CN114032314A - Method for detecting river-type buffalo beta-casein genotype based on SNP technology - Google Patents
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Abstract
The invention relates to a method for breeding a good river type buffalo variety by typing a beta-casein gene based on detection of SNP polymorphism. The beta-casein gene has polymorphism in different cattle, different genotypes and obvious difference in milk yield, milk quality and period. The method comprises the following steps: extracting sample DNA, detecting DNA quality, designing and synthesizing parting beta-casein gene primer, preparing KASP liquid to carry out KASP reaction, detecting fluorescent signal by PCR reaction and parting gene. The method realizes the screening of four beta-casein genotypes of the river buffalo, and provides a convenient technical method for exploring special germplasm resources and excellent buffalo varieties of dairy cows.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of biology, and particularly relates to a method for typing and breeding excellent river-type buffalo by fluorescent PCR amplification and identification of target gene SNP.
[ background of the invention ]
Milk proteins are also considered to be one of the most important sources of bioactive peptides in milk, and peptides with potent biological activity may be released from milk proteins by the action of proteolytic enzymes in the gut, thereby affecting major body systems including endocrine, neurological, digestive, cardiovascular and immune systems. More than 50 years ago, the gene polymorphism of milk protein is found for the first time by Aschaffernburg and Drewry, and the gene polymorphism of milk protein is found out by the Aschaffernburg and the Drewry, and the two genotypes and different genotypes are found out by the Aschaffernburg and the Drewry and are obviously different in milk yield, milk quality and period. Since then, more and more researchers began to study the polymorphism of milk protein gene, and in recent years, the research on the polymorphism of milk protein gene has been rapidly developed due to the application of new detection methods, and up to 9 kinds of allosteric forms have been found, and river buffalo is mainly of four milk protein gene types (A, B, C, D). Therefore, the superior milk source variety can be screened by screening the dominant milk protein genotype.
The current genotype sorting method of milk protein mainly comprises the following steps:
high Performance Liquid Chromatography (HPLC), immunoblotting, capillary electrophoresis, dimensional electrophoresis, and isopoint focusing.
Polymerase chain reaction single-strand conformation polymorphism of DNA level, restriction fragment length polymorphism polymerase chain reaction, microsatellite, pyrosequencing and other technologies.
The above method is time consuming, laborious and costly.
The sensitivity and stability of the protein level method are inferior to those of the DNA level, and the method for typing the SNP by adopting the KASP at the DNA level realizes the typing of the beta-casein gene with high flux, high accuracy and low cost.
[ summary of the invention ]
The invention aims to: aiming at the problems, the method for typing the SNP by adopting the KASP at the DNA level realizes the genotyping of the milk protein with high flux, high accuracy and low cost.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for detecting river buffalo beta-casein genotype based on SNP technology comprises the following steps:
step S1: extracting sample DNA;
step S2: electrophoresis of DNA to detect the quality of DNA;
step S3: preparing a PCR reaction system, wherein the system comprises a DNA sample with volume fraction of more than 20%, mixed primers with volume fraction of 1.4% and the balance of KASP mixed solution;
step S4: performing PCR amplification;
wherein, the mixed primers added in the step S3 include 6 KASP primers, which are three types of β -casein genotype SNP1 and three types of SNP4, respectively, that is, SNP1 has one non-mutated genotype and two mutated genotypes, and SNP4 has one non-mutated genotype and two mutated genotypes; one of the mutant gene primer and the non-mutant gene primer is provided with a HEX fluorescent label, and the other is provided with a FAM fluorescent label, and after PCR amplification, the fluorescence intensity of a DNA sample is detected, so that genotyping is carried out.
Further, in step S2, the mixed primers added include four groups for detecting the base types of four mutation sites, and the primers are designed for different mutation sites as follows:
(1) when the base type on the beta-casein-80 site is to be detected, the primer sequence is as follows:
SNP1-Primerl：
GAAGGTGACCAAGTTCATGCTTTGCAGAGCTCAGAAGCGC；
SNP1-Pr imer2：
GAAGGTCGGAGTCAACGGATTTTGCAGAGCTCAGAAGCGT；
SNP1-Pr imerCommon：
AGGCCCAGGGCAAGCAGGAG；
(2) when the base type on the beta-casein-55 site is to be detected, the primer sequence is as follows:
SNP4-Primerl：
GAAGGTGACCAAGTTCATGCTAGGCCCAGGGCAAGCAGGAGG；
SNP4-Pr imer2：
GAAGGTCGGAGTCAACGGATTAGGCCCAGGGCAAGCAGGAGA；
SNP4-Pr imerCommon：
TTGCAGAGCTCAGAAGCG。
further explaining that (1) the base type on the beta-casein-80 site is detected, and if the amplified DNA only has the corresponding FAM fluorescence intensity, no mutation exists in two chains on the site; if only the corresponding HEX fluorescence intensity appears after amplification, then both strands on the site mutate; if two mixed fluorescence intensities appear after amplification, one of the two strands at the site is mutated, and the other strand is not mutated;
(2) when the base type on the beta-casein-55 site is to be detected, if the amplified DNA only has the corresponding FAM fluorescence intensity, no mutation exists in two chains on the site; if only the corresponding HEX fluorescence intensity appears after amplification, then both strands on the site mutate; if two mixed fluorescence intensities appear after amplification, one of two strands at the site is mutated, and the other strand is not mutated.
Further, in step S4, the PCR amplification step includes:
step S41: pre-denaturation at 94 ℃ for 15 minutes for 1 cycle;
step S42: denaturation at 94 ℃ for 20 seconds; extension, at 61-55 ℃ for 60 seconds, and performing 10 cycles;
step S43: denaturation at 94 ℃ for 20 seconds, annealing at 55 ℃ and extension for 60 seconds, 26 cycles.
Further, in step S1, the method for extracting sample DNA (phenol chloroform extraction method) includes:
step S11: pre-treating the tissue or blood material;
step S12: the tissue needs to be ground, and the blood is firstly digested by a Porteinase K solution and a buffer solution GB, so that a solution system is clarified;
step S13: adding Tris saturated phenol and shaking for 10 min;
step S14: centrifuging at 12000R for 7min at 4 ℃, wherein the supernatant is DNA;
step S15: preparing Tris saturated phenol: chloroform: isoamyl alcohol solution (25: 24: 1);
step S16: adding phenol chloroform isoamyl alcohol solution into a test tube containing the supernatant, centrifuging, taking the supernatant, adding-20 frozen alcohol, and placing in a-20 refrigerator for overnight;
step S17: centrifuging at 12000R for 7min at 4 ℃;
step S18: discarding the supernatant, and obtaining DNA by white precipitation;
step S19: the integrity of the DNA sample was checked by agarose gel electrophoresis and the purity of the DNA was checked by absorbance.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
the invention adopts the DNA-level KASP to carry out SNP genotyping to realize genotyping of river buffalo milk protein with high flux, high accuracy and low cost. Resources and technical advantages in the fields of gene detection and the like can be fully utilized to quickly screen and cultivate high-quality river type buffalo, the special germplasm resources of the cows are explored, the appropriate cows are selected for a pasture, and the milk quality is improved.
[ description of the drawings ]
FIG. 1 is a diagram showing the typing results of SNP 1.
FIG. 2 is a diagram showing the typing results of SNP 4.
FIG. 3 shows the results of the genotyping of milk proteins judged comprehensively from the results of SNP1+ SNP 4.
[ detailed description ] embodiments
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures 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 invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Example (b):
firstly, preparing a DNA sample:
the blood of buffalo is used as the material for extracting and preparing DNA sample.
1. Pretreatment of blood materials
Step S1: pre-treating the tissue or blood material;
step S2: the tissue needs to be ground, and the blood is firstly digested by a Porteinase K solution and a buffer solution GB, so that a solution system is clarified.
2. Phenol chloroform method for extracting DNA
Step S1: adding Tris saturated phenol and shaking for 10 min;
step S2: centrifuging at 12000R for 7min at 4 ℃, wherein the supernatant is DNA;
step S3: preparing Tris saturated phenol: chloroform: isoamyl alcohol solution (25: 24: 1);
step S4: adding phenol chloroform isoamyl alcohol solution into a test tube containing the supernatant, centrifuging, taking the supernatant, adding-20 frozen alcohol, and placing in a-20 refrigerator for overnight;
step S5: centrifuging at 12000R for 7min at 4 ℃;
step S6: discarding the supernatant, and obtaining DNA by white precipitation;
3. electrophoresis detection of DNA quality
The integrity of the DNA sample was checked by agarose gel electrophoresis and the purity of the DNA was checked by absorbance.
Second, design of primers
The invention relates to 2 mutation points on river buffalo beta-casein.
1. Primers designed for different mutation points
SNP1-Primerl：
GAAGGTGACCAAGTTCATGCTTTGCAGAGCTCAGAAGCGCSNP1-Pr imer2：
GAAGGTCGAGTCAACGGATTTTGCAGAGCTCAGAAGCGTSNP1-Pr imerCommon：
AGGCCCAGGGCAAGCAGGAG。
SNP4-Primerl:：
GAAGGTGACCAAGTTCATGCTAGGCCCAGGCAAGCAGGAGGSNP4-Primer2：
GAAGGTCGAGTCAACGGATTAGGCCCAGGGCAAGCAGGAGASNP4-PrimerCommon：
TTGCAGAGCTCAGAAGCG。
Table 1: the base types corresponding to different mutation sites determined by the invention
| Genotype(s) | Site SNP1 | Site SNP4 |
| A | C | C |
| B | C | T |
| C | T | C |
| D | T | T |
Second, PCR amplification
1) DNA samples were applied to 96-well or 384-well PCR plates, 2 and so on for each PCR plate
The above negative control (NTC). The KASP reaction using 8-tube or single tube was avoided as much as possible, and the number of DNA samples was 20 or more as much as possible.
2) Preparing KASP gene typing mixed solution
| Reagent composition | Volume of |
| DNA | 2 |
| 2×KASP Master Mix | 5 |
| Primer Mix | 0.14 |
| Water (W) | 2.86 |
| Total reaction volume | 10 |
3) Adding the KASP genotyping mix to a PCR plate containing DNA template and using a pipette or a microtube
And (4) a quantitative liquid-splitting instrument, namely adding the required gene-splitting mixed liquid into the PCR plate.
4) The reaction plate was sealed and the fluoroscopically transparent membrane was centrifuged to seal the PCR plate, which was then centrifuged.
5) PCR cycling reactions and fluorescence readings were performed.
In the present invention, the total volume of the PCR system for amplification is 10. mu.L, and the PCR system comprises 2. mu.L of DNA, 5. mu.L of KASP Master Mix (LGC Genomics, Hoddeson, UK, KASP mixture), 0.14. mu.L of mixed primers, and the balance of water. Blank control, DNA template for ddH2O (double distilled water). Thermally activating PCR amplification conditions at 94 deg.C for 15min, denaturing at 94 deg.C for 20s, annealing at 61-55 deg.C and extending for 60s, and performing 10 cycles; denaturation at 94 ℃ for 20s, annealing and extension at 55 ℃ for 60s, and 26 cycles.
Fourth, typing results
As described above, the primers of the present invention are designed such that each PCR amplification primer set includes three amplification bands: 6 KASP primers which are respectively three types of beta-casein genotype SNP1 and three types of SNP4, namely SNP1 has one type of non-mutated genotype and two types of mutated genotypes, and SNP4 has one type of non-mutated genotype and two types of mutated genotypes; one of the mutant gene primer and the non-mutant gene primer is provided with a HEX fluorescent label, and the other is provided with a FAM fluorescent label, after PCR amplification, the fluorescence intensity of the DNA sample is detected, so that genotyping is carried out, because different DNA samples show different fluorescence intensities after PCR amplification.
Taking the β -casein _80 mutation point as an example, please refer to fig. 1, assuming that a cow is not mutated at this point (at this time, the genotype of the cow may be AA), the base type at this position is C, and only an amplification band corresponding to the mutated gene primer appears after DNA amplification, that is, only fluorescence intensity corresponding to FAM appears, that is, the color (red) at the bottom right corner of fig. 1; assuming that a cow has a chain mutation at this position (at this time, the genotype of the cow may be AD or BB), the base type of the chain at this position is C, and the other chain is T, and after DNA amplification, amplification bands corresponding to a mutated gene primer and an unmutated gene primer will appear at the same time, that is, the finally amplified DNA shows a mixed color of fluorescent intensity corresponding to HEX and fluorescent intensity corresponding to FAM, that is, the fluorescent color in the middle of fig. 1; assuming that a cow has mutations in both strands at this position (at this time, the genotype of the cow is DD), the base type at this position is T, and only an amplification band corresponding to the upstream mutant gene primer appears after DNA amplification, that is, the finally amplified DNA shows a color corresponding to the fluorescence intensity of HEX, that is, the color of the upper left corner of fig. 1 (dark blue).
And then, the genotype of the river-type buffalo beta-casein can be comprehensively judged by combining the amplification results of other mutation points. Beta-casein is common to three subtypes, AA, AB and BB, with BB accounting for 64.38% of the total most common. The protein content of the BB type buffalo milk is obviously higher than that of the AA subtype, and the fat content of the AA subtype buffalo milk is obviously higher than that of the BB subtype. The AB subtype has the fastest digestion performance, the AA subtype exists secondly, and the BB subtype is the most difficult to digest. The DPPH removing capability of the AA subtype is superior to that of the BB subtype in the aspect of oxidation resistance; the BB subtype has better OH free radical scavenging capability than the AA subtype; the BB subtype has a better reducibility than the AA subtype. Therefore, the buffalo with high-quality milk source can be bred according to the genotype of the beta-casein.
In conclusion, the method for typing the SNP by adopting the KASP at the DNA level realizes the genotyping of the milk protein with high flux, high accuracy and low cost. Can make full use of the resource and technical advantages in the fields of gene detection and the like to quickly screen and culture high-quality river buffalo.
The above examples merely represent some embodiments of the present invention and are described in more detail and detail but are not to be construed as limiting the scope of the invention. It should be noted that it is within the scope of the present invention for a person skilled in the art to make several variations and modifications without departing from the spirit of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Sequence listing
<110> research institute of Calf in autonomous region of Guangxi Zhuang nationality
<120> method for detecting river buffalo beta-casein genotype based on SNP technology
<160> 6
<170> SIPOSequenceListing 1.0
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<213> Artificial sequence (rengongxulie)
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gaaggtgacc aagttcatgc tttgcagagc tcagaagcgc 40
<210> 2
<211> 40
<212> DNA
<213> Artificial sequence (rengongxulie)
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gaaggtcgga gtcaacggat tttgcagagc tcagaagcgt 40
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gaaggtgacc aagttcatgc taggcccagg gcaagcagga gg 42
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gaaggtcgga gtcaacggat taggcccagg gcaagcagga ga 42
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Claims (5)
1. The method for detecting the river buffalo beta-casein genotype based on the SNP technology is from KASP in principle and mainly comprises the following steps:
step S1: extracting sample DNA;
step S2: electrophoresis of DNA to detect the quality of DNA;
step S3: preparing a PCR reaction system, wherein the system comprises a DNA sample with volume fraction of more than 20%, mixed primers with volume fraction of 1.4% and the balance of KASP mixed solution;
step S4: performing PCR amplification;
wherein, the mixed primers added in the step S3 include 6 KASP primers, which are three types of β -casein genotype SNP1 and three types of SNP4, respectively, that is, SNP1 has one non-mutated genotype and two mutated genotypes, and SNP4 has one non-mutated genotype and two mutated genotypes; one of the mutant gene primer and the non-mutant gene primer is provided with a HEX fluorescent label, and the other is provided with a FAM fluorescent label, and after PCR amplification, the fluorescence intensity of a DNA sample is detected, so that genotyping is carried out.
2. The method for detecting the beta-casein genotype of the river buffalo according to the SNP technology as claimed in claim 1, wherein in the step S3, the mixed primers are added to detect the base types of four mutation sites, and the primers are designed for different mutation sites as follows:
(1) when the base type on the beta-casein-80 site is to be detected, the primer sequence is as follows:
SNP1-Primerl：
gaaggtgaccaagttcatgctttgcagagctcagaagcgc；
SNP1-Primer2：
gaaggtcggagtcaacggattttgcagagctcagaagcgt；
SNP1-PrimerCommon：
aggcccagggcaagcaggag；
(2) when the base type on the beta-casein-55 site is to be detected, the primer sequence is as follows:
SNP4-Primerl：
gaaggtgaccaagttcatgctaggcccagggcaagcaggagg；
SNP4-Primer2：
gaaggtcggagtcaacggattaggcccagggcaagcaggaga；
SNP4-PrimerCommon：
ttgcagagctcagaagcg。
3. the method for detecting the beta-casein genotype of the river buffalo based on the SNP technology as claimed in claim 2 is mainly characterized in that:
(1) when the base type on the beta-casein-80 site is to be detected, if the amplified DNA only has the corresponding FAM fluorescence intensity, no mutation exists in two chains on the site; if only the corresponding HEX fluorescence intensity appears after amplification, then both strands on the site mutate; if two mixed fluorescence intensities appear after amplification, one of the two strands at the site is mutated, and the other strand is not mutated;
(2) when the base type on the beta-casein-55 site is to be detected, if the amplified DNA only has the corresponding FAM fluorescence intensity, no mutation exists in two chains on the site; if only the corresponding HEX fluorescence intensity appears after amplification, then both strands on the site mutate; if two mixed fluorescence intensities appear after amplification, one of two strands at the site is mutated, and the other strand is not mutated.
4. The method for detecting beta-casein genotype of river buffalo based on SNP technology according to claim 1, wherein in the step S4, the step of PCR amplification comprises:
step S41: pre-denaturation at 94 ℃ for 15 minutes for 1 cycle;
step S42: denaturation at 94 ℃ for 20 seconds; extension, at 61-55 ℃ for 60 seconds, and performing 10 cycles;
step S43: denaturation at 94 ℃ for 20 seconds, annealing at 55 ℃ and extension for 60 seconds, 26 cycles.
5. The method for detecting beta-casein genotype of river buffalo based on SNP technology as claimed in claim 1, wherein in the main step S1, the extraction method of sample DNA comprises:
step S11: pre-treating the tissue or blood material;
step S12: the tissue needs to be ground, and the blood is firstly digested by a Porteinase K solution and a buffer solution GB, so that a solution system is clarified;
step S13: adding Tris saturated phenol and shaking for 10 min;
step S14: centrifuging at 12000R for 7min at 4 ℃, wherein the supernatant is DNA;
step S15: preparing Tris saturated phenol: chloroform: isoamyl alcohol solution, phenol: chloroform: the volume ratio of isoamyl alcohol is 25: 24: 1;
step S16: adding phenol chloroform isoamyl alcohol solution into a test tube containing the supernatant, centrifuging, taking the supernatant, adding-20 frozen alcohol, and placing in a-20 refrigerator for overnight;
step S17: centrifuging at 12000R for 7min at 4 ℃;
step S18: discarding the supernatant, and obtaining DNA by white precipitation;
step S19: the integrity of the DNA sample was checked by agarose gel electrophoresis and the purity of the DNA was checked by absorbance.
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| WO2007107862A2 (en) * | 2006-03-22 | 2007-09-27 | Universita' Degli Studi Di Milano | Method for the identification of bovine milk protein genetic polymorphisms |
| CN105647911A (en) * | 2016-04-08 | 2016-06-08 | 甘肃农业大学 | Method for rapidly and efficiently extracting genomic DNA of mammal ear tissue |
| CN109055566A (en) * | 2018-07-11 | 2018-12-21 | 北京奶牛中心 | The detection method and application thereof of cow group HH type genetic defect gene |
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2021
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007107862A2 (en) * | 2006-03-22 | 2007-09-27 | Universita' Degli Studi Di Milano | Method for the identification of bovine milk protein genetic polymorphisms |
| CN105647911A (en) * | 2016-04-08 | 2016-06-08 | 甘肃农业大学 | Method for rapidly and efficiently extracting genomic DNA of mammal ear tissue |
| CN109055566A (en) * | 2018-07-11 | 2018-12-21 | 北京奶牛中心 | The detection method and application thereof of cow group HH type genetic defect gene |
Non-Patent Citations (2)
| Title |
|---|
| VIKAS VOHRA等: "Genetic variants of beta-lactoglobulin gene and its association with milk composition traits in riverine buffalo", JOURNAL OF DAIRY RESEARCH, vol. 73, pages 1 * |
| 刘亚楠;任大喜;刘建新;李玲;曾庆坤;: "中国水牛乳蛋白基因多态性与乳蛋白组成的关联研究", 中国畜牧杂志, no. 05, pages 8 - 12 * |
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