CN109517910B - Method for detecting single nucleotide polymorphism of Zaishen cattle CAPN1 gene based on DNA mixed pool - Google Patents
Method for detecting single nucleotide polymorphism of Zaishen cattle CAPN1 gene based on DNA mixed pool Download PDFInfo
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Abstract
The invention discloses a method for detecting single nucleotide polymorphism of a Zaosheng cattle CAPN1 gene based on a DNA mixed pool, which comprises the following steps: (1) taking the whole genome DNA of the early-win cattle to be detected containing the CAPN1 gene as a template, taking a primer pair P as a primer to carry out PCR specific amplification, wherein the amplification area is exon5-exon6, namely 3594-4094 bp, and an upstream primer P1: 5'-GCTGTGGCAGTTTGGTGAGTGG-3', downstream primer P2: 5'-TGAGGCAAAGGACAGGGT-3', respectively; (2) constructing a DNA mixing pool: adopting an orthogonal test, establishing DNA mixing pools by taking the number of DNA mixing samples and the final concentration of the mixed DNA as a combination factor, simultaneously carrying out 3 times of PCR amplification by taking each mixing pool as a single template, and carrying out sequencing analysis on an amplification product; the number of DNA mixed samples for optimal screening was 150, and the final concentration of DNA was 100 ng/. mu.L. The invention provides a method for detecting single nucleotide polymorphism of the CAPN1 gene of the early-win cattle by designing a specific primer and analyzing and researching the influence of a DNA mixing pool on PCR amplification quality and detection rate of SNPs, and provides a basis for carrying out rapid screening of the SNPs sites of the trait associated gene for the early-win cattle in the follow-up process.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a method for detecting single nucleotide polymorphism of a Zasheng cattle CAPN1 gene based on a DNA mixed pool.
Background
With the development of molecular biology technology, the technologies such as RAPD, RFLP, SSR, PCR-SSCP and the like are widely applied to the field of main economic character marking and development of livestock and poultry, but the technologies need large sample screening in the application process, and have large workload and higher cost. The DNA mixing pool is used as a rapid and efficient detection technology, is suitable for genotype typing, mutation detection, linkage analysis, gene positioning and the like, different pool building methods have certain influence on the detection accuracy of SNPs, and the reaction conditions in detection also directly influence the test effect.
The early-success cattle, as a special livestock breeding resource in the Longdong region of Gansu province, has been improved for years, better inherits the advantages of rapid growth and development, good fat deposition and tender meat quality of Qinchuan cattle, and has great potential for meat development. CAPN1 is one of calpain gene family components, is an important candidate gene influencing meat quality, and is closely related to processes such as beef tenderization and fat deposition. The PCR amplification quality and the SNPs detection rate of the exon5-exon6 region of the Zaishen cattle CAPN1 gene are closely related to the DNA mixing pool, but no related report about the relationship between the DNA mixing pool and the amplification quality and the SNPs detection rate of the Zaishen cattle CAPN1 gene is found at present, so that a suitable DNA mixing pool building method is researched and screened, and a reference can be provided for further carrying out rapid screening and genetic diversity research of the trait associated gene polymorphic sites for the Zaishen cattle.
Disclosure of Invention
The invention aims to provide a method for detecting single nucleotide polymorphism of the CAPN1 gene of the Zaishen cattle based on a DNA mixing pool, and provides reference for further carrying out rapid screening and genetic diversity research on polymorphic sites of trait associated genes for the Zaishen cattle.
The technical scheme of the invention is that a method for detecting single nucleotide polymorphism of CAPN1 gene of Zaishen cattle based on a DNA mixed pool comprises the following steps:
(1) the method comprises the steps of carrying out PCR amplification on an early-wining cattle CAPN1 gene by using a to-be-detected early-wining cattle whole genome DNA containing a CAPN1 gene as a template and a primer pair P as primers, wherein the amplification area is exon5-exon6, namely 3594-4094 bp, the size of a product is 500bp, and the sequence of the primer pair P is as follows:
upstream primer P1: 5'-GCTGTGGCAGTTTGGTGAGTGG-3', and the adhesive tape is used for adhering the film to a substrate,
the downstream primer P2: 5'-TGAGGCAAAGGACAGGGT-3', respectively;
(2) constructing a DNA mixing pool: adopting 2 factor Xn horizontal design orthogonal test, respectively using the quantity of DNA mixed samples and the final concentration of the mixed DNA as A factor and B factor, wherein the A factor is designed by increasing the quantity of the DNA mixed samples, the A1, A2 and A3 … An factors are designed in the A factor, n is the number of the test level, the A2 factor contains An A1 factor individual, the A3 factor contains An A1 and A2 factor individual, the An factor contains An A1, A2 and A3 … An-1 factor individual, a DNA mixed pool is established by using the A factor and the B factor as combined factors, 3 times of PCR amplification are simultaneously carried out by using each DNA mixed pool as a single template, and the amplified products are subjected to sequencing analysis; detecting DNA purity by agarose gel electrophoresis, and detecting OD by spectrophotometer260/OD280The value is obtained.
Preferably, the DNA mixing pool is 150 DNA mixing samples, and the final concentration of DNA is 100 ng/. mu.L.
Preferably, the reaction system for PCR amplification is: the total volume is 25 μ L, wherein the 2 XTaq Master Mix mixture is 12 μ L, the concentrations of the upstream primer and the downstream primer are 10 pmol/. mu.L and 1 μ L respectively, the concentration of the DNA template is 100 ng/. mu.L and 1 μ L, ddH2O10 mu L; reaction conditions are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 50s, annealing at 65 deg.C for 35s, extension at 72 deg.C for 35s, 30 cycles, extension at 72 deg.C for 8min, and storage at 4 deg.C.
The invention further provides application of the method for constructing the DNA mixing pool in rapidly screening the SNPs sites of the trait associated genes for the early-win beef.
Compared with the defects and shortcomings of the prior art, the invention has the following beneficial effects: the invention adopts 2 factor multiplied by 3 horizontal test design, takes the quantity of DNA mixed samples and the final concentration of DNA as combined factors, and analyzes and compares the influence of different mixing pools on the PCR amplification quality and the detection rate of SNPs. Obtains a DNA mixing pool with the best PCR amplification quality and the best SNPs detection rate, and provides reference for further carrying out the rapid screening of the trait associated gene polymorphic sites for the early-surpassing beef and the research of genetic diversity.
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FIG. 1 shows PCR products amplified by different DNA mixing pools provided in the embodiments of the present invention; in the figure, M: DL 2000 molecular weight standard; 1-3: respectively, A1B3, A2B3 and A3B3 amplification PCR products; 4-6: respectively, A1B2, A2B2 and A3B2 amplification PCR products; 7-9: the PCR products were A1B1, A2B1, and A3B1, respectively.
FIG. 2 is a diagram showing the sequencing result of the polymorphism of the DNA mixed pool amplified sequence under factor A3 provided by the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
1. Materials and methods
1.1 blood sample Collection
150 samples of the morning-winy cattle blood were collected from the company of Fuchun animal husbandry, Ning county, Qingyang city. Blood is collected from the jugular vein of each cow by 6mL, and the cow is stored at the temperature of 20 ℃ below zero for later use after citric acid glucose is anticoagulated.
1.2 Primary reagents
The D3392-01 type blood DNA extraction kit was purchased from omega Biotek, 2 XTAQ Master Mix, DL 2000 Marker was purchased from Nanjing Novozam Biotech, Inc.
1.3 primer design and PCR amplification
According to the nucleotide sequence of common cattle CAPN1 gene (Genbank ID: AH 009246), specific primers were designed by using Prime 5.0 software and synthesized by Beijing Yihuiyuan Biotech, Inc. The sequence of primer pair P is:
upstream primer P1: 5'-GCTGTGGCAGTTTGGTGAGTGG-3' the flow of the air in the air conditioner,
the downstream primer P2: 5'-TGAGGCAAAGGACAGGGT-3';
the method comprises the steps of carrying out PCR amplification on the early-wining cattle CAPN1 gene by taking the whole genome DNA of the to-be-detected early-wining cattle containing the CAPN1 gene as a template and the primer pair P as primers, wherein the amplification region is exon5-exon6, namely 3594-4094 bp, and the size of a product is 500 bp.
The reaction system of PCR amplification is as follows: the total volume is 25 μ L, wherein the 2 XTaq Master Mix mixture is 12 μ L, the concentrations of the upstream primer and the downstream primer are 10 pmol/. mu.L and 1 μ L respectively, the concentration of the DNA template is 100 ng/. mu.L and 1 μ L, ddH2O10 mu L; reaction conditions are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 50s, annealing at 65 deg.C for 35s, extension at 72 deg.C for 35s, 30 cycles, extension at 72 deg.C for 8min, and storage at 4 deg.C. Detecting DNA purity by agarose gel electrophoresis, and detecting OD by spectrophotometer260/OD280The value is obtained.
1.4 construction of DNA mixing pool and sequencing analysis
The number of DNA mixed samples and the final concentration of the mixed DNA are 2 factors, 3 DNA mixed samples are designed, 2 factor x 3 horizontal orthogonal test design is adopted, 9 DNA mixed pools are respectively established, as shown in table 1, in order to improve the reliability of test data and reduce statistical errors, 3 repetitions are arranged in each DNA mixed pool, each repeated DNA mixed pool is used as a single template to simultaneously carry out 3 times of PCR amplification, and 3 times of PCR amplification products are used as evaluation basis of PCR amplification effect.
TABLE 1 different DNA mixing tanks
In table 1, the number of DNA mixed samples was designated as a factor a, the final concentration of DNA after mixing was designated as B factor, the a factors were a1 (20), a2 (60), and A3 (150), respectively, and the a2 factor contained individuals with a1 factor, and the A3 factor contained individuals with a1 and a2 factor. And 5 mu L of the qualified PCR product is taken and sent to Beijing Tianyihui-Chiyowa Biotechnology Co., Ltd for sequencing, and DNAMAN 7.0 software is used for comparing and analyzing the sequence variation condition.
2. Results and analysis
2.1 Effect of different DNA mixing pools on PCR amplification quality
As shown in FIG. 1, the results of PCR amplification products were detected to show that, at the level of factor B1 (final DNA concentration 50 ng/. mu.L), the PCR amplification products A1B1, A2B1 and A3B1 showed bright bands and OD260/OD280The values are 1.6, 1.6 and 1.7 in sequence; in thatAt the level of B2 factor (final DNA concentration 100 ng/. mu.L), the bands of A1B2, A2B2 and A3B2 are clear, the sizes of the bands are consistent with the size of the target fragment, and the OD is260/OD280Values of 1.9, 2.0, 1.9, respectively; at the level of factor B3 (final DNA concentration 150 ng/. mu.L), nonspecific fragments of A1B3, A2B3 and A3B3 appeared.
2.2 influence of different DNA mixing pools on the detection Rate of SNPs
Directly sequencing PCR products amplified by different DNA mixing pools, wherein sequencing results show that single base mutation of C-T is detected at 4085bp positions of A1B1 and A1B2 under the level of A1 factors (20 DNA mixing samples); at the level of A2 factor (60 DNA mixed samples), the single base mutation of C-G, T-C occurs at 4091bp and 4093bp for A2B1 and A2B 2; the direct sequencing results of the PCR products amplified by the DNA mixing pool of A3B1 and A3B2 are shown in FIG. 2, wherein FIG. 2A is a nucleotide sequence diagram of CSPN1 gene, and FIG. 2B is a sequence diagram of the PCR products amplified by the DNA mixing pool under A3 factor. As can be seen from FIG. 2, at the level of A3 factor (150 DNA mixed samples), both A3B1 and A3B2 generated single-base mutation of C-T, G-T, C-G, T-C, C-T at 4085bp, 4090bp, 4091bp, 4093bp and 4094 bp. The PCR products amplified by A1B3, A2B3 and A3B3 have more bands and do not meet the test requirements.
2.3 conclusion
DNA mixing pool established at final concentration of 100 ng/. mu.L under the same DNA mixing sample quantity, amplified PCR product band purity and OD260/OD280The values are all better than the final concentration of 50 ng/. mu.L, but have no significant influence on the sequencing result; the DNA mixing pool established by the sample concentration of 150 ng/. mu.L has the advantages that the mismatching probability in the PCR amplification reaction is increased due to the overlarge template DNA concentration, so that non-specific fragments appear, the test requirements are not met, and sequencing analysis is not carried out. The DNA mixing pool has low dependence on the sample mixing quantity in a proper sample final concentration range, and the DNA mixing pool is as small as dozens or hundreds of DNA mixing pools. Under the same DNA mixed sample final concentration, 1 single base mutation is detected in the exon5-exon6 region of CAPN1 gene by using a DNA mixed pool established by 20 samples; 2 and 5 single base mutations are detected in DNA mixing pools established by 60 and 150 samples respectively; the more the number of the samples in the mixing pool is, the more accurate the mutant samples can be screenedThe higher the detection rate of SNPs. In conclusion, from the analysis of PCR amplification quality, SNPs detection rate and experiment cost, under the present experiment condition, the DNA mixing pool established by combining 150 mixed samples and 100 ng/. mu.L final concentration is the best.
By applying the DNA mixing pool method established by the research, the SNPs sites of the trait associated genes affecting the early-surpassing beef can be quickly screened, so that the dominant genotype is determined, and the method has important significance for early selection and reservation of excellent individuals for seed selection and matching.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (3)
1. A method for detecting single nucleotide polymorphism of CAPN1 gene of Zaishen cattle based on a DNA mixed pool is characterized by comprising the following steps:
(1) the method comprises the steps of carrying out PCR amplification on an early-wining cattle CAPN1 gene by using a to-be-detected early-wining cattle whole genome DNA containing a CAPN1 gene as a template and a primer pair P as primers, wherein the amplification area is exon5-exon6, namely 3594-4094 bp, the size of a product is 500bp, and the sequence of the primer pair P is as follows:
upstream primer P1: 5'-GCTGTGGCAGTTTGGTGAGTGG-3' the flow of the air in the air conditioner,
the downstream primer P2: 5'-TGAGGCAAAGGACAGGGT-3', respectively;
(2) constructing a DNA mixing pool: using factor 2nDesigning orthogonal test horizontally, and respectively taking the number of DNA mixed samples and the final concentration of DNA after mixing as A factor and B factor, wherein A1, A2 and A3 … A are designed according to the increasing number of the DNA mixed samples in the A factornThe factor(s) is (are),nfor the test level number, the A2 factor contains A1 factor individuals, the A3 factor contains A1 and A2 factor individuals, and AnThe factor contains A1, A2, A3 … An1 factor individual, establishing a DNA mixing pool by taking A factor and B factor as combined factors, simultaneously carrying out 3 times of PCR amplification by taking each DNA mixing pool as a single template, and carrying out sequencing analysis on an amplification product; detecting DNA purity by agarose gel electrophoresis, and detecting OD by spectrophotometer260/OD280A value;
the DNA mixing pool is 150 DNA mixing samples, and the final DNA concentration is 100 ng/muL.
2. The method for detecting single nucleotide polymorphism of the early win bovine CAPN1 gene based on the DNA mixed pool as claimed in claim 1, wherein the reaction system of the PCR amplification is as follows: the total volume is 25 μ L, wherein the 2 XTaq Master Mix mixture is 12 μ L, the concentrations of the upstream primer and the downstream primer are 10 pmol/. mu.L and 1 μ L respectively, the concentration of the DNA template is 100 ng/. mu.L and 1 μ L, ddH2O10 mu L; reaction conditions are as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 50s, annealing at 65 deg.C for 35s, extension at 72 deg.C for 35s, 30 cycles, extension at 72 deg.C for 8min, and storage at 4 deg.C.
3. The use of the method for detecting single nucleotide polymorphism of CAPN1 gene of Zaishen cattle based on DNA mixed pool as claimed in claim 1 or 2 for rapidly screening SNPs locus of Zaishen cattle.
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Citations (2)
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|---|---|---|---|---|
| CN101624598A (en) * | 2009-08-17 | 2010-01-13 | 吉林大学 | Authentication method of cattle CAPN1 gene as longisimus dorsi tenderness molecular marker and application |
| WO2011032243A1 (en) * | 2009-09-15 | 2011-03-24 | Empresa Brasileira De Pesquisa Agropecuária - Embrapa | Methods and kits for the identification of animals having a greater potential for desirable characteristics, and for the early identification of fat deposits in bovines |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101624598A (en) * | 2009-08-17 | 2010-01-13 | 吉林大学 | Authentication method of cattle CAPN1 gene as longisimus dorsi tenderness molecular marker and application |
| WO2011032243A1 (en) * | 2009-09-15 | 2011-03-24 | Empresa Brasileira De Pesquisa Agropecuária - Embrapa | Methods and kits for the identification of animals having a greater potential for desirable characteristics, and for the early identification of fat deposits in bovines |
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