KR20110060156A - Composition for extracting nucleic acid, nucleic acid extracting method and nucleic acid amplifying method using the same - Google Patents
Composition for extracting nucleic acid, nucleic acid extracting method and nucleic acid amplifying method using the same Download PDFInfo
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Abstract
Description
The present invention relates to a nucleic acid extracting composition and a nucleic acid extracting method using the same, and more particularly, it is possible to quickly diagnose the genetic modification and low diagnostic cost, nucleic acid extraction that can omit the culture and centrifugation process of the sample The present invention relates to a composition for extracting nucleic acids and a method for extracting the same.
Traditionally, ethanol precipitation methods (Lis and Schleif, Nucleic Acids Res., 2: 383-389 (1975)) have been used after using three buffers represented by dilution, disruption and neutralization of human blood-derived cells. However, because the method is inconvenient to handle and low in purity, US Pat. No. 5,707,812 has developed a nucleic acid extraction and purification kit comprising a column. (Horn et al., Human Gene Ther., 6: 565-573 (1995). Chandra et al., Anal.Biochem., 203: 169-172 (1992); Marquet et al., BioPharm.:26-37 (1995) et al.)
The principle of nucleic acid extraction using such columns is the purification of nucleic acid molecules from proteins and other intracellular materials using the principle of structural interaction between water molecules and nucleic acids using glass fibers or silica membranes that specifically bind to nucleic acids. For this purpose, a centrifugation process was necessary.
Therefore, conventional DNA extraction kits currently used include (1) injecting blood into binding buffer, (2) first centrifugation, (3) combining centrifuged samples, and (4) second centrifugation. DNA extraction through a separation step, (5) washing the centrifuged sample, (6) a third centrifugation step, (7) eluting DNA from the sample, and (8) a fourth centrifugation step It was.
The above-mentioned method has been spotlighted as a high purity DNA extraction method, but it requires a lot of time and cost because it has to go through a culturing step and a centrifugation step, and there is a problem that a relatively large amount of blood is required for DNA extraction.
In addition, conventionally, organic solvents such as phenol and chloroform have been used to extract nucleic acids from prokaryotic and eukaryotic or complex biological samples. Nucleic acid extraction using the enzyme was digested enzymatically with proteolytic enzymes, cells were lysed with ionic surfactant, and nucleic acid was extracted with phenol or phenol / chloroform mixture. According to the extraction, the organic solvent phase and the water phase were separated, and the extracted nucleic acid could be separated by precipitating the nucleic acid distributed toward the water with alcohol. However, phenol or phenol / chloroform mixture has a dangerous effect on life or the environment and is considered a dangerous waste, so there is a problem that must be handled with care.
Accordingly, there is a need for development of a composition and method for rapidly extracting target nucleic acids from cell walls, amplifying the target nucleic acids quickly and accurately, and detecting amplification products at the same time as the target nucleic acids.
Accordingly, a first object of the present invention is to provide a nucleic acid extracting composition for extracting nucleic acids from cell walls simply and quickly.
In addition, a second object of the present invention is to provide a nucleic acid extraction method for extracting nucleic acids from cell walls simply and quickly by using the nucleic acid extracting composition.
In addition, a third object of the present invention is to provide a nucleic acid amplification method that can shorten the amplification time of the nucleic acid, the cost required to identify the amplification product through the amplification reaction.
In order to achieve the first object of the present invention described above, the present invention provides a nucleic acid extract composition comprising 50 to 60% by volume PEG, 38 to 49% by volume KOH, and 0.5 to 2% by volume surfactant.
And in order to achieve the second object of the present invention, the present invention comprises a first step of adding a nucleic acid extraction composition to a sample; And a second step of extracting the nucleic acid of the sample by reacting the sample solution in which the sample and the nucleic acid extracting composition are mixed at room temperature to 100 ° C, and decomposing the cell wall of the sample. Provide a method.
In addition, in order to achieve the third object of the present invention, the present invention comprises a first step of adding a nucleic acid extraction composition to a sample; A second step of reacting the sample solution in which the sample and the nucleic acid extracting composition are mixed at room temperature to 100 ° C., decomposing the cell wall of the sample to extract the nucleic acid of the sample; And it provides a simple nucleic acid amplification method comprising a third step of performing a nucleic acid amplification reaction by adding a nucleic acid amplification composition to the sample solution passed through the second step.
If the composition according to the present invention is used, unlike the conventional organic solvent extraction method, since it does not use phenol, it is not harmful to the human body and has an effect of minimizing problems such as environmental pollution.
The nucleic acid extraction method of the present invention has excellent field applicability compared to the conventional column type nucleic acid extraction method, and the extraction cost and extraction time are remarkable since the initial culture of cells and the centrifugation process for obtaining the supernatant can be omitted. To reduce the effect.
In addition, the nucleic acid extraction method according to the present invention not only simplifies the procedure, but also reduces the exposure time to nuclease, thereby contributing to stabilization of the nucleic acid, thereby increasing the yield of nucleic acid having a more complete nucleic acid sequence. In addition, it can be seen that the nucleic acid extracting method according to the present invention can obtain nucleic acid having high purity without contamination by protein, RNA, and the like, without causing mutation.
In addition, the nucleic acid extraction method according to the present invention has the effect of obtaining a nucleic acid economically and simply by using a small amount of a liquid sample or a solid sample such as blood, saliva, which is added.
In addition, the nucleic acid amplification method according to the present invention reduces the cost of identifying the amplification product through the amplification reaction, and is simple and light in use, and can be examined in a clinical diagnostic laboratory and any other place regardless of the location, and the target The presence of DNA can be determined simply and quickly.
Hereinafter, a nucleic acid extracting composition, a nucleic acid extracting method using the same, and a nucleic acid amplifying method according to preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
The nucleic acid extracting composition according to an embodiment of the present invention is used to rapidly extract nucleic acids from a cell wall, and is preferably formed of an alkaline solution having a pH of 12.5 to 13.5 to smoothly decompose the cell wall. PEG) and potassium hydroxide (KOH) and surfactants.
The nucleic acid extracting composition may reduce the time required to extract the nucleic acid, and the time required to extract the nucleic acid, and cell lysis and nucleic acid extraction is possible at the same time.
First, the nucleic acid extracting composition according to an embodiment of the present invention includes PEG.
As the PEG, any PEG may be used, but PEG 200 is preferably used. In general, the nucleic acid extracting composition formed of the alkaline solution performs the task of extracting the nucleic acid by destroying the cells, but may be an inhibitor upon entering the reagent for amplifying the nucleic acid. However, in the nucleic acid extracting composition according to the present invention, since PEG is added to the nucleic acid amplification reagent, which does not cause an inhibitory reaction, it does not become an inhibitor of the nucleic acid amplification reagent.
PEG contained in the nucleic acid extracting composition according to the present invention contains 50 to 60% by volume based on the total 100% by volume. At this time, if the content of PEG contained in the nucleic acid extraction composition is less than 50% by volume, nucleic acid extraction is not smooth, if the content of PEG exceeds 60% by volume may not be amplified gene.
And the nucleic acid extracting composition according to an embodiment of the present invention comprises KOH.
The KOH allows the entire solution to be alkaline so that cell walls can be smoothly removed. At this time, it is preferable to use the final concentration of 100 to 200mM.
KOH contained in the nucleic acid extracting composition according to the present invention contains 38 to 49% by volume based on the total 100% by volume. At this time, if the content of KOH contained in the nucleic acid extracting composition is less than 38% by volume, the pH is lowered. If the content of KOH is more than 49% by volume, the pH is increased, resulting in an inhibitor in the nucleic acid amplification reagent.
Subsequently, the nucleic acid extracting composition according to one embodiment of the present invention includes a surfactant.
The surfactant is added to assist the decomposition of the cell wall, fatty acid ester of d-sorbitol, such as Span 20, Span 40, Span 60, Span 65, Span 80, Span 85, etc. , Tween 21, Tween 40, Tween 60, Tween 65, Tween 80, Tween 81, Tween 85 and the like (Nakaray Tesque company name), polyoxytetylene glycol sorbitan alkyl ester, TritonX-100, etc. Polyoxyethylene glycol pt-octylphenyl ether, 2-mercaptoethanol, sarcosyl sulfate, sodium dodecyl sulfate (SDS), and the like. These may be used independently and may be used in combination of 2 or more types. In particular, it is preferable to use TritonX-100 and Tween 20, and it is more preferable to use TritonX-100.
Surfactant included in the nucleic acid extracting composition according to the present invention contains 0.5 to 2% by volume based on the total 100% by volume. In this case, when the content of the surfactant contained in the nucleic acid extracting composition is less than 0.5% by volume, the cell wall may not be degraded smoothly, which may cause a problem of decreasing the amount of nucleic acid extracted. In addition, when the content of the nucleic acid extracting composition is more than 2% by volume may inhibit the nucleic acid amplification reaction.
In addition, the present invention provides a nucleic acid extraction method using a nucleic acid extraction composition.
The nucleic acid extraction method according to an embodiment of the present invention includes a first step of adding a nucleic acid extracting composition to a sample, and a second step of reacting a mixture of the sample and the nucleic acid extracting composition.
The first step is a step of adding a nucleic acid extracting composition to a sample, 100 to 300 μl of the nucleic acid extracting composition is added to 1 to 10 μl (mg) of the sample to be extracted. In this case, the nucleic acid extraction composition may include polyethylene glycol (PEG), potassium hydroxide (KOH) having a concentration of 100 to 200mM, and a surfactant. Here, in the case of using a liquid sample such as blood or saliva as the sample, about 100 μl of the nucleic acid extracting composition is preferably used. In the case of using a solid sample such as a hard tissue or a plant as the sample, the nucleic acid is extracted. It is preferable that 200-300 microliters are used for the composition.
The target nucleic acid to be extracted by the nucleic acid extraction method may be any of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Since the cells contain these nucleic acids (DNA and RNA), the target nucleic acid therein can be extracted by the present nucleic acid extraction method. At this time, the kind of cells is not limited, and all cells such as animal cells, plant cells, microbial cells and the like can be targeted. As a sample to be used for the nucleic acid extracting method, a biologically-derived sample containing cells is suitable, but the present invention is not limited thereto, and the nucleic acid extracting method can be applied to a sample not derived from a living body.
As a sample which does not originate in a living body, the foodstuff containing a cell, soil, water, fiber, dust, etc. are mentioned, for example. As the bioderived sample, biological components of animals and plants can be appropriately used. Examples of animal-derived samples including humans include, for example, blood, tissue fluid, lymph fluid, cerebrospinal fluid, pus, mucus, runny nose, sputum, urine, feces, and fluids such as ascites, skin, lungs, liver, mucus, various And a cleaning liquid after washing tissues such as organs and bones, and nasal cavity, bronchus, skin, and various organs. In humans, dialysis drainage may also be used as a sample.
In addition, the nucleic acid targeted by the extraction method of this nucleic acid is not limited to the nucleic acid native to the cell contained in a sample, but can be applied also to nucleic acid, such as a nucleic acid of the virus infected by the cell, and the microorganisms in which the cell was taken. Therefore, biologically-derived samples containing phagocytic cells (white blood cells, etc.) of infectious patients are also suitable as samples of the hexin extraction method.
On the other hand, the second step is a step of reacting the sample solution mixed with the sample and the nucleic acid extracting composition at room temperature to 100 ℃ for a predetermined time, by extracting the nucleic acid of the sample by decomposing the cell wall of the sample through the reaction. . At this time, the predetermined time is preferably 10 to 15 minutes. In addition, the liquid sample is preferably reacted at room temperature, and the solid sample is preferably reacted with boiling water in boiling water (about 100 ° C.).
The second step will be described in more detail according to the sample.
As a first embodiment, if a liquid sample such as blood or saliva is used as the sample, it is preferable to add about 100 μl of the nucleic acid extracting composition to 1 to 10 μl of the liquid sample, and react for 10 to 15 minutes at room temperature. It is preferable.
As a second embodiment, when a solid sample such as tissue or plant is used as the sample, it is preferable to add about 100 μl of the nucleic acid extracting composition to a solid sample of 1 to 10 mg, and react for 10 to 15 minutes at room temperature. It is preferable. However, in the case of using a solid tissue or fabric as a solid sample, it is preferable to add 200 to 300 μl of the nucleic acid extracting composition to the 1 to 10 mg solid sample, and 10 to the boiling water (about 100 ° C.) in a bath. It is preferred to react for 15 minutes.
As a third embodiment, when sputum is used as the sample, it is preferable to add about 100 to 200 μl of the nucleic acid extracting composition to 10 to 50 μl of sputum, and 10 to 15 in a boiling water in boiling water (about 100 ° C.). It is preferred to react for minutes.
Conventional nucleic acid extraction methods are divided into the steps of lysing cells using a variety of solutions, separating the proteins after lysis, and in the step of protein separation, reacting the enzyme to show the enzyme activity for a certain time by treating the reaction enzyme. This is time consuming and requires several steps, which increases the possibility of contamination of impurities and has difficulty in obtaining reproducible results when performing repeated experiments. However, the nucleic acid extracting method using the nucleic acid extracting composition according to the present invention reduces the step of extracting nucleic acids by simultaneously allowing cell lysis, protein separation, and nucleic acid extraction with one composition, and at the same time reduces the time required to extract nucleic acids. Can be reduced. In addition, by making it possible to divide the two stages in both stages, it is possible to obtain reproducible results by reducing mistakes and errors that can be made by the operator.
In addition, the nucleic acid extracting composition of the present invention can extract DNA even with a very small amount of cells, and the extraction efficiency is constant even if the number of cells is increased.
Furthermore, since the DNA extracted using the nucleic acid extracting composition of the present invention is precisely cleaved by restriction enzymes and is not contaminated by proteins, RNA and other enzymes, the DNA may be used in other experiments involving enzymatic reactions. No problem, no problem even if you perform PCR reaction
In addition, the present invention provides a nucleic acid amplification composition used for nucleic acid amplification reaction.
The nucleic acid amplification composition according to the present invention is used to perform amplification of a target nucleic acid without purifying a nucleic acid in a sample, and a gene amplification reaction buffer (10X reaction buffer), 8 to 12 mM deoxyribonucleotide (dNTP), 80 to Magnesium chloride (MgCl 2), polymerase, and purified water of 120mM, and may further include a stabilizer. If such a nucleic acid amplification composition is used, no loss of extraction and purification occurs even when only a small amount of the target nucleic acid is present in the sample. Therefore, it is possible to detect a small amount of target nucleic acid without causing a decrease in detection sensitivity. As a result, the work can be simplified, and the work time can be shortened.
Nucleic acid amplification composition according to an embodiment of the present invention comprises a gene amplification reaction buffer.
As the gene amplification reaction buffer, it is preferable to use a buffer containing Tris-HCl and betaine, bovine serum albumin (BSA), and Tween 20.
In one embodiment, the gene amplification buffer according to the present invention consists of 600-650 mM Tris-HCl, 150-200 mM ammonium sulfate, 0.5-1 M betaine, 10-20 mM bovine serum albumin, 20 mM BSA, Tween 20.
In another embodiment, the gene amplification buffer according to the invention consists of 80-120 mM Tris-HCl, 350-450 mM potassium chloride, 0.5-1 M betaine, 10-20 mM bovine serum albumin, Tween 20.
Tween 20 used in each embodiment is preferably used 0.1 to 0.2% based on 100% by volume gene amplification buffer.
Gene amplification reaction buffer contained in the nucleic acid amplification composition according to the present invention contains 15 to 25% by volume based on the total 100% by volume. In this case, when the content of the gene amplification reaction buffer contained in the nucleic acid amplification composition is less than 15% by volume, nucleic acid amplification by the nucleic acid amplification reaction may not be performed smoothly. In addition, when the content of the gene amplification buffer exceeds 25% by volume, nucleic acid amplification by the nucleic acid amplification reaction may not be performed smoothly or a nonspecific reaction may occur.
Nucleic acid amplification composition according to an embodiment of the present invention includes dNTP.
The dNTP is added for GATC synthesis in the amplification reaction of the nucleic acid, The final concentration is preferably 8 to 12 mM, more preferably 10 mM.
DNTP included in the nucleic acid amplification composition according to the present invention contains 15 to 25% by volume based on the total 100% by volume. At this time, when the content of the restriction dNTP contained in the nucleic acid amplification composition is less than 15% by volume or the content of dNTP exceeds 25% by volume, nucleic acid amplification by a nucleic acid amplification reaction may not be performed smoothly.
Nucleic acid amplification composition according to an embodiment of the present invention comprises magnesium chloride.
The magnesium chloride is added to promote the activity of the nucleic acid amplification composition, the final concentration of magnesium chloride is preferably 80 to 120mM, more preferably 100mM.
Magnesium chloride contained in the nucleic acid amplification composition according to the present invention contains 3 to 5% by volume based on 100% by volume. At this time, when the content of magnesium chloride contained in the nucleic acid amplification composition is less than 3% by volume, the polymerase is not activated. In addition, when the content of magnesium chloride exceeds 5% by volume non-specific reaction may occur.
Nucleic acid amplification composition according to an embodiment of the present invention comprises a polymerase.
The polymerase is added for the gene amplification reaction, and any polymerase can be used as long as it can achieve this purpose, but it is preferable to use a polymerase using a monoclonal antibody. In particular, it is recommended to use Hot start Taq DNA Polymerase (2.5Unit / ml).
The polymerase included in the nucleic acid amplification composition according to the present invention contains 2 to 3% by volume based on 100% by volume. At this time, if the content of the polymerase contained in the nucleic acid amplification composition is less than 2% by volume, the gene amplification reaction does not proceed, and if the content of the polymerase exceeds 4% by volume, a nonspecific reaction may occur.
Nucleic acid amplification composition according to an embodiment of the present invention comprises distilled water.
The distilled water is added to adjust the final volume and concentration of the nucleic acid amplification composition, it is preferable to use the third distilled water. Distilled water included in the nucleic acid amplification composition according to the present invention contains 41 to 51% by volume based on the total 100% by volume.
Furthermore, the present invention provides a nucleic acid amplification method using the nucleic acid amplification composition.
Here, nucleic acid amplification means amplifying a nucleic acid in a sample by using a primer specific for a random arrangement of a target nucleic acid or a primer of a random arrangement and a polymerase (DNA polymerase or RNA polymerase).
As the amplification method, a conventionally known amplification method can be appropriately used. Specifically, PCR method, Nested-PCR method, RT-PCR method, ICAN method, UCAN method, LAMP method, primer extension method, transcription (translation), replication (replication), etc. can be used. It is preferable to use the LAMP method.
Hereinafter, the case where the PCR method is used for the nucleic acid amplification step of the nucleic acid amplification method will be described.
The PCR method is a method of amplifying a characteristic DNA region by in vitro repetition of a DNA synthesis reaction by two kinds of primers having a specific DNA region and a DNA polymerase (heat resistant DNA polymerase). It is a well-known technique generally used in the engineering field. PCR methods include Nested-PCR, RT (Real-Time) -PCR, and the like.
The Nested-PCR method is a method of performing two-step PCR using an outer primer and an inner primer, and using the first amplification product from a desired region as a template, both primers are inward from both primer positions. How to set up and run. By using Nested-PCR, even if the target nucleic acid is not sufficiently amplified to the detectable amount in the first PCR, it is possible to amplify it to the detectable amount by the second PCR. In addition, when nonspecific amplification products are generated in the first PCR, these nonspecific amplification products are less likely to have an arrangement similar to the primers in the second PCR. Therefore, in the second PCR, since the probability of amplifying only fragments having a target array is increased, the damage caused by the generation of nonspecific amplified products can be eliminated, and the target nucleic acid can be detected more accurately.
The RT-PCR method is a variation for applying PCR to mRNA, and is a PCR method including a step of performing reverse transcription reaction using reverse transcriptase as a previous step of PCR. When the RT-PCR method is used in the nucleic acid amplification method, it is possible to make the target nucleic acid mRNA. That is, the nucleic acid amplification method can be applied to the detection of gene expression.
Nucleic acid amplification method according to the present invention is a first step of adding a nucleic acid extracting composition to a sample, a second step of extracting a nucleic acid by reacting a sample solution mixed with a sample and a nucleic acid extracting composition, and the nucleic acid to the sample solution A third step of performing a nucleic acid amplification reaction by adding a composition for amplification.
The first step is a step of adding a nucleic acid extracting composition to a sample, 100 to 300 μl of the nucleic acid extracting composition is added to 1 to 10 μl (mg) of the sample to be extracted. At this time, the nucleic acid extraction composition may include polyethylene glycol (PEG), 100 to 200mM potassium hydroxide, and a surfactant. Here, in the case of using a liquid sample such as blood or saliva as the sample, about 100 μl of the nucleic acid extracting composition is preferably used. In the case of using a solid sample such as a hard tissue or a plant as the sample, the nucleic acid is extracted. It is preferable that 200-300 microliters are used for the composition.
The second step is a step of extracting the target nucleic acid of the sample by reacting the sample solution mixed with the sample and the nucleic acid extracting composition for a predetermined time at room temperature to 100 ℃, decomposing the cell wall of the sample. At this time, the predetermined time is preferably 10 to 15 minutes. In addition, the liquid sample is preferably reacted at room temperature, and the solid sample is preferably reacted with boiling water in boiling water (about 100 ° C.).
The third step is a step of performing a nucleic acid amplification reaction by adding a nucleic acid amplification composition to the sample solution passed through the second step, the nucleic acid amplification composition is preferably added 2 to 5μl. If necessary, a stabilizer may be further added to the sample solution to stabilize the nucleic acid amplification composition. It is preferable to use a stabilizer commonly used in the art as such a stabilizer, but it is more preferable to use trihalose, Methyl-α-D-Gluco-pyranoside. In addition, in the third step, a primer for amplifying a specific gene may be added before performing the nucleic acid amplification reaction.
On the other hand, after the third step may further comprise the step of detecting the presence of the target DNA in the sample solution by adding a reagent to the sample solution passed through the third step.
Here, the reagent is conventional in various PCR (polymerase chain reaction), LCR (ligase chain reaction), oligonucleotide ligation assay (oligonucleotide ligation assay), hybridization assay, various probe assay (probe assay) It may mean an analyte used as. Specifically, the reagent is a reagent for observing the presence of the target DNA in the biological sample solution is complete nucleic acid amplification reaction, a fluorescent reagent is usually used as a reagent for generating optically detectable light. When the fluorescent reagent is used, it is possible to observe whether the target DNA is present in the biological sample solution through fluorescence generated after mixing with the biological sample solution. Examples of such nucleic acid analysis reagents include Sybr Green I (Molecular probe, USA), which is embedded in double-stranded DNA. Here, Sybr Green I is a reagent that inserts double-stranded DNA amplified during nucleic acid amplification and shows fluorescence of a specific wavelength.
In addition, the detection of the target DNA may be performed through DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.
More specifically, gel electrophoresis may use agarose gel electrophoresis or acrylamide gel electrophoresis depending on the size of the amplification product. In addition, in the fluorescence measuring method, when amplification is performed by labeling Cy-5 or Cy-3 at the 5'-end of a primer, a target sequence is labeled with a detectable fluorescent labeling substance, and the labeled fluorescence is measured using a fluorimeter. can do. In addition, the radiometric method of labeling the amplification product by adding a radioisotope such as 32 P or 35 S to the amplification reaction liquid when amplification is performed, and then radioactive measuring apparatus, for example, a Geiger counter or liquid flash The radioactivity can be measured using a liquid scintillation counter.
The present invention will be described in more detail with reference to the following examples and comparative examples. However, an Example is for illustrating this invention and is not limited only to these.
Agarose gel manufacturing
First, 2 g of agarose [QA-Agarose ™, Q-bio gene, USA] was added to a Erlenmeyer flask (250 ml), and 0.5X TBE (tris boric acid EDTA) buffer solution [Tris-Base, Q-biogene, USA] 100 ml.
Then, the mixture of the Erlenmeyer flask is dissolved in the microwave for 2 minutes and then the mixture is placed in a gel container.
The mixture was then cured for 30 minutes to prepare a 2% agarose gel.
Identification of Amplified Products Using Electrophoresis Device
After filling 0.5X TBE buffer solution with electrophoretic device [Mupid-α, Advance, Japan], 4 μl of amplification product and 6X Loading dye (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol) [Bio Basic, CANADA 0.8 μl was mixed and loaded in 4.8 μl.
Then, electrophoresis was performed for 25 minutes at 100V.
Then, after taking off the gel stained for 10 minutes (ethidium bromide) EtBr [SIGMA ® , USA], found to wash did not bind EtBr and the DNA for 10 minutes with distilled water.
Then, the agarose gel was placed on a UV transilluminator [CTOC KOREA, Korea] to confirm the reaction, and finally confirmed by sequencing.
Preparation of the first gene amplification reaction buffer
A buffer comprising 600 mM Tris-HCl, 200 mM (NH 2 ) 4 SO 4 , 1M Betaine, 20 mM BSA, 0.2% Tween 20 was prepared.
Preparation of the Second Gene Amplification Buffer
Buffer containing 100mM Tris-HCl, 400mM KCl, 20mM BSA, 0.2% Tween 20, 1M Betaine was prepared.
Example 1 Preparation of Nucleic Acid Extraction Composition
Into the Erlenmeyer flask (250 mL), 55 mL of PEG 200 [SIGMA ® , USA] and 44 mL of 150 mM KOH [Duchefa Bichemical, Netherlands], and 1 mL of Tween 20 [Nakarai Tesque, Japan] were injected. Mixing to prepare a composition for nucleic acid extraction.
Example 2 Preparation of Nucleic Acid Amplification Composition
10 mL of the first gene amplification buffer, 10 mM dNTP [SIGMA ® , USA] 10 ml, 100 mM MgCl 2 [SIGMA ® , USA] 0.4 ml, polymerase [HS Prime Taq DNA Polymerase, Gennet Bio, Korea] 2 ml, tertiary distilled water 27.6 ml is added to prepare a nucleic acid amplification composition.
Example 3 Preparation of Nucleic Acid Amplification Composition
10 ml of the second gene amplification buffer, 10 mM dNTP [SIGMA ® , USA] 10 ml, 100 mM MgCl 2 [SIGMA ® , USA] 0.4 ml, polymerase [HS Prime Taq DNA Polymerase, Genenet Bio, Korea] 2 ml, tertiary distilled water 27.6 ml is added to prepare a nucleic acid amplification composition.
Comparative Example 1 Preparation of Nucleic Acid Amplification Composition
A nucleic acid amplification composition was prepared under the same experimental conditions as in Example 2, except that 2 ml of the polymerase [Prime Taq DNA Polymerase, Genet Bio, Korea] was used instead of 2 ml of the polymerase [HS Prime Taq DNA Polymerase, Genet Bio, Korea]. The nucleic acid amplification composition is prepared.
Experimental Example 1
Extraction of Nucleic Acids in Blood
First, 10 µl of whole blood and 100 µl of a nucleic acid extraction composition were injected into a Erlenmeyer flask (250 ml).
Subsequently, the whole blood and nucleic acid extracting compositions were reacted at room temperature for 5 minutes, followed by nucleic acid amplification using a primer for amplifying
Then, the amplified product was observed using a 2% agarose gel and electrophoresis device.
Comparative Example 1
Extraction of Nucleic Acids in Blood
The nucleic acid was extracted under the same experimental conditions as in [Experimental Example 1], but the nucleic acid amplification reaction was performed using the nucleic acid amplification composition of [Comparative Example 1] instead of the nucleic acid amplification composition of [Example 2].
1 is an electrophoretic image of a nucleic acid amplification product according to a first embodiment of the present invention.
Referring to Figure 1, it was confirmed that using a hot start taq DNA polymerase when preparing a nucleic acid amplification composition is more effective.
At this time, M is a 100bp marker, 1, 2 and 3 are samples mixed in three primers, A represents Experimental Example 1, B represents Comparative Experimental Example 1.
Experimental Example 2
Nucleic Acid Extraction in Tissues
First, 10 mg of beef tissue and 100 μl of a nucleic acid extraction composition were injected into an Erlenmeyer flask (250 ml).
Next, the beef tissue and nucleic acid extract composition was reacted with boiling water at 100 ° C. for 10 minutes. 5 μl of solution was added to the nucleic acid amplification composition of [Example 2] Nucleic acid amplification reactions were performed.
Then, the amplified product was observed using a 2% agarose gel and electrophoresis device.
Figure 2 is an electrophoretic picture of the nucleic acid amplification product according to a second embodiment of the present invention.
Referring to FIG. 2, in the case of tissues, the time taken for the nucleic acid extraction process is longer than that in the blood. When the nucleic acid extracting composition and the nucleic acid amplifying composition according to the present invention are used, the time required for extracting and amplifying nucleic acids from the tissues is measured. It was confirmed that it can be shortened.
Where M is a 100bp marker, P is an amplified nucleic acid, 1 and 2 are beef tissues, and 3 and 4 are stored blood .
[Experimental Example 3]
GMO Extract in Soybeans
First, 10 mg of finely ground soybeans, 10 mg of wheat flour, and 100 μl of a nucleic acid extraction composition were injected into an Erlenmeyer flask (250 mL).
Next, the soybean flour, wheat flour and nucleic acid extracting composition were reacted in boiling water at 100 ° C. for 15 minutes, and 5 μl of solution was taken to identify the lectin gene, which is an intrinsic gene of soybean, and the Nos gene. A nucleic acid amplification reaction was performed using the primers to be amplified and the nucleic acid amplification composition of [Example 2].
Then, the amplified product was observed using a 2% agarose gel and electrophoresis device.
Comparative Example 2
Nucleic Acid Extraction Using Soybean
Amplifying the nucleic acid under the same experimental conditions as Experimental Example 3, but instead of the nucleic acid amplifying composition of [Example 2], the nucleic acid amplifying composition of [Comparative Example 1] Nucleic acid amplification reaction was performed.
Figure 3 is an electrophoretic picture of the nucleic acid amplification product according to a third embodiment of the present invention.
Referring to Figure 3, it was confirmed that it is more effective to use a hot start taq DNA polymerase as a polymerase when preparing a nucleic acid amplification composition.
In this case, M is a 100 bp marker, P is a GMO positive control group, N is a negative control group, 1 to 4 is unfloured powder, and 5 to 6 are soybeans. In addition, A represents Experimental Example 3, and B represents Comparative Experimental Example 2.
[Experimental Example 4]
Tuberculosis strain extraction included in sputum
First, the Mycobacterium tuberculosis bacteria grown in Ogawa media of the hospital were killed by NaOH treatment, and then diluted 10-fold using Tris-HCl buffer.
Subsequently, 10 µl of sputum of a normal person to which tuberculosis bacteria diluted 10-fold was added to a Erlenmeyer flask (250 ml) and 100 µl of a nucleic acid extraction composition were injected.
Next, the composition for extracting sputum and nucleic acid was reacted with boiling water at 100 ° C. for 10 minutes, and then 5 μl of solution was taken. A primer for amplifying the IS6110 gene site for detecting human-type tuberculosis and the nucleic acid amplification composition of [Example 2] Nucleic acid amplification reaction was performed.
Then, the amplified product was observed using a 2% agarose gel and electrophoresis device.
Comparative Example 3
Tuberculosis strain extraction included in sputum
The nucleic acid was amplified under the same experimental conditions as in Experimental Example 4, but the nucleic acid amplification reaction was performed using the nucleic acid amplification composition of [Comparative Example 1] instead of the nucleic acid amplification composition of [Example 2].
4 is an electrophoretic image of the nucleic acid amplification product according to the fourth embodiment of the present invention.
Referring to Figure 4, it was confirmed that the tuberculosis strain can be detected through a simple process.
At this time, M is a 100bp marker, N is a negative control, and 1 to 5 are sputum added with Mycobacterium tuberculosis diluted 10-fold. In addition, A represents Experimental Example 4, and B represents Comparative Experimental Example 3.
[Experimental Example 5]
Apoe Gene Extraction in Oral Epithelial Cell Capture
First, the oral epithelial cells collected by washing the subject's mouth was transferred to a 1.5 ml centrifuge tube, centrifuged at 12,000 rpm for 2 minutes at room temperature, and then the pellets were not attached. The supernatant was removed.
Then, centrifugation and supernatant removal were performed twice.
Then, 100 μl of the nucleic acid extracting composition was injected into the collected pellets.
Then, the pellet and nucleic acid extracting composition was reacted with boiling water for 5 minutes in boiling water, 5 μl of the reaction solution was added, and the nucleic acid amplification composition of [Example 2] was added to perform nucleic acid amplification.
Then, the restriction enzyme (Hha1) was processed and genotype was analyzed by DHPLC analysis. Here, the restriction enzyme (HhaI) is a restriction enzyme that cuts between the first G and C by recognizing GCGC, and distinguishes the E2, E3, and E4 genotypes by analyzing different types of GCGC genes among ApoE genes.
5 is a photograph showing a sequencing result of the nucleic acid amplification product according to the fifth embodiment of the present invention.
Referring to Figure 5, the genotype of the ApoE gene for the subject could be confirmed.
As described above, the nucleic acid extracting composition according to the present invention has an effect of shortening the time required for extracting nucleic acids and eliminating various procedures as compared with the case of using a general nucleic acid extracting composition. The existing alkaline method using an alkaline solution that destroys the cell wall adversely affects the nucleic acid amplification reaction because of the alkaline solution, but the nucleic acid extracting composition according to the present invention does not include an inhibitory factor for the nucleic acid extraction reaction.
While the above has been described with reference to a preferred embodiment of the present invention, those skilled in the art will be able to variously modify and change the present invention without departing from the spirit and scope of the invention as set forth in the claims below. It will be appreciated.
1 is an electrophoretic image of a nucleic acid amplification product according to a first embodiment of the present invention.
Figure 2 is an electrophoretic picture of the nucleic acid amplification product according to a second embodiment of the present invention.
Figure 3 is an electrophoretic picture of the nucleic acid amplification product according to a third embodiment of the present invention.
4 is an electrophoretic image of the nucleic acid amplification product according to the fourth embodiment of the present invention.
5 is a photograph showing a sequencing result of the nucleic acid amplification product according to the fifth embodiment of the present invention.
Claims (10)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190052524A (en) * | 2017-11-08 | 2019-05-16 | 대한민국(농촌진흥청장) | Composition for extracting nucleic acids form mushroom and its processed goods, and Method for extracting nucleic acids using the same |
| CN113462742A (en) * | 2021-08-04 | 2021-10-01 | 江苏臻石生物科技有限公司 | Biological sample nucleic acid release preservative |
| KR102310223B1 (en) * | 2021-07-21 | 2021-10-08 | 주식회사 에이아이더뉴트리진 | Composition for Cell-lysis to apply to a structure with a lab-on-paper chip |
| WO2022115450A3 (en) * | 2020-11-24 | 2022-07-07 | Piotr Chomczynski | Rapid processing of biological samples using glycols and alkaline ph for sars-cov-2-rna detection |
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2009
- 2009-11-30 KR KR1020090116661A patent/KR20110060156A/en not_active Application Discontinuation
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190052524A (en) * | 2017-11-08 | 2019-05-16 | 대한민국(농촌진흥청장) | Composition for extracting nucleic acids form mushroom and its processed goods, and Method for extracting nucleic acids using the same |
| WO2022115450A3 (en) * | 2020-11-24 | 2022-07-07 | Piotr Chomczynski | Rapid processing of biological samples using glycols and alkaline ph for sars-cov-2-rna detection |
| KR102310223B1 (en) * | 2021-07-21 | 2021-10-08 | 주식회사 에이아이더뉴트리진 | Composition for Cell-lysis to apply to a structure with a lab-on-paper chip |
| WO2023003137A1 (en) * | 2021-07-21 | 2023-01-26 | 주식회사 에이아이더뉴트리진 | Composition for cell lysis to be applied to structure including lab-on-paper chip |
| CN113462742A (en) * | 2021-08-04 | 2021-10-01 | 江苏臻石生物科技有限公司 | Biological sample nucleic acid release preservative |
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