CN111187837A - Kit for detecting Eps8 gene expression level - Google Patents
Kit for detecting Eps8 gene expression level Download PDFInfo
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- CN111187837A CN111187837A CN201911233706.1A CN201911233706A CN111187837A CN 111187837 A CN111187837 A CN 111187837A CN 201911233706 A CN201911233706 A CN 201911233706A CN 111187837 A CN111187837 A CN 111187837A
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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
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention discloses a kit for detecting Eps8 gene expression level, which comprises PCR reaction buffer solution, wherein the PCR reaction buffer solution comprises the following raw materials in parts by weight: 3-5 parts of dNTP mixture, 2-4 parts of magnesium chloride solution, 1-3 parts of DNA polymerase, 1-3 parts of Taq enzyme, 2-4 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 4-6 parts of ammonium sulfate, 2-4 parts of sodium nitride and 5-7 parts of deionized water. According to the kit for detecting the expression level of the Eps8 gene, the DNA polymerase is heat-resistant DNA polymerase, the enzyme activity in the heat-resistant DNA polymerase is 2-8U/mu L, the heat-resistant DNA polymerase is one or a combination of chemically modified hot-start heat-resistant DNA enzyme and antibody modified hot-start heat-resistant DNA enzyme, and ammonium sulfate and sodium nitride are added into the raw materials of the PCR reaction buffer solution raw materials, so that the reaction efficiency of PCR can be effectively improved, and the kit has the advantages of accuracy, sensitivity, simplicity in operation, time and labor saving, reduction in detection cost and improvement of inspection efficiency.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a kit for detecting the expression level of an Eps8 gene.
Background
Eps8 is a tyrosine kinase activation substrate of an epidermal growth factor receptor, can be phosphorylated by EGFR tyrosine, and can also be phosphorylated by other receptor tyrosine kinases, a human Eps8 gene is positioned on a chromosome 12q23-q24, two subtypes of p97Eps8 and p68Eps8 and three family members of Eps8L1-3 are provided, the human Eps8 gene is mainly distributed in cytoplasm around a nucleus in a circular shape, recently researches show that Eps8 is over-expressed in various human tumor specimens and cell lines, the specific activity of Rac is endowed in the form of a complex of Eps8-E3b1-Sos1, Eps8-Abi1-p85-Sos1 and IRSp53-Eps8, actin cytoskeleton rearrangement is induced, the mitosis and transformation capability of cells are promoted through multiple signal paths such as EGFR, PI3K/Akt, MAPK and the like, and the protein tyrosine kinase activation substrate can be involved in various tumors such as pancreatic cancer, thyroid cancer, papillary cancer, breast cancer and papillary cancer, Intestinal cancer, head and neck squamous cell carcinoma, oral squamous cell carcinoma and malignant hematological tumor, so that the detection of the gene Eps8 can provide a reference basis for diagnosis and treatment of various cancers.
At present, a kit for detecting the expression level of a gene generally comprises HER2 gene PCR reaction liquid and ACTB gene PCR reaction liquid, wherein the general HER2 gene PCR reaction liquid comprises PCR reaction buffer liquid, HER2 gene specific upstream primer, HER2 gene specific downstream primer and EVA Green fluorescent dye, the ACTB gene PCR reaction liquid comprises PCR reaction buffer liquid, ACTB gene specific upstream primer, ACTB gene specific downstream primer and EVA Green fluorescent dye, most of the PCR reaction buffer liquid comprises KCL, MgSO4 and dNTPs, the detection efficiency of the Eps8 in the process of using the PCR reaction buffer liquid in the kit is low, certain errors exist in the detection accuracy of the expression level of the Eps8 gene, and the use effect of the gene detection kit is reduced.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides the reagent kit for detecting the expression level of the Eps8 gene, and solves the problems that the detection efficiency of the PCR reaction buffer solution used in the gene detection kit on Eps8 is low, a certain error exists in the detection accuracy of the expression level of the Eps8 gene, and the use effect of the gene detection kit is reduced.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a kit for detecting the expression level of an Eps8 gene comprises a PCR reaction buffer solution, wherein the PCR reaction buffer solution comprises the following raw materials in parts by weight: 3-5 parts of dNTP mixture, 2-4 parts of magnesium chloride solution, 1-3 parts of DNA polymerase, 1-3 parts of Taq enzyme, 2-4 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 4-6 parts of ammonium sulfate, 2-4 parts of sodium nitride and 5-7 parts of deionized water.
Preferably, the PCR reaction buffer solution raw materials comprise the following components in parts by weight: 3 parts of dNTP mixture, 2 parts of magnesium chloride solution, 1 part of DNA polymerase, 1 part of Taq enzyme, 2 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 4 parts of ammonium sulfate, 2 parts of sodium nitride and 5 parts of deionized water.
Preferably, the PCR reaction buffer solution raw materials comprise the following components in parts by weight: 5 parts of dNTP mixture, 4 parts of magnesium chloride solution, 3 parts of DNA polymerase, 3 parts of Taq enzyme, 4 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 6 parts of ammonium sulfate, 4 parts of sodium nitride and 7 parts of deionized water.
Preferably, the PCR reaction buffer solution raw materials comprise the following components in parts by weight: 4 parts of dNTP mixture, 3 parts of magnesium chloride solution, 2 parts of DNA polymerase, 2 parts of Taq enzyme, 3 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 5 parts of ammonium sulfate, 3 parts of sodium nitride and 6 parts of deionized water.
Preferably, the DNA polymerase is thermostable DNA polymerase, the enzyme activity in the thermostable DNA polymerase is 2-8U/μ L, and the thermostable DNA polymerase is one or more of chemically modified hot-start thermostable DNA polymerase and antibody modified hot-start thermostable DNA polymerase.
Preferably, the Taq enzyme is a real-time hot start Taq enzyme, the real-time hot start Taq enzyme is prepared by mixing phage, trypsin and ammonium bicarbonate, an enzymolysis solution is obtained by ultrafiltration to obtain a filtrate containing affinity ligand peptide, the filtrate is diluted to 1000 times, and the diluted filtrate is mixed with a common Taq enzyme 1: 1 mixing to obtain the real-time hot start Taq enzyme.
Preferably, the concentration of the tris hydrochloric acid solution is 8mol/L, and the pH of the tris hydrochloric acid solution is 8.8.
The thermostable DNA polymerase is a protein resistant to high temperature, and has 5'-3' polymerase activity, including Taq DNA polymerase, Tth DNA polymerase, Bca Best DNA polymerase, and Sac DNA polymerase.
The pure product of ammonium sulfate is colorless transparent orthorhombic crystal, the water solution is acidic, insoluble in alcohol, acetone and ammonia water, has hygroscopicity, is solidified into blocks after moisture absorption, is heated to 513 ℃ and is completely decomposed into ammonia gas, nitrogen gas, sulfur dioxide and water, the ammonia gas is released under the action of alkali, and reacts with barium chloride solution to generate barium sulfate precipitate, and the protein can also be salted out.
Sodium nitride is white hexagonal crystal, is unstable to heat, and the water solution releases toxic HN3 (hydrogen azide) when meeting acid, can be heated to 300 ℃ in vacuum without explosion and decomposed into sodium and nitrogen, and is heated to 300 ℃ in air to release a large amount of nitrogen, and the solubility in water is as follows: 40.16% at 10 ℃ and 41.7% at 17 ℃, solubility in ethanol: 0.3% at 25 deg.C, soluble in liquid ammonia, insoluble in diethyl ether, and examined for N3-ion with 100g/L ferric chloride solution, the reaction of ferric trichloride with N3-ion produces a vivid reddish blood color, which is very sensitive.
Preferably, the preparation method of the PCR reaction buffer solution specifically comprises the following steps:
s1, mixing the dNTP mixture, the magnesium chloride solution, the DNA polymerase and the tris (hydroxymethyl) aminomethane hydrochloric acid solution to obtain a mixed solution, placing the mixed solution in a water bath at 45 ℃, stirring the mixed solution by using a glass stirring rod at a stirring speed of 150r/min at a constant speed for 30 minutes;
s2, adding Taq enzyme into the mixed solution, and continuing stirring the mixed solution at a stirring speed of 50r/min for 15 minutes;
and S3, cooling the mixed solution to 25 ℃, adding ammonium sulfate and sodium nitride into the mixed solution, adding deionized water into the mixed solution, stirring at a constant speed of 100r/min for 20 minutes, and standing for 5 minutes to obtain a PCR reaction buffer solution.
(III) advantageous effects
The invention provides a kit for detecting the expression level of Eps8 gene. Compared with the prior art, the method has the following beneficial effects:
(1) the kit for detecting the expression level of the Eps8 gene comprises the following raw materials in parts by weight through PCR reaction buffer solution: 3-5 parts of dNTP mixture, 2-4 parts of magnesium chloride solution, 1-3 parts of DNA polymerase, 1-3 parts of Taq enzyme, 2-4 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 4-6 parts of ammonium sulfate, 2-4 parts of sodium trinitride and 5-7 parts of deionized water, wherein the DNA polymerase is heat-resistant DNA polymerase, the enzyme activity in the heat-resistant DNA polymerase is 2-8U/mu L, the heat-resistant DNA polymerase is one or the combination of a chemically modified heat-starting heat-resistant DNA enzyme and an antibody modified heat-starting heat-resistant DNA enzyme, ammonium sulfate and sodium nitride are added into the raw materials of the PCR reaction buffer solution, the method can effectively improve the reaction efficiency of PCR, and has the advantages of reliable and accurate result, sensitivity, simple operation, time and labor saving, detection cost reduction, inspection efficiency improvement and the like.
(2) This detect kit of Eps8 gene expression level, through Taq enzyme for real-time hot start Taq enzyme, real-time hot start Taq enzyme obtains the enzymolysis liquid through phage and trypsin and ammonium bicarbonate mixture, ultrafiltrates the enzymolysis liquid and obtains the filtrate that contains affinity ligand peptide, dilutes the filtrate to 1000 times again, dilutes filtrate and ordinary Taq enzyme 1: 1, the real-time hot start Taq enzyme is obtained, and the real-time hot start Taq enzyme can be utilized to enable the PCR reaction buffer solution to be combined with the ligand more rapidly when the expression level of the Eps8 gene is detected, so that the efficiency of detecting the expression level of the Eps8 gene is effectively improved.
(3) The kit for detecting the expression level of the Eps8 gene is prepared by S1, mixing a dNTP mixture, a magnesium chloride solution, DNA polymerase and a tris (hydroxymethyl) aminomethane hydrochloric acid solution to obtain a mixed solution, placing the mixed solution in a water bath at 45 ℃, stirring the mixed solution by using a glass stirring rod at a stirring speed of 150r/min for 30 minutes, S2, adding Taq enzyme into the mixed solution, continuing stirring the mixed solution at a stirring speed of 50r/min for 15 minutes, S3, cooling the mixed solution to 25 ℃, adding ammonium sulfate and sodium nitride into the mixed solution, adding deionized water into the mixed solution, stirring at a stirring speed of 100r/min for 20 minutes, standing for 5 minutes to obtain a PCR reaction buffer solution, and having a simple preparation process for the PCR reaction buffer solution, the prepared PCR reaction buffer solution has good detection effect on the expression level of the Eps8 gene and high detection efficiency.
Drawings
FIG. 1 is a statistical table of comparative experimental data according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, the embodiment of the present invention provides three technical solutions: a kit for detecting the expression level of Eps8 gene specifically comprises the following embodiments:
example 1
S1, selecting 3 parts of a dNTP mixture, 2 parts of a magnesium chloride solution, 1 part of DNA polymerase and 2 parts of a tris (hydroxymethyl) aminomethane hydrochloric acid solution, mixing the raw materials to obtain a mixed solution, placing the mixed solution in a water bath at 45 ℃, and stirring the mixed solution by using a glass stirring rod at a constant stirring speed of 150r/min for 30 minutes;
s2, adding 1 part of Taq enzyme into the mixed solution, and continuing stirring the mixed solution at a stirring speed of 50r/min for 15 minutes;
and S3, cooling the mixed solution to 25 ℃, adding 4 parts of ammonium sulfate and 2 parts of sodium nitride into the mixed solution, simultaneously adding 5 parts of deionized water, stirring at a constant speed of 100r/min for 20 minutes, and standing for 5 minutes to obtain the PCR reaction buffer solution.
Example 2
S1, selecting 5 parts of a dNTP mixture, 4 parts of a magnesium chloride solution, 3 parts of DNA polymerase and 4 parts of a tris (hydroxymethyl) aminomethane hydrochloric acid solution, mixing the raw materials to obtain a mixed solution, placing the mixed solution in a water bath at 45 ℃, and stirring the mixed solution by using a glass stirring rod at a constant stirring speed of 150r/min for 30 minutes;
s2, adding 3 parts of Taq enzyme into the mixed solution, and continuing stirring the mixed solution at a stirring speed of 50r/min for 15 minutes;
and S3, cooling the mixed solution to 25 ℃, adding 6 parts of ammonium sulfate and 4 parts of sodium nitride into the mixed solution, simultaneously adding 6 parts of deionized water, stirring at a constant speed of 100r/min for 20 minutes, and standing for 5 minutes to obtain the PCR reaction buffer solution.
Example 3
S1, selecting 4 parts of a dNTP mixture, 3 parts of a magnesium chloride solution, 2 parts of DNA polymerase and 3 parts of a tris (hydroxymethyl) aminomethane hydrochloric acid solution, mixing the raw materials to obtain a mixed solution, placing the mixed solution in a water bath at 45 ℃, and stirring the mixed solution by using a glass stirring rod at a constant stirring speed of 150r/min for 30 minutes;
s2, adding 2 parts of Taq enzyme into the mixed solution, and continuing stirring the mixed solution at a stirring speed of 50r/min for 15 minutes;
and S3, cooling the mixed solution to 25 ℃, adding 5 parts of ammonium sulfate and 3 parts of sodium nitride into the mixed solution, simultaneously adding 6 parts of deionized water, stirring at a constant speed of 100r/min for 20 minutes, and standing for 5 minutes to obtain the PCR reaction buffer solution.
Comparative experiment
In a gene detection laboratory, the PCR reaction buffers prepared in examples 1 to 3 are respectively selected to perform a reaction rate comparison experiment, as can be seen from fig. 1, the reaction rate of the PCR reaction buffer prepared in example 1 is 95, the reaction rate of the PCR reaction buffer prepared in example 2 is 93, and the reaction rate of the PCR reaction buffer prepared in example 3 is 97.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A kit for detecting the expression level of Eps8 gene, which comprises PCR reaction buffer solution, and is characterized in that: the PCR reaction buffer solution comprises the following raw materials in parts by weight: 3-5 parts of dNTP mixture, 2-4 parts of magnesium chloride solution, 1-3 parts of DNA polymerase, 1-3 parts of Taq enzyme, 2-4 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 4-6 parts of ammonium sulfate, 2-4 parts of sodium nitride and 5-7 parts of deionized water.
2. The kit for detecting the expression level of Eps8 gene according to claim 1, wherein: the PCR reaction buffer solution comprises the following raw materials in parts by weight: 3 parts of dNTP mixture, 2 parts of magnesium chloride solution, 1 part of DNA polymerase, 1 part of Taq enzyme, 2 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 4 parts of ammonium sulfate, 2 parts of sodium nitride and 5 parts of deionized water.
3. The kit for detecting the expression level of Eps8 gene according to claim 1, wherein: the PCR reaction buffer solution comprises the following raw materials in parts by weight: 5 parts of dNTP mixture, 4 parts of magnesium chloride solution, 3 parts of DNA polymerase, 3 parts of Taq enzyme, 4 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 6 parts of ammonium sulfate, 4 parts of sodium nitride and 7 parts of deionized water.
4. The kit for detecting the expression level of Eps8 gene according to claim 1, wherein: the PCR reaction buffer solution comprises the following raw materials in parts by weight: 4 parts of dNTP mixture, 3 parts of magnesium chloride solution, 2 parts of DNA polymerase, 2 parts of Taq enzyme, 3 parts of tris (hydroxymethyl) aminomethane hydrochloric acid solution, 5 parts of ammonium sulfate, 3 parts of sodium nitride and 6 parts of deionized water.
5. The kit for detecting the expression level of Eps8 gene according to any one of claims 1-4, wherein: the DNA polymerase is heat-resistant DNA polymerase, the enzyme activity in the heat-resistant DNA polymerase is 2-8U/muL, and the heat-resistant DNA polymerase is one or more of chemically modified hot-start heat-resistant DNA enzyme and antibody modified hot-start heat-resistant DNA enzyme.
6. The kit for detecting the expression level of Eps8 gene according to any one of claims 1-4, wherein: the Taq enzyme is a real-time hot start Taq enzyme, the real-time hot start Taq enzyme is mixed with trypsin and ammonium bicarbonate through bacteriophage to obtain an enzymolysis solution, the enzymolysis solution is ultrafiltered to obtain a filtrate containing affinity ligand peptide, the filtrate is diluted to 1000 times, and the diluted filtrate is mixed with a common Taq enzyme 1: 1 mixing to obtain the real-time hot start Taq enzyme.
7. The kit for detecting the expression level of Eps8 gene according to any one of claims 1-4, wherein: the concentration of the tris hydrochloric acid solution was 8mol/L, and the pH of the tris hydrochloric acid solution was 8.8.
8. The kit for detecting the expression level of Eps8 gene according to claims 1-4, wherein: the preparation method of the PCR reaction buffer solution specifically comprises the following steps:
s1, mixing the dNTP mixture, the magnesium chloride solution, the DNA polymerase and the tris (hydroxymethyl) aminomethane hydrochloric acid solution to obtain a mixed solution, placing the mixed solution in a water bath at 45 ℃, stirring the mixed solution by using a glass stirring rod at a stirring speed of 150r/min at a constant speed for 30 minutes;
s2, adding Taq enzyme into the mixed solution, and continuing stirring the mixed solution at a stirring speed of 50r/min for 15 minutes;
and S3, cooling the mixed solution to 25 ℃, adding ammonium sulfate and sodium nitride into the mixed solution, adding deionized water, stirring at a constant speed of 100r/min for 20 minutes, and standing for 5 minutes to obtain a PCR reaction buffer solution.
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