CN117867130A - PCR kit and method for detecting mouse genotype - Google Patents
PCR kit and method for detecting mouse genotype Download PDFInfo
- Publication number
- CN117867130A CN117867130A CN202410023981.5A CN202410023981A CN117867130A CN 117867130 A CN117867130 A CN 117867130A CN 202410023981 A CN202410023981 A CN 202410023981A CN 117867130 A CN117867130 A CN 117867130A
- Authority
- CN
- China
- Prior art keywords
- pcr
- concentration
- mouse
- lysate
- genotype
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 46
- 238000009004 PCR Kit Methods 0.000 title claims abstract description 12
- 239000006166 lysate Substances 0.000 claims abstract description 76
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 74
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 54
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 45
- 238000004062 sedimentation Methods 0.000 claims abstract description 25
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims abstract description 21
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 21
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims abstract description 21
- 229960003237 betaine Drugs 0.000 claims abstract description 21
- 239000004005 microsphere Substances 0.000 claims abstract description 21
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 20
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 20
- 108010002747 Pfu DNA polymerase Proteins 0.000 claims abstract description 20
- 239000004094 surface-active agent Substances 0.000 claims abstract description 20
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical group [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims abstract description 18
- 102000052510 DNA-Binding Proteins Human genes 0.000 claims abstract description 13
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 12
- 239000011324 bead Substances 0.000 claims abstract description 12
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 12
- 108700020911 DNA-Binding Proteins Proteins 0.000 claims abstract description 7
- 108010003723 Single-Domain Antibodies Proteins 0.000 claims abstract description 7
- 108700014590 single-stranded DNA binding proteins Proteins 0.000 claims abstract description 7
- 230000003321 amplification Effects 0.000 claims description 53
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 53
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 45
- 238000006243 chemical reaction Methods 0.000 claims description 38
- 108020004414 DNA Proteins 0.000 claims description 28
- 241000699670 Mus sp. Species 0.000 claims description 26
- 238000012408 PCR amplification Methods 0.000 claims description 23
- 239000011734 sodium Substances 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 238000000137 annealing Methods 0.000 claims description 7
- 101710116602 DNA-Binding protein G5P Proteins 0.000 claims description 6
- 101710162453 Replication factor A Proteins 0.000 claims description 6
- 101710176758 Replication protein A 70 kDa DNA-binding subunit Proteins 0.000 claims description 6
- 101710176276 SSB protein Proteins 0.000 claims description 6
- 101710126859 Single-stranded DNA-binding protein Proteins 0.000 claims description 6
- 238000001962 electrophoresis Methods 0.000 claims description 6
- 238000011144 upstream manufacturing Methods 0.000 claims description 6
- 238000012257 pre-denaturation Methods 0.000 claims description 5
- 238000004925 denaturation Methods 0.000 claims description 4
- 230000036425 denaturation Effects 0.000 claims description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 125
- 239000000203 mixture Substances 0.000 description 30
- 239000000047 product Substances 0.000 description 29
- 239000006228 supernatant Substances 0.000 description 16
- 238000010362 genome editing Methods 0.000 description 15
- 238000000246 agarose gel electrophoresis Methods 0.000 description 14
- 238000003776 cleavage reaction Methods 0.000 description 11
- 230000000694 effects Effects 0.000 description 11
- 230000007017 scission Effects 0.000 description 11
- 238000005336 cracking Methods 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 241001417092 Macrouridae Species 0.000 description 8
- 235000009233 Stachytarpheta cayennensis Nutrition 0.000 description 8
- 238000001502 gel electrophoresis Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- 241000581650 Ivesia Species 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 108010067770 Endopeptidase K Proteins 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 238000006386 neutralization reaction Methods 0.000 description 4
- 230000006920 protein precipitation Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 3
- 101150068332 KIT gene Proteins 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 101150014691 PPARA gene Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000012197 amplification kit Methods 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000001651 autotrophic effect Effects 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- MOTZDAYCYVMXPC-UHFFFAOYSA-N dodecyl hydrogen sulfate Chemical compound CCCCCCCCCCCCOS(O)(=O)=O MOTZDAYCYVMXPC-UHFFFAOYSA-N 0.000 description 1
- 229940043264 dodecyl sulfate Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- -1 sodium alkyl sulfate Chemical class 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
Classifications
-
- 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
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- 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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present application relates to a PCR kit and method for detecting a mouse genotype. The PCR kit for detecting the genotype of the mouse comprises a lysate and a PCR premix; the lysate comprises Tris-HCl, na 2 EDTA and a protein sedimentation aid, wherein the protein sedimentation aid comprises magnetic bead microspheres with surfaces modified with sodium dodecyl sulfate; the PCR premix comprises Tris-HCl and Mg 2+ 、NH 4 + 、K + Surfactants, bovine serum albumin, betaine, dntps, single-stranded DNA binding proteins, taq mut4 DNA polymerase, pfu DNA polymerase, and nanobodies.
Description
Technical Field
The application relates to the field of molecular biology detection, in particular to a PCR kit and a method for detecting mouse genotypes.
Background
Transgenic mice are often used as important animal models for the study of human diseases, which are widely studied in the fields of life sciences and medicine. The transgenic mouse model constructed in most laboratories is autotrophic and self-propagating and has a variety of strains. Prior to the development of scientific studies using these mice, it is often necessary to identify and group the mice genotypes, which are common procedures including: (1) obtaining mouse genome DNA; (2) Conventional PCR amplification, the genotype of the mice is ultimately determined by comparison of the amplified bands of a particular gene.
At present, two common mouse genotype identification methods exist, the first method is to extract mouse DNA by using an animal genome kit method for conventional PCR mix amplification, and the method has the advantages of high purity of extracted genome DNA, less impurities, long time, complicated steps and slightly high cost; the second is to extract rat tail DNA by alkali cleavage method to perform conventional PCR mix amplification, the method has the advantages of short extraction time and simple operation, and the defects of low purity of extracted genome DNA, more impurities and low identification success rate. The second method has the disadvantage that, on the one hand, direct cleavage of the NaOH solution leads to cleavage of the genomic DNA, thus generally requiring an additional step of neutralization of the alkalinity; on the other hand, the crude DNA templates sometimes contain more impurities or PCR inhibitors, which are only suitable for partial PCR reactions with DNA polymerases that are more tolerant to multiple inhibitors, and have poor compatibility with most DNA polymerases; and part of the kit needs to adopt the steps of adding precipitation or adsorbing PCR inhibitor (such as adding acetate precipitation SDS and centrifuging to remove), and some of the kit needs to dilute the template, so that the concentration of the PCR inhibitor is reduced, and the normal operation of PCR amplification is ensured. Either way, however, is cumbersome and multiple additions of reagents increase the probability of cross-contamination between different samples.
Thus, the conventional technology has yet to be improved.
Disclosure of Invention
Based on this, it is necessary to provide a PCR kit and a method for detecting a mouse genotype with rapid identification, high amplification success rate and high yield.
The technical scheme for solving the technical problems is as follows:
in a first aspect of the present application, there is provided a PCR kit for detecting a mouse genotype, comprising a lysate and a PCR premix;
the lysate comprises Tris-HCl, na 2 EDTA and a protein sedimentation aid, wherein the protein sedimentation aid comprises magnetic bead microspheres with surfaces modified with sodium dodecyl sulfate;
the PCR premix comprises Tris-HCl and Mg 2+ 、NH 4 + 、K + Surfactants, bovine serum albumin, betaine, dntps, single-stranded DNA binding proteins, taq mut4 DNA polymerase, pfu DNA polymerase, and nanobodies.
In one embodiment, the concentration of Tris-HCl in the lysate is 20 mM-100 mM, and the Na is 2 The concentration of EDTA is 0.5 mM-1 mM, and the mass percentage of the protein sedimentation assisting agent is 1% -7%.
In one embodiment, the concentration of Tris-HCl in the lysate is 20 mM-100 mM, and the Na is 2 The concentration of EDTA is 0.5 mM-1 mM, and the mass percentage of the protein sedimentation assisting agent is 3% -5%.
In one embodiment, the concentration of Tris-HCl in the PCR premix is 40 mM-60 mM, and the concentration of Mg is 2+ The concentration of (C) is 1 mM-3 mM, the NH is 4 + The concentration of K is 10 mM-30 mM + The concentration of the surface active agent is 5 mM-20 mM, the volume percentage of the surface active agent is 0.2% -0.5%, the mass percentage of the bovine serum albumin is 0.01% -0.1%, the concentration of the betaine is 0.3-0.8M, the concentration of the dNTP is 10-50 mu M, the concentration of the single-stranded DNA binding protein is 5 ng/. Mu.L-20 ng/. Mu.L, the concentration of the Taqmut4DNA polymerase is 90 ng/. Mu.L-200 ng/. Mu.L, the concentration of the PfeuDNA polymerase is 5 ng/. Mu.L-20 ng/. Mu.L, and the concentration of the nanobody is 0.2 mg/mL-0.8 mg/mL.
The application also provides a method for detecting the genotype of the mouse, which comprises the following steps:
adding a lysate into a sample of the mice to be tested for heating treatment to obtain a DNA sample of the mice to be tested; the lysate comprises Tris-HCl, na 2 EDTA and a protein sedimentation aid, wherein the protein sedimentation aid comprises magnetic bead microspheres with surfaces modified with sodium dodecyl sulfate;
taking the mouse DNA sample to be detected as a template, adding a PCR premix and an amplification primer, and carrying out PCR amplification reaction to obtain an amplification product; the PCR premix comprises Tris-HCl and Mg 2+ 、NH 4 + 、K + Surfactants, bovine serum albumin, betaine, dntps, single-stranded DNA binding proteins, taqmut4DNA polymerase, pfu DNA polymerase, and nanobodies; and
The amplified products were analyzed to determine the mouse genotype.
In one embodiment, the heating treatment satisfies at least one condition of (1) to (2):
(1) The temperature of the heating treatment is 95-100 ℃;
(2) The heating treatment time is 8-12 min.
In one embodiment, each 25. Mu.L of the reaction system comprises the PCR premix, 9.5. Mu.M to 10.5. Mu.M upstream primer 0.9. Mu.L to 1. Mu.L, 9.5. Mu.M to 10.5. Mu.M downstream primer 0.9. Mu.L to 1. Mu.L, and 0.9. Mu.L to 1. Mu.L of the test mouse DNA sample.
In one embodiment, each 25 mu L of the reaction system comprises 12.4 mu L to 12.5 mu L of PCR premix, 0.9 mu L to 1 mu L of 10 mu M upstream primer, 0.9 mu L to 1 mu L of 10 mu M downstream primer and 0.9 mu L to 1 mu L of DNA of the mouse DNA sample to be tested.
In one embodiment, the PCR amplification reaction is programmed to: pre-denaturation at 94-96 ℃ for 2-4 min; denaturation at 97-99 ℃ for 8-12 s, annealing at 58-65 ℃ for 18-22 s, annealing at 70-74 ℃ for 18-22 s, and circulation for 30-40 times and extension at 70-74 ℃ for 3 min-5 min.
In one embodiment, the step of analyzing the amplification product comprises:
and obtaining an electrophoresis band of the amplification product, and comparing the electrophoresis band with a wild type target sequence band to determine the genotype of the mouse.
The kit for detecting the PCR direct amplification of the mouse genotype comprises a lysate and a PCR premix, wherein the lysate comprises Tris-HCl and Na 2 EDTA and protein deposition aid including twelve surface modificationsThe magnetic bead microsphere of sodium alkyl sulfate, the kit does not need the steps of neutralization alkalinity and centrifugation, proteinase K is not needed, a sample can be directly cracked by using a cracking liquid, SDS (sodium dodecyl sulfate) can denature protein in the cracking process, then the magnetic bead microsphere can gradually adsorb denatured protein, finally the clearance effect on PCR (polymerase chain reaction) inhibitors is completed along with the sedimentation effect of the microsphere, and the template has few impurities after cracking, is simple to operate and can realize rapid detection; and then, by adjusting the formula components of the PCR premix, adding the nano antibody to modify the Taq mut4+pfu double-enzyme system and the single-stranded DNA binding protein, the dirt resistance of the premix is improved, and the lysate and the premix are mutually matched, so that the success rate of the PCR direct amplification reaction is ensured, and the PCR yield is improved.
Drawings
FIG. 1 is a gel electrophoresis chart of PCR products of different concentrations of protein precipitants in example 1;
FIG. 2 is a graph showing the blocking activity of the nanobody of example 1 on Taq mut4 enzyme under different reaction conditions, wherein the abscissa represents the length of base pairs (bp) and the ordinate represents the fluorescence signal value;
FIG. 3 is a gel electrophoresis chart of PCR products of the PCR premix solution of example 1 using different enzyme systems;
FIG. 4 is a gel electrophoresis diagram of PCR products of different genes of interest in a gene editing mouse using the kit of example 1;
FIG. 5 is a gel electrophoresis diagram of PCR products of the Ppara gene in the gene editing mouse by the kit of example 2;
FIG. 6 is a gel electrophoresis diagram of PCR products of the Ifih1-KO gene in a kit gene editing mouse in example 3;
FIG. 7 is a gel electrophoresis diagram of PCR products of different genes of interest in a kit gene editing mouse in example 4;
FIG. 8 is a gel electrophoresis diagram of PCR products of different target genes in a gene editing mouse using the kit of example 2 and the A company direct amplification kit;
FIG. 9 is a gel electrophoresis chart of PCR products of different target genes in the kit gene editing mice of example 6 and comparative examples 1 to 2.
Detailed Description
In order to make the above objects, features and advantages of the present application more comprehensible, embodiments accompanied with preferred embodiments accompanied with the present application are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is, however, susceptible of embodiment in many other forms than those described herein and similar modifications can be made by those skilled in the art without departing from the spirit of the application, and therefore the application is not to be limited to the specific embodiments disclosed below.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items. The reagents or apparatus used herein are not intended to be manufacturer specific and are conventional products available from commercial sources.
Concentration values are referred to in this application, the meaning of which includes fluctuations within a certain range. For example, it may fluctuate within a corresponding accuracy range. For example, 2%, may allow fluctuations within + -0.1%. For values that are larger or do not require finer control, it is also permissible for the meaning to include larger fluctuations. For example, 100mM, fluctuations in the range of.+ -. 1%,.+ -. 2%,.+ -. 5%, etc. can be tolerated. Molecular weight is referred to, allowing its meaning to include fluctuations of + -10%.
In the present application, m/v represents the mass-volume ratio, and% (m/v) represents the mass percentage contained in a mixed system of a certain volume. For example, a concentration of 5% (m/v) of substance A in the mixed system means that 5 g of substance A is contained per 100 ml of the mixed system.
In this application, mmol/L, mM are all millimoles per liter and are used interchangeably. mol/L, M each represents a mole per liter and can be used interchangeably.
In the present application, unless otherwise specified, the concentrations or amounts of the respective components in the lysate component refer to the final concentrations or final amounts in the lysate component, which correspond to the final concentrations or final amounts in the lysate component constituting the lysate reagent.
In the present application, unless otherwise specified, the concentrations or amounts of the respective components in the PCR premix composition refer to the final concentrations or final amounts in the PCR premix composition, which correspond to the final concentrations or final amounts in the PCR premix composition constituting the PCR premix reagent.
In the present application, the technical features described in an open manner include a closed technical scheme composed of the listed features, and also include an open technical scheme including the listed features.
An embodiment of the application provides a PCR kit for detecting the genotype of a mouse, which comprises a lysate and a PCR premix; the lysate comprises Tris-HCl, na 2 EDTA and a protein sedimentation aid, wherein the protein sedimentation aid comprises magnetic bead microspheres with surfaces modified with sodium dodecyl sulfate; the PCR premix comprises Tris-HCl and Mg 2+ 、NH 4 + 、K + Surfactants, bovine serum albumin, betaine, dntps, single-stranded DNA binding proteins, taq mut4 DNA polymerase, pfu DNA polymerase, and nanobodies.
It can be understood that the protein precipitation aid is formed by modifying a layer of cationic polymer on the surface of the silicon dioxide microsphere to form a cationic microsphere; mixing cationic microsphere with sodium dodecyl sulfate solution for reaction, adsorbing sodium dodecyl sulfate on the surface of cationic microsphere by electrostatic adsorption to form microsphere with hydrophobic end of dodecyl sulfate outside, and combining the hydrophobic end with protein to adsorb protein onto microsphere, wherein SDS denatures protein during cracking, then magnetic bead microsphere can gradually adsorb denatured protein, and finally, with sedimentation of microsphere, removing PCR inhibitor (SDS, protein substance, etc.) is completed.
The kit for detecting the PCR direct amplification of the mouse genotype comprises a lysate and a PCR premix, wherein the lysate comprises Tris-HCl and Na 2 EDTA and protein sedimentation aid, the protein sedimentation aid comprises magnetic bead microspheres with surfaces modified by sodium dodecyl sulfate, and the kit does not need the steps of neutralization alkalinity and centrifugation The method comprises the steps of directly cracking a sample by using a cracking solution without using proteinase K, wherein in the cracking process, SDS (sodium dodecyl sulfate) denatures the protein, then magnetic bead microspheres can gradually adsorb the denaturated protein, finally, along with the sedimentation of the microspheres, the removal of PCR (polymerase chain reaction) inhibitors is completed, template impurities are less after cracking, the operation is simple, and the rapid detection can be realized; and then, by adjusting the formula components of the PCR premix, adding the nano antibody to modify the Taq mut4+pfu double-enzyme system and the single-stranded DNA binding protein, the dirt resistance of the premix is improved, and the lysate and the premix are mutually matched, so that the success rate of the PCR direct amplification reaction is ensured, and the PCR yield is improved.
In some of these embodiments, the concentration of Tris-HCl in the above lysate is 20 mM-100 mM, na 2 The concentration of EDTA is 0.5 mM-1 mM, and the mass percentage of the protein sedimentation assisting agent is 1% -7%.
It is to be understood that when a range of values is disclosed herein, the range is to be regarded as continuous, and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The concentration of Tris-HCl may be in the range of "20 mM-100 mM", that is, the minimum value and the maximum value in the range of 20 mM-100 mM, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the point values in the embodiments: 20mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM. Or a range of any two of these values, including, by way of example: 50 mM-80-mM.
Na 2 The EDTA concentration ranges from "0.5mM to 1mM", i.e., the minimum and maximum values in the range of 0.5mM to 1mM, and each value between the minimum and maximum values. Specific examples include, but are not limited to, the point values in the embodiments: 0.5mM, 0.55 mM, 0.6 mM, 0.65 mM, 0.7 mM, 0.75 mM, 0.8 mM, 0.85 mM, 0.9 mM, 0.95 mM or 1 mM. Or a range of any two of these values, including, by way of example: 0.6 mM-0.8. 0.8 mM.
The protein sedimentation agent has a value range of 1% -7%, namely a minimum value and a maximum value in the range of 1% -7%, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the point values in the embodiments: 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6.7%, 6.8%, 6.9% or 7%. Or a range of any two of these values, including, by way of example: 2% -5%.
In some of these embodiments, the concentration of Tris-HCl in the above lysate is 20 mM-100 mM, na 2 The concentration of EDTA is 0.5 mM-1 mM, and the mass percentage of the protein sedimentation assisting agent is 3% -5%.
In some embodiments, the concentration of Tris-HCl in the PCR premix is 40 mM-60 mM, mg 2+ Is 1 mM-3 mM, NH 4 + The concentration of (C) is 10 mM-30 mM, K + The concentration of the (2) is 5 mM-20 mM, the volume percentage of the surfactant is 0.2% -0.5%, the mass percentage of the bovine serum albumin is 0.01% -0.1%, the concentration of the betaine is 0.3M-0.8M, the concentration of the dNTP is 10 mu M-50 mu M, the concentration of the single-stranded DNA binding protein is 5 ng/. Mu.L-20 ng/. Mu.L, the concentration of the Taq mut4 DNA polymerase is 90 ng/. Mu.L-200 ng/. Mu.L, the concentration of the PfuDNA polymerase is 5 ng/. Mu.L-20 ng/. Mu.L, and the concentration of the nano antibody is 0.2 mg/mL-0.8 mg/mL.
Alternatively, mg 2+ Derived from Mg 2 SO 4 ，K + Derived from K 2 SO 4 ，NH 4 + Derived from (NH) 4 ) 2 SO 4 . It will be appreciated that Mg 2+ 、K + And NH 4 + The source of (a) is not limited to the above, but may be other substances.
The concentration of Tris-HCl may be in the range of "40 mM-60 mM", that is, the minimum value and the maximum value in the range of 40 mM-60 mM, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the point values in the embodiments: 40mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, or 60mM. Or a range of any two of these values, including, by way of example: 40 mM-50 and mM.
Mg 2+ The concentration of (C) may be in the range of "1 mM-3 mM", that is, the minimum and maximum values in the range of 1 mM-3 mM, and each value between the minimum and maximum values. Specific examples include, but are not limited to, the point values in the embodiments: 1.1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2 mM, 2.1 mM, 2.2 mM, 2.3 mM, 2.4 mM, 2.5 mM, 2.6 mM, 2.7 mM, 2.8 mM, 2.9 mM, or 3 mM. Or a range of any two of these values, including, by way of example: 1.5 mM-3. 3 mM.
NH 4 + The concentration of (C) may be in the range of "10 mM-30 mM", that is, the minimum and maximum values in the range of 10 mM-30 mM, and each value between the minimum and maximum values. Specific examples include, but are not limited to, the point values in the embodiments: 10mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM. Or a range of any two of these values, including, by way of example: 15 mM-30 and mM.
K + The concentration of (C) is within the range of "5 mM-20 mM", i.e., the minimum and maximum values within the range of 5 mM-20 mM, and each of the minimum and maximum values A value. Specific examples include, but are not limited to, the point values in the embodiments: 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM. Or a range of any two of these values, including, by way of example: 15 mM-20. 20 mM.
The minimum value and the maximum value of the range of 0.2% -0.5% can be taken by the range of 0.2% -0.5% of the volume percentage of the surfactant, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the point values in the embodiments: 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.4%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49% or 0.5%. Or a range of any two of these values, including, by way of example: 0.3% -0.5%.
The bovine serum albumin can take the minimum value and the maximum value of the range of 0.01% -0.1% and each value between the minimum value and the maximum value when the value range of the bovine serum albumin is 0.01% -0.1%. Specific examples include, but are not limited to, the point values in the embodiments: 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.1%. Or a range of any two of these values, including, by way of example: 0.05% -0.1%.
The betaine concentration can be in the range of "0.3M to 0.8M", i.e., the minimum and maximum values in the range of 0.3M to 0.8M, and each value between the minimum and maximum values. Specific examples include, but are not limited to, the point values in the embodiments: 0.3M, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79 or 0.8. Or a range of any two of these values, including, by way of example: 0.5 M-0.7. 0.7M.
The concentration of dNTPs can be set to a value ranging from "10. Mu.M to 50. Mu.M", i.e., a minimum value and a maximum value ranging from 10. Mu.M to 50. Mu.M, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the point values in the embodiments: 10. Mu.M, 11. Mu.M, 12. Mu.M, 13. Mu.M, 14. Mu.M, 15. Mu.M, 16. Mu.M, 17. Mu.M, 18. Mu.M, 19. Mu.M, 20. Mu.M, 21. Mu.M, 22. Mu.M, 23. Mu.M, 24. Mu.M, 25. Mu.M, 26. Mu.M, 27. Mu.M, 28. Mu.M, 29. Mu.M, 30. Mu.M, 31. Mu.M, 32. Mu.M, 33. Mu.M, 34. Mu.M, 35. Mu.M, 36. Mu.M, 37. Mu.M, 38. Mu.M, 39. Mu.M, 40. Mu.M, 41. Mu.M, 42. Mu.M, 43. Mu.M, 44. Mu.M, 45. Mu.M, 46. Mu.M, 47. Mu.M, 48. Mu.M, 49. Mu.M, or 50. Mu.M. Or a range of any two of these values, including, by way of example: 20 mu M to 30 mu M.
The concentration of the single-stranded DNA binding protein can be within the range of 5 ng/-20 ng/-L, and the minimum value and the maximum value of the range of 5 ng/-20 ng/-L can be obtained, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the point values in the embodiments: 5ng/μl, 6 ng/μl, 7 ng/μl, 8 ng/μl, 9 ng/μl, 10 ng/μl, 11 ng/μl, 12 ng/μl, 13 ng/μl, 14 ng/μl, 15ng/μl, 16 ng/μl, 17 ng/μl, 18 ng/μl, 19 ng/μl, or 20ng/μl. Or a range of any two of these values, including, by way of example: 5 ng/. Mu.L to 15 ng/. Mu.L.
The concentration of Taq mut4 DNA polymerase can be in the range of "90 ng/. Mu.L to 200 ng/. Mu.L" to take the minimum and maximum values of the range of 90 ng/. Mu.L to 200 ng/. Mu.L, and each value between the minimum and maximum values. Specific examples include, but are not limited to, the point values in the embodiments: 90ng/μL, 95 ng/μL, 100 ng/μL, 105 ng/μL, 110 ng/μL, 115 ng/μL, 120 ng/μL, 125 ng/μL, 130 ng/μL, 135 ng/μL, 140 ng/μL, 145 ng/μL, 150 ng/μL, 155 ng/μL, 160 ng/μL, 165 ng/μL, 170 ng/μL, 175 ng/μL, 180 ng/μL, 185 ng/μL, 190 ng/μL, 195 ng/μL, or 200ng/μL. Or a range of any two of these values, including, by way of example: 100 ng/. Mu.L to 180 ng/. Mu.L.
The concentration of PfuDNA polymerase can be in the range of "5 ng/-20 ng/-L", and the minimum and maximum values of the range of 5 ng/-20 ng/-L can be taken, and each value between the minimum and maximum values. Specific examples include, but are not limited to, the point values in the embodiments: 5ng/μl, 6 ng/μl, 7 ng/μl, 8 ng/μl, 9 ng/μl, 10 ng/μl, 11 ng/μl, 12 ng/μl, 13 ng/μl, 14 ng/μl, 15 ng/μl, 16 ng/μl, 17 ng/μl, 18 ng/μl, 19 ng/μl, or 20ng/μl. Or a range of any two of these values, including, by way of example: 10 ng/. Mu.L to 20 ng/. Mu.L.
The concentration of the nano antibody is in the range of 0.2 mg/mL-0.8 mg/mL, namely, the minimum value and the maximum value in the range of 0.2 mg/mL-0.8 mg/mL can be taken, and each value between the minimum value and the maximum value. Specific examples include, but are not limited to, the point values in the embodiments: 0.2mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6mg/mL, 0.7 mg/mL, or 0.8 mg/mL. Or a range of any two of these values, including, by way of example: 0.2 mg/mL-0.6 mg/mL.
In some embodiments, the surfactant is selected from one of Tween-20 and Triton X-100.
Further embodiments of the present application provide a method for detecting a genotype of a mouse, and in a specific example, the method includes the following steps S10, S20, and S30.
Step S10: adding a lysate into a sample of the mice to be tested for heating treatment to obtain a DNA sample of the mice to be tested; the lysate comprises Tris-HCl, na 2 EDTA and protein deposition aid, which includes magnetic bead microsphere with surface modified with sodium dodecyl sulfate.
In some embodiments, the temperature of the heating process is 95 ℃ to 100 ℃.
In some embodiments, the heating treatment is performed for 8-12 min.
Compared with the traditional proteinase K-containing lysate (the lysate time is about 35-40 min), the lysate provided by the application has the advantages that the lysate time is only 8-12 min, and the time is obviously shortened; and the steps of neutralization, alkalinity and centrifugation are not needed in the cracking process, a dilution step is not needed, and the high-flux automatic cracking detection is convenient.
Step S20: taking the mouse DNA sample to be detected obtained in the step S10 as a template, adding a PCR premix and an amplification primer, and carrying out PCR amplification reaction to obtain an amplification product; the PCR premix comprises Tris-HCl and Mg 2+ 、NH 4 + 、K + Surfactants, bovine serum albumin, betaine, dntps, single-stranded DNA binding proteins, taq mut4 DNA polymerase, pfu DNA polymerase, and nanobodies.
In some of these embodiments, in step S20, each 25. Mu.L of the reaction system comprises a PCR premix, 9.5. Mu.M to 10.5. Mu.M upstream primer 0.9. Mu.L to 1. Mu.L, 9.5. Mu.M to 10.5. Mu.M downstream primer 0.9. Mu.L to 1. Mu.L, and 0.9. Mu.L to 1. Mu.L of the test mouse DNA sample.
In a specific example, in step S20, each 25. Mu.L of the reaction system comprises 12.4. Mu.L-12.5. Mu.L of the PCR premix, 0.9. Mu.L-1. Mu.L of the 10. Mu.M upstream primer, 0.9. Mu.L-1. Mu.L of the 10. Mu.M downstream primer, and 0.9. Mu.L-1. Mu.L of the DNA of the mouse DNA sample to be tested.
In some of these embodiments, in step S20, the PCR amplification reaction is programmed to: pre-denaturation at 94-96 ℃ for 2-4 min; denaturation at 97-99 ℃ for 8-12 s, annealing at 58-65 ℃ for 18-22 s, annealing at 70-74 ℃ for 18-22 s, and circulation for 30-40 times and extension at 70-74 ℃ for 3 min-5 min.
In a specific example, in step S20, the procedure of the PCR amplification reaction is set as follows: pre-denaturation at 95℃for 3min; denaturation at 98℃for 10s, annealing at 60℃for 20s, extension at 72℃for 20s, cycling for 35 times, extension at 72℃for 5min.
Step S30: the amplification product obtained in step S20 is analyzed to determine the genotype of the mouse.
In some of these embodiments, the step of analyzing the stimulation agent comprises: and a step of obtaining an electrophoresis band of the amplification product obtained in the step S20, and comparing the electrophoresis band with a wild-type target sequence band to determine the genotype of the mouse.
The method for detecting the genotype of the mouse is simple and easy to operate, is used for detecting the genotype of the mouse, and the prepared mouse DNA template is high in purity, and is high in amplification yield and amplification specificity, and the success rate of the genotype identification of the mouse is high.
The following is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer.
Example 1
1. Concentration screening of protein precipitants in lysate
1. The formula of the kit comprises:
protein precipitants with different concentrations are arranged in the existing lysate, and the composition of the lysate is as follows: 100mM Tris-HCl (pH 8.0), 0.5 mM Na 2 EDTA, 3-5 m/v% protein sedimentation aid.
Composition of PCR premix: 60mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、20 mM NH 4 + 、10 mM K + 0.2v/v% surfactant (Tween-20), 0.1M/v% BSA, 0.75M betaine, 12.5. Mu.M dNTP, 5 ng/. Mu.L Taq-SSB protein, 120 ng/. Mu.L Taq mut4 DNA polymerase, 20 ng/. Mu.L Pfu DNA polymerase, 0.2mg/mL nanobody (available from Wohangfan Van. With the designation VAPN 0001).
PCR method:
2-4 mm mice to be identified were rat tails were placed in PCR tubes, 50. Mu.L of lysate was then added and placed in a PCR instrument and heated at 98℃for 10min. After completion of the cleavage, 1. Mu.L of the supernatant was directly taken as an amplification template.
16 pairs of amplification primers were selected, and a PCR amplification reaction system was prepared according to Table 1, and the amplified template DNA was 1. Mu.L of the supernatant.
TABLE 1 PCR reaction System
After the preparation of the PCR amplification reaction system was completed, 25. Mu.L of the system was used, and amplification was performed according to the PCR reaction conditions recommended in Table 2, thereby obtaining a PCR product required for the experiment. The PCR product is subjected to agarose gel electrophoresis to judge the amplification accuracy.
TABLE 2 PCR reaction conditions
As a result, as shown in FIG. 1, the amplification effect was in the following order from strong to weak: 3% protein precipitant = 5% protein precipitant +.7% protein precipitant >1% protein precipitant, the preferred range of protein precipitant concentration is 3% -5%.
2. STR method for detecting blocking activity of nano antibody to Taq mut4 enzyme under different reaction conditions
Reaction conditions: (1) amplifying for 1 hour at different temperatures of 25 ℃,40 ℃ and 50 ℃; (2) amplifying for 1 hour under different temperature conditions at 25 ℃,40 ℃ and 50 ℃ respectively on the basis of 5min pre-denaturation conditions at 95 ℃.
The results are shown in FIG. 2, the abscissa represents base pair length (bp), the ordinate represents fluorescence signal value, A in FIG. 2 is the result of STR sequencing of a control group (nanobody-modified Taq mut4 enzyme), B in FIG. 2 is the result of STR sequencing of an experimental group (nanobody-modified Taq mut4 enzyme), the sample unmodified by nanobody (control group) shows the existence of enzyme activity at 25℃or 40℃and the sample modified by nanobody (experimental group) can completely block the enzyme activity at 25℃or 40℃and normally release the enzyme activity under the condition of treating at 95℃for 5 min.
3. Performance test of Pfu DNA polymerase in Mix
The composition of the lysate was the same as in step 1, and the enzyme system of different PCR premix was tested for performance, wherein scheme 1: composition of PCR premix: 60mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、20 mM NH 4 + 、10 mM K + 0.2v/v% surface active, 0.1M/v% BSA, 0.75. 0.75M betaine, 12.5. Mu.M dNTP, 5 ng/. Mu.L Taq-SSB protein, 20 ng/. Mu.L Pfu DNA polymerase.
Scheme 2: composition of PCR premix: 60mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、20 mM NH 4 + 、10 mM K + 0.2v/v% surface activity, 0.1M/v% BSA, 0.75M betaine, 12.5. Mu.M dNTP, 5 ng/. Mu.L Taq-SSB protein, 120 ng/. Mu.L Taq mut4 DNA polymerase, 0.2mg/mL nanobody.
Scheme 3: composition of PCR premix: 60mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、20 mM NH 4 + 、10 mM K + 0.2v/v% surface activity, 0.1M/v% BSA, 0.75M betaine, 12.5. Mu.M dNTP, 5 ng/. Mu.L Taq-SSB protein, 120 ng/. Mu.L Taq mut4 DNA polymerase, 20 ng/. Mu.L Pfu DNA polymerase, 0.2mg/mL nanobody.
2-4 mm mice to be identified were rat tails were placed in PCR tubes, 50. Mu.L of lysate was then added and placed in a PCR instrument and heated at 98℃for 10min. After completion of the cleavage, 1. Mu.L of the supernatant was directly taken as an amplification template.
The corresponding primers were selected, and a PCR amplification reaction system was prepared according to Table 1, and the amplified template DNA was 1. Mu.L of the supernatant. After the preparation of the PCR amplification reaction system was completed, 25. Mu.L of the system was used, and amplification was performed according to the PCR reaction conditions recommended in Table 2, thereby obtaining a PCR product required for the experiment. The PCR product is subjected to agarose gel electrophoresis to judge the amplification accuracy.
As shown in FIG. 3, the performance of amplifying 2-3kb fragment by Taq DNA polymerase can be enhanced by adding a small amount of Pfu DNA polymerase to the nanobody modified Taq mut4 polymerase system.
4. PCR method for identifying different target genes in gene editing mice
1. The formula of the kit comprises:
composition of lysate: 100mM Tris-HCl (pH 8.0), 0.5 mM Na 2 EDTA, 3 m/v% protein sink aid.
Composition of PCR premix: 60mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、20 mM NH 4 + 、10 mM K + 0.2v/v% surface Activity0.1M/v% BSA, 0.75M betaine, 12.5. Mu.M dNTP, 5 ng/. Mu.L Taq-SSB protein, 120 ng/. Mu.L Taq mut4 DNA polymerase, 20 ng/. Mu.L Pfu DNA polymerase, 0.2mg/mL nanobody.
PCR method:
2-4 mm mice to be identified were rat tails were placed in PCR tubes, 50. Mu.L of lysate was then added and placed in a PCR instrument and heated at 98℃for 10min. After completion of the cleavage, 1. Mu.L of the supernatant was directly taken as an amplification template.
The corresponding amplification primers were selected, and a PCR amplification reaction system was prepared according to Table 1, and the amplified template DNA was 1. Mu.L of the supernatant. And (3) using a 25 mu L system, after all preparation is completed, amplifying according to the PCR reaction conditions recommended in Table 2, and finally obtaining the PCR product required by the experiment. The PCR product is subjected to agarose gel electrophoresis to judge the amplification accuracy.
The agarose gel electrophoresis result is shown in fig. 4, and the mouse tail treated by the lysate and the PCR premix solution is amplified by PCR, and the corresponding genes are amplified to be consistent with the target sequence strips.
Example 2
Example 2 provides a PCR method for identifying a Ppara knockout mouse.
1. The formula of the kit comprises:
composition of lysate: 80 mM Tris-HCl (pH 8.0), 0.5 mM Na2EDTA, 5 m/v% protein precipitation aid.
Composition of PCR premix: 50mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、20 mM NH 4 + 、5 mM K + 0.5v/v% surfactant, 0.1M/v% BSA, 0.3M betaine, 12.5. Mu.M dNTP, 10 ng/. Mu.L Taq-SSB protein, 150 ng/. Mu.L Taq mut4 DNA polymerase, 5 ng/. Mu.L Pfu DNA polymerase, 0.2mg/mL nanobody.
PCR method:
2-4 mm mice to be identified were rat tails were placed in PCR tubes, 50. Mu.L of lysate was then added and placed in a PCR instrument and heated at 98℃for 10min. After completion of the cleavage, 1. Mu.L of the supernatant was directly taken as an amplification template.
The corresponding amplification primers were selected, and a PCR amplification reaction system was prepared according to Table 1, and the amplified template DNA was 1. Mu.L of the supernatant. And (3) using a 25 mu L system, after all preparation is completed, amplifying according to the PCR reaction conditions recommended in Table 2, and finally obtaining the PCR product required by the experiment. The PCR product is subjected to agarose gel electrophoresis to judge the amplification accuracy.
The agarose gel electrophoresis result is shown in fig. 5, and the mouse tail treated by the lysate and the PCR premix solution is amplified by PCR, and the corresponding genes are amplified to be consistent with the target sequence strips.
Example 3
Example 3 provides a PCR method for identification of Ifih1-KO mice.
1. The formula of the kit comprises:
composition of lysate: 100mM Tris-HCl (pH 8.0), 1 mM Na2EDTA, 4 m/v% protein precipitation aid.
Composition of PCR premix: 100mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、30 mM NH 4 + 、5 mM K + 0.2v/v% surfactant, 0.1M/v% BSA, 0.5M betaine, 12.5. Mu.M dNTP, 12.5 ng/. Mu.L Taq-SSB protein, 90 ng/. Mu.L Taq mut4 DNA polymerase, 5 ng/. Mu.L Pfu DNA polymerase, 0.4mg/mL nanobody.
PCR method:
2-4 mm mice to be identified were rat tails were placed in PCR tubes, 50. Mu.L of lysate was then added and placed in a PCR instrument and heated at 98℃for 10min. After completion of the cleavage, 1. Mu.L of the supernatant was directly taken as an amplification template.
The corresponding primers were selected, and a PCR amplification reaction system was prepared according to Table 1, and the amplified template DNA was 1. Mu.L of the supernatant. And (3) using a 25 mu L system, after all preparation is completed, amplifying according to the PCR reaction conditions recommended in Table 2, and finally obtaining the PCR product required by the experiment. The PCR product is subjected to agarose gel electrophoresis to judge the amplification accuracy.
The agarose gel electrophoresis results are shown in fig. 6, and the mouse tail treated by the lysate and the PCR premix solution is amplified by PCR, and the corresponding genes are amplified to be consistent with the target sequence strips.
Example 4
Example 4 provides a PCR method for amplifying multiple genes of interest in a common mouse.
1. The formula of the kit comprises:
composition of lysate: 50 mM Tris-HCl (pH 8.0), 1 mM Na2EDTA, 5 m/v% protein precipitation aid.
Composition of PCR premix: 100mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、25 mM NH 4 + 、10 mM K + 0.2v/v% surfactant, 0.1M/v% BSA, 0.5M betaine, 12.5. Mu.M dNTP, 20 ng/. Mu.L Taq-SSB protein, 180 ng/. Mu.L Taq mut4 DNA polymerase, 9 ng/. Mu.L Pfu DNA polymerase, 0.8mg/mL nanobody.
PCR method:
2-4 mm mice to be identified were rat tails were placed in PCR tubes, 50. Mu.L of lysate was then added and placed in a PCR instrument and heated at 98℃for 10min. After completion of the cleavage, 1. Mu.L of the supernatant was directly taken as an amplification template.
The corresponding primers 1 to 8 were selected, and a PCR amplification reaction system was prepared according to Table 1, and the amplified template DNA was 1. Mu.L of the supernatant. And (3) using a 25 mu L system, after all preparation is completed, amplifying according to the PCR reaction conditions recommended in Table 2, and finally obtaining the PCR product required by the experiment. The PCR product is subjected to agarose gel electrophoresis to judge the amplification accuracy.
The agarose gel electrophoresis results are shown in FIG. 7, and the mouse tail treated by the lysate and the PCR premix solution is amplified by PCR, and the corresponding genes are amplified to be consistent with the target sequence strips.
Example 5
Example 5 provides a PCR method for identifying different genes of interest in Gene editing mice
1. The formula of the kit comprises:
composition of lysate: 100mM Tris-HCl (pH 8.0), 0.5 mM Na 2 EDTA, 3 m/v% protein sink aid.
Composition of PCR premix: 60mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、20 mM NH 4 + 、10 mM K + 、0.2v/v％Surfactant, 0.1M/v% BSA, 0.75M betaine, 12.5. Mu.M dNTP, 5 ng/. Mu.L Taq-SSB protein, 120 ng/. Mu.L Taq mut4 DNA polymerase, 20 ng/. Mu.L Pfu DNA polymerase, 0.2mg/mL nanobody.
PCR method:
2-4 mm mice to be identified were rat tails were placed in PCR tubes, 50. Mu.L of lysate was then added and placed in a PCR instrument and heated at 98℃for 10min. After completion of the cleavage, 1. Mu.L of the supernatant was directly taken as an amplification template.
The PCR amplification of different target genes in the gene editing mice is carried out by using 16 amplification primers and adopting the kit and the kit of company A (purchased from Beijing full-scale gold organism, with the product number of AD 501), the result of agarose gel electrophoresis is shown in figure 8, and the result shows that the amplification yield and the amplification specificity of the rat tail direct amplification kit scheme are superior to those of company A.
Example 6
Example 6 provides a PCR method for the identification of different genes of interest in a gene editing mouse.
1. The formula of the kit comprises:
composition of lysate: 100mM Tris-HCl (pH 8.0), 0.5mM Na 2 EDTA, 3 m/v% protein sink aid.
Composition of PCR premix: 60mM Tris-HCl (pH 8.8), 2 mM Mg 2+ 、20 mM NH 4 + 、10 mM K + 0.2v/v% surfactant, 0.1M/v% BSA, 0.75M betaine, 12.5. Mu.M dNTP, 5 ng/. Mu.L Taq-SSB protein, 120 ng/. Mu.L Taq mut4 DNA polymerase, 20 ng/. Mu.L Pfu DNA polymerase, 0.2mg/mL nanobody.
PCR method:
2-4 mm mice to be identified were rat tails were placed in PCR tubes, 50. Mu.L of lysate was then added and placed in a PCR instrument and heated at 98℃for 10min. After completion of the cleavage, 1. Mu.L of the supernatant was directly taken as an amplification template.
The results of PCR amplification of different target genes in the gene editing mice using the kit of the present application using 16 amplification primers, respectively, and agarose gel electrophoresis are shown in FIG. 9.
Comparative example 1
Comparative example 1 provides a PCR method for identifying different genes of interest in a gene editing mouse.
The PCR method in comparative example 1 was substantially identical to that of example 6, except that: the lysates used were different, in particular as follows:
composition of lysate A: 50mM Tris-HCl, 0.5mM Na 2 EDTA, 0.25% (w/v) SDS, lysate pH 8.0.
2-4 mm mouse tails to be identified are taken and put into a PCR tube, then 200ul of A lysate and 4ul of proteinase K are mixed and then added into the PCR tube, the mixture is placed into a PCR instrument and heated for 30min at 95 ℃ and 3min at 98 ℃ in sequence, after the lysate is uniformly mixed, the mixture is centrifuged at 12,000 rpm for 5 min, and 1 ul of supernatant is taken as an amplification template.
The results of PCR amplification of different target genes in the gene editing mice using the kit of the present application using 16 amplification primers, respectively, and agarose gel electrophoresis are shown in FIG. 9.
Comparative example 2
Comparative example 2 provides a PCR method for identifying different genes of interest in a gene editing mouse.
The PCR method in comparative example 2 was substantially identical to that of example 6, except that: the lysates used were different, in particular as follows:
composition of lysate B: 50mM NaOH solution.
2-4 mm mouse tail to be identified is taken to a PCR tube, 50 mu L of lysate is added, the mixture is placed in a PCR instrument and heated for 10min at 95 ℃ in sequence, after the lysate is uniformly mixed, the mixture is centrifuged at 12,000 rpm for 11min, and 1 mu L of supernatant is taken as an amplification template.
The results of PCR amplification of different target genes in the gene editing mice using the kit of the present application using 16 amplification primers, respectively, and agarose gel electrophoresis are shown in FIG. 9. The lysate of the application does not need centrifugal operation, the lysis time is only 10min, and the lysate B is basically level, but is obviously superior to the lysate A (38 min) containing proteinase K. The gel image results show that partial bands in the PCR amplification results corresponding to the lysate B cannot be amplified or are obviously weaker, which indicates that some inhibition possibly exists to cause the amplification effect to be poor. The template cracked by the lysate has an unobvious inhibition effect, and the amplification success rate and the amplification yield are optimal. The amplification success rate and the yield are arranged in the order from high to low: example 6 (lysate of this application) > comparative example 1 (A lysate) > comparative example 2 (B lysate).
Conclusion: the lysate has short lysis time and convenient operation, the prepared template has no obvious inhibition on the subsequent PCR amplification, and the amplification effect is superior to that of other two lysates (A/B lysates).
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples only represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.
Claims (10)
1. A PCR kit for detecting the genotype of a mouse, which is characterized by comprising a lysate and a PCR premix;
the lysate comprises Tris-HCl, na 2 EDTA and a protein sedimentation aid, wherein the protein sedimentation aid comprises magnetic bead microspheres with surfaces modified with sodium dodecyl sulfate;
The PCR premix comprises Tris-HCl and Mg 2+ 、NH 4 + 、K + Surfactants, bovine serum albumin, betaine, dntps, single-stranded DNA binding proteins, taq mut4 DNA polymerase, pfu DNA polymerase, and nanobodies.
2. The PCR kit for detecting a genotype of mice as claimed in claim 1, whereinIn the lysate, the concentration of Tris-HCl is 20 mM-100 mM, and the concentration of Na is the same as that of Na 2 The concentration of EDTA is 0.5 mM-1 mM, and the mass percentage of the protein sedimentation assisting agent is 1% -7%.
3. The PCR kit for detecting a mouse genotype as in claim 2, wherein in said lysate, the concentration of Tris-HCl is 20 mM-100 mM and the Na 2 The concentration of EDTA is 0.5 mM-1 mM, and the mass percentage of the protein sedimentation assisting agent is 3% -5%.
4. The PCR kit for detecting a genotype of a mouse of any one of claims 1-3, wherein in the PCR premix solution, the concentration of Tris-HCl is 40 mM-60 mM, and the concentration of Mg is 40 mM-60 mM 2+ The concentration of (C) is 1 mM-3 mM, the NH is 4 + The concentration of K is 10 mM-30 mM + The concentration of the surface active agent is 5 mM-20 mM, the volume percentage content of the surface active agent is 0.2% -0.5%, the mass percentage content of the bovine serum albumin is 0.01% -0.1%, the concentration of the betaine is 0.3-0.8M, the concentration of the dNTP is 10-50 mu M, the concentration of the single-stranded DNA binding protein is 5 ng/. Mu.L-20 ng/. Mu.L, the concentration of the Taq mut4 DNA polymerase is 90 ng/. Mu.L-200 ng/. Mu.L, the concentration of the PfeDNA polymerase is 5 ng/. Mu.L-20 ng/. Mu.L, and the concentration of the nano antibody is 0.2 mg/mL-0.8 mg/mL.
5. A method for detecting a genotype of a mouse, comprising the steps of:
adding a lysate into a sample of the mice to be tested for heating treatment to obtain a DNA sample of the mice to be tested; the lysate comprises Tris-HCl, na 2 EDTA and a protein sedimentation aid, wherein the protein sedimentation aid comprises magnetic bead microspheres with surfaces modified with sodium dodecyl sulfate;
taking the mouse DNA sample to be detected as a template, adding a PCR premix and an amplification primer, and carrying out PCR amplification reaction to obtain an amplification product; the PCR premix comprises Tris-HCl and Mg 2+ 、NH 4 + 、K + Surfactants, bovine serum albumin, betaine, dntps, single-stranded DNA binding proteins, taq mut4 DNA polymerase, pfu DNA polymerase, and nanobodies; and
The amplified products were analyzed to determine the mouse genotype.
6. The method for detecting a genotype of a mouse according to claim 5, wherein the heating treatment satisfies at least one condition of (1) to (2):
(1) The temperature of the heating treatment is 95-100 ℃;
(2) The heating treatment time is 8-12 min.
7. The method according to any one of claims 5 to 6, wherein the reaction system comprises the PCR premix, 9.5. Mu.M to 10.5. Mu.M upstream primer 0.9. Mu.L to 1. Mu.L, 9.5. Mu.M to 10.5. Mu.M downstream primer 0.9. Mu.L to 1. Mu.L and 0.9. Mu.L to 1. Mu.L of the sample of mouse DNA to be tested per 25. Mu.L of the reaction system.
8. The method according to claim 7, wherein the reaction system comprises 12.4. Mu.L to 12.5. Mu.L of the PCR premix, 0.9. Mu.L to 1. Mu.L of the 10. Mu.M upstream primer, 0.9. Mu.L to 1. Mu.L of the 10. Mu.M downstream primer, and 0.9. Mu.L to 1. Mu.L of the DNA of the sample of the mouse to be tested per 25. Mu.L of the reaction system.
9. The method for detecting a mouse genotype according to any one of claims 5-6, wherein the PCR amplification reaction is programmed to: pre-denaturation at 94-96 ℃ for 2-4 min; denaturation at 97-99 ℃ for 8-12 s, annealing at 58-65 ℃ for 18-22 s, annealing at 70-74 ℃ for 18-22 s, and circulation for 30-40 times and extension at 70-74 ℃ for 3 min-5 min.
10. The method for detecting a genotype of a mouse according to any one of claims 5-6, wherein the step of analyzing the amplified product comprises:
and obtaining an electrophoresis band of the amplification product, and comparing the electrophoresis band with a wild type target sequence band to determine the genotype of the mouse.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410023981.5A CN117867130A (en) | 2024-01-05 | 2024-01-05 | PCR kit and method for detecting mouse genotype |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410023981.5A CN117867130A (en) | 2024-01-05 | 2024-01-05 | PCR kit and method for detecting mouse genotype |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117867130A true CN117867130A (en) | 2024-04-12 |
Family
ID=90596520
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410023981.5A Pending CN117867130A (en) | 2024-01-05 | 2024-01-05 | PCR kit and method for detecting mouse genotype |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117867130A (en) |
-
2024
- 2024-01-05 CN CN202410023981.5A patent/CN117867130A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Diegoli et al. | An optimized protocol for forensic application of the PreCR™ Repair Mix to multiplex STR amplification of UV-damaged DNA | |
| Gallup et al. | Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system ‘FocusField2-6GallupqPCRSet-upTool-001’to attain consistently high fidelity qPCR reactions | |
| US20080166771A1 (en) | Method and system for detection of nucleic acids | |
| CA2102784A1 (en) | Identifying, isolating and cloning messenger rnas | |
| US20070072229A1 (en) | Method for isolation, amplification and quantitation of ribonucleic acid | |
| WO2011023131A1 (en) | Composite amplification kits of 20 short tandem repeats | |
| CN114921524A (en) | Method for detecting and neutralizing sample nucleic acid | |
| CN107475252B (en) | Nucleic acid releasing agent, method for rapidly releasing nucleic acid and application thereof | |
| JP2001029078A (en) | Amplification of rna | |
| Kubo et al. | Improved reverse transcription-recombinase polymerase amplification assay for blood mRNA screening: comparison with one-step RT-qPCR assay | |
| EP3810805A1 (en) | Method for detection and quantification of genetic alterations | |
| CN117867130A (en) | PCR kit and method for detecting mouse genotype | |
| WO2020118543A1 (en) | Method for separating and/or enriching host source nucleic acid and pathogenic nucleic acid, and reagent and preparation method therefor | |
| Connelly et al. | PCR amplification of a triple-repeat genetic target directly from whole blood in 15 minutes as a proof-of-principle PCR study for direct sample analysis for a clinically relevant target | |
| CN113564157B (en) | PCR reaction system, kit and PCR method | |
| CN113862394B (en) | RPA detection method for tomato infertility virus | |
| Mandal et al. | Development of a membrane-based method for isolation of genomic DNA from human blood | |
| CN110305947B (en) | Detection method for chromosome long fragment insertion and long fragment insertion detection method based on MassARRAY platform | |
| CN106701949A (en) | Gene mutation detection method for reducing amplification bias and reagent | |
| CN113621607A (en) | Lysis solution and application thereof | |
| CN107904297B (en) | Primer group, joint group and sequencing method for microbial diversity research | |
| CN112063701A (en) | Nucleic acid composition for detecting KRAS gene mutation, kit and detection method thereof | |
| CN106222164B (en) | Methods, compositions and kits for unidirectional amplification of nucleic acids in vitro using transposase | |
| US10144951B2 (en) | Method and materials to deplete impurities for extraction and purification of nucleic acids from stool | |
| JP2001008685A (en) | Method for synthesizing nucleic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |