using fluorescent and biotin-labeled primers shown in the present invention, followed by amplification by nucleic acid amplification using electrophoresis.
FIG. 6 is a graph showing the result of analyzing nucleic acid amplification products amplified using fluorescent and biotin-labeled primers according to the present invention to a nucleic acid analysis strip and then analyzing them with a fluorescent scanner. FIG. In the figure, Control is the test result of the nucleic acid amplification product of the non-amplified negative nucleic acid with the nucleic acid assay strip, and Test is the result of analyzing the positive nucleic acid amplification result of the nucleic acid amplification into the nucleic acid analysis strip.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are merely examples of the present invention, and the scope of the present invention is not limited to these examples.
Example 1: Fabrication of fluorescent labels and biotin-labeled primers
For the implementation of the present invention, experiments were conducted using Mycobacterium tuberculosis. Primers for testing mycobacteria were prepared with reference to the following articles. (Mukesh Jain et al., Validation of housekeeping genes as internal control for gene expression in rice by quantitative real-time PCR. Biochemical and Biophysical Research Communications 345 (2006) 646-651
The fluorescent label and biotin-labeled primer used in the present invention were custom-made and used in Macrogen (Korea). The fluorescent-labeled primer was labeled with Cy5 at the 5 'end of SEQ ID NO: 1, and the biotin-labeled primer labeled with biotin at the 5' end of SEQ ID NO: 2 (Table 1).
(Sense)
(Anti-sense)
Example 2: Nucleic acid amplification using a labeled primer
The template DNA used in the present invention was extracted from rice using genomic cell / Tissue Mini Kit of Nucleosin Co., Ltd. (genus).
PCR was carried out under the conditions shown in Table 2 using the primers prepared in Example 1 with the genomic DNA extracted as a template. At this time, distilled water was added instead of the genomic DNA, which was used as a negative control.
The result of PCR was confirmed to be amplified by electrophoresis using 1% agarose gel (FIG. 5)
Example 3: Preparation of sample pad
The sample pad is a place where the nucleic acid amplified product is put, and preprocessing is performed so that the nucleic acid product can be absorbed well. The sample pads were thoroughly wetted with the pretreatment solution (100 mM Borax, 0.2% TX-100, 0.1% NaN3, pH 9.0) using a standard 14 product from Whatman Inc. (USA), completely dried and stored in a dehumidifying container Respectively.
Example 4: Avidin fixation on nitrocellulose membrane
The nitrocellulose membrane used was Immunopore FP product of Whatman (USA), and Avidin was purchased from Sigma-Aldrich (USA). The prepared Avidin was diluted to a concentration of 1 mg / ml using a dilution buffer (10 mM Tris-HCl, pH 7.6), and then 1 μl was added to nitrocellulose using a micropipette. The dispensed nitrocellulose membrane was dried in a 37 ° C dryer for 1 hour and stored in a dehumidifier until use.
Example 5: Preparation of chromatographic strips for nucleic acid detection
A product of Laminate Type L-P25 from Advanced Microdevices (India) was used as a plastic support for the production of chromatographic strips for nucleic acid detection. The sample pads and nitrocellulose membranes prepared in Examples 3 and 4, and an adsorption pad (Whatman Co., USA, CF5) were adhered to the respective positions of a plastic support to produce strips (see FIG. 3). The cut pieces were cut at intervals of 4 mm using a cutter, and each cut strip was assembled in a plastic housing.
Example 6: Development test of chromatographic strip of PCR product
20 μl of the PCR amplification product prepared in Example 2 was added to 20 μl of the sample pad of the chromatographic strip for nucleic acid detection prepared in Example 5, and then 100 μl of sample developing buffer (10
Example 7: Nucleic acid amplification product detection test
The strips completed in Example 6 were scanned using a fluorescence detection device (WizScan, Wise Bio Solution, Korea). As a result, it was confirmed that the fluorescent signal was detected in the scanner when the amplified gene was applied using PCR, and no fluorescence signal was observed in the negative control containing only distilled water.
The results of the present invention are summarized as follows. It is confirmed that the principle of nucleic acid analysis proposed in the present invention is very simple and can confirm the amplification of nucleic acid rapidly. Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
Particularly, when the present invention is used in nucleic acid amplification experiments, preparation and analysis processes such as electrophoresis, which is a conventional nucleic acid analysis method, are long, and it is unnecessary to be exposed to harmful environments such as ethidium bromide and ultraviolet (UV) do. In addition, the present invention can be applied to various fields such as detection and identification of pathogens, detection and identification of genetic diseases, and the like because the analysis procedure is simple and rapid. Therefore, when the nucleic acid amplification and analysis method is replaced, It is useful for experimental methods.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
The present invention provides a method for analyzing nucleic acid amplification more easily by providing a method and apparatus for rapidly and easily analyzing nucleic acid amplification and analyzing the resultant nucleic acid. This rapid and accurate detection method can easily confirm whether or not the amplification can be performed, and thus it can be very usefully used in modern biology processing a large amount of genetic information.
<110> Wizbiosolution <120> Device for rapid nucleic acid amplification and detection method using <130> P20121224 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tggtggaggc tcgaagcgat actg 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 atgcctctaa tcattggctt tacc 24
Claims (9)
Introducing the amplification product into a device coupled with avidin or streptavidin; And
And detecting whether the amplification product is attached to the avidin or streptavidin-coupled membrane through detection of the fluorescent substance.
Introducing the amplification product into a device coupled with avidin or streptavidin;
Confirming whether the amplified product is attached to the avidin or streptavidin coupled membrane through detection of the fluorescent substance; And
And isolating the amplified product from avidin or streptavidin.
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| KR1020130003758A KR20140094043A (en) | 2013-01-14 | 2013-01-14 | Oligo device for rapidly detecting nucleic acid amplification and detection method using thereof |
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| KR1020130003758A KR20140094043A (en) | 2013-01-14 | 2013-01-14 | Oligo device for rapidly detecting nucleic acid amplification and detection method using thereof |
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