CN103031363A - Method for detecting gene polymorphism of main meat quality of pigs through multiplex-PCR (Polymerase Chain Reaction) and multiple enzyme digestion - Google Patents
Method for detecting gene polymorphism of main meat quality of pigs through multiplex-PCR (Polymerase Chain Reaction) and multiple enzyme digestion Download PDFInfo
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Abstract
The invention belongs to the technical field of gene detection, and relates to a method for detecting gene polymorphism of main meat quality of pigs through multiplex-PCR (Polymerase Chain Reaction) and multiple enzyme digestion. The method comprises the following steps of: adopting Alw 21 I incision enzyme and Hinf I incision enzyme to carry out double enzyme digestion on double PCR amplification products of a halothane gene and heart fatty acid gene 5' -upstream region; adopting BsrBr I incision enzyme and Msp I incision enzyme to carry out double enzyme digestion on double PCR amplification products of an acid meat gene and heart fatty acid gene secondary intron region; and finally, using Hae III incision enzyme to carry out single enzyme digestion on the double PCR amplification products of the acid meat gene and heart fatty acid gene secondary intron region. Through the detection method, one reaction system can be used to carry out amplification and enzyme digestion on two sections of DNA sequences simultaneously, and the detection efficiency of halothane, acid meat and heart fatty acid gene polymorphism is greatly improved.
Description
Technical field
The present invention relates to a kind of method of using multiple PCR method and multiple restriction enzyme method to detect the main meat gene pleiomorphism of pig, belong to the technique of gene detection field.
Background technology
Meat is permitted polygenic regulation and control as the quantitative character of pig, and wherein, fluothane, sour meat and heart fat acid gene are three important meat genes, and the sudden change of these three genes all can produce a very large impact the meat of pig.Wherein, the sudden change of halothane is because the C1843-G among the halothane cDNA sports T1843-A, makes the 615th arginine of receptor protein become halfcystine, thereby causes the change of expression product structure and function, and then cause the generation of porcine stress syndrome; The sudden change of acid meat gene be since in sour meat gene extron 3 inhibition from mutation of bases G → A the activity of phosphoric acid protein kinase, caused whole pH value low, color is pale, " the hampshire type pork " that has moisture to ooze out; There is restriction enzyme on the pig heart fatty acid gene
MspI,
HaeIII,
Hin3 enzymes of f I are cut pleomorphism site, the polymorphism in these three sites all with flesh between lipid content be significantly relevant.
At present, these three genes have become the main dna marker in pig DNA marker assisted selection (MAS) breeding; The method that detects these three genes is traditional PCR-RFLP.And traditional PCR-RFLP method can only detect a gene at every turn, detection efficiency is lower, the method that patent of the present invention uses double PCR and double enzyme to cut increases simultaneously at every turn and enzyme is cut two segment DNA fragments, thereby has greatly improved the efficient that the Meat gene pleiomorphism detects.
Summary of the invention
Goal of the invention:
The present invention is directed to the variety of issue that exists in the above-mentioned prior art, proposed the method that a kind of multiplex PCR and multiple restriction enzyme detect the main meat gene pleiomorphism of pig, overcome defects, improved detection efficiency.
Technical scheme:
A kind of multiplex PCR and multiple restriction enzyme detect the method for the main meat gene pleiomorphism of pig, it is characterized in that: carry out according to the following steps:
(1), at first selects primer and cut the corresponding restriction endonuclease of pleomorphism site with enzyme for the mutational site of fluothane, sour meat and these three meat genes of heart fat acid, search the enzyme of restriction endonuclease under various enzyme cutting buffering liquid conditions and cut efficient, and list accordingly enzyme and cut the more much higher heavy enzymes combinations of efficient.
(2), according to selected primer in the step (1) and all multiple restriction enzyme combinations, calculate each enzymes combinations for the sudden change of corresponding DNA cloning fragment before and the endonuclease bamhi length after the sudden change, analyze and whether can recognize that the electrophoresis result that causes because of sudden change changes.
(3), filter out the enzymes combinations that to recognize sudden change in the step (2), select in these combinations several, and Multiplex PCR amplification system and the multiple restriction enzyme system of the structure dna fragmentation corresponding with the selected enzymes combinations that can recognize sudden change.
Adopt in the above-mentioned steps (3)
AlwThe 21l restriction endonuclease and
HinThe fl restriction endonuclease carries out double enzyme to the double PCR amplified production of halothane and heart fat acid gene 5 '-upstream and cuts; Adopt
BsrThe Brl restriction endonuclease and
MspThe l restriction endonuclease carries out double digestion to the double PCR amplified production in sour meat gene and heart fat acid gene intron 2 district; Use at last
HaeThe lll restriction endonuclease carries out single endonuclease digestion to the double PCR amplified production in sour meat gene and heart fat acid gene intron 2 district.
At first the double PCR amplification is carried out in the specific fragment of halothane and heart fat acid gene 5 '-upstream, then the double PCR amplification is carried out in the specific fragment in sour meat gene and heart fat acid gene intron 2 district;
Multiplex PCR reaction system following (μ L):
1. reaction system: 50;
2.ddH
2O： 25；
3.buffer： 8；
4.dNTP： 8；
5. upstream primer 1:1;
6. upstream primer 2:1;
7. downstream primer 1:1;
8. downstream primer 2:1;
9.
TaqEnzyme: 2;
10.DNA template: 3;
Reaction conditions is: 94 ℃/10min denaturation; 94 ℃/35sec-61 ℃/35sec-72 ℃/35 circulations of 35sec; 72 ℃/10min extends; Detected result is used 140V voltage, and 2.5% agarose gel electrophoresis, electrophoresis time are 35 minutes.
Described multiple restriction enzyme total reaction system is 10 μ l, PCR product 5 μ l wherein, and two kinds of each 0.5 μ l of restriction enzyme, distilled water 3 μ l, enzyme Qie Wendu is 37 ℃, enzyme is cut time 12h; Detected result is used 140V voltage, and 3% agarose gel electrophoresis, electrophoresis time are 22 minutes.
Two kinds of above-mentioned restriction enzymes are
Alw21l+
HinFl or
BsrBrl+
MspL.
Advantage and effect:
The present invention proposes the method that a kind of multiplex PCR and multiple restriction enzyme detect the main meat gene pleiomorphism of pig, compared with prior art, have following advantage:
By detection method of the present invention, amplification and the enzyme that can use simultaneously a reaction system to carry out two segment DNA sequences are cut, and have improved the detection efficiency of fluothane, sour meat and heart fat acid gene polymorphism.
Description of drawings:
Fig. 1 is halothane and heart fat acid gene 5 '-upstream specific fragment double PCR amplification.
Fig. 2 is sour meat gene and heart fat acid gene intron 2 district specific fragment double PCR amplification.
Fig. 3 is that halothane and heart fat acid gene 5 '-upstream specific fragment double enzyme are cut the result.
Fig. 4 is that sour meat gene and heart fat acid gene intron 2 district specific fragment double enzyme are cut the result.
Fig. 5 is sour meat gene and heart fat acid gene intron 2 district specific fragment
HaeThe III enzyme is cut the result.
Fig. 6 is the technology of the present invention route synoptic diagram.
Embodiment:
Terminological interpretation:
Meat: the organoleptic quality of meat, Further processing, nutritive value and hygienic quality.Wherein, organoleptic quality is that people's the sensory organ such as vision, sense of smell, the sense of taste and sense of touch are to the comprehensive impression of pork.The organoleptic quality of pork is commonly used the indexs such as muscle pH value, yellowish pink, drip loss, tender degree, succulence, flavour mark and is evaluated.
The meat gene: the gene of regulation and control Meat Quality, Meat Quality is generally quantitative character, by a plurality of major genes and candidate gene regulation and control.Molecular marker assisted selection: molecular marker assisted selection (MAS, marker-assisted selection) be the new technology that produces along with developing rapidly of modern molecular biology technique, it can analyze individual genetic composition rapidly and accurately from molecular level, thereby realize genotypic direct selection, carry out molecular breeding.
Multiple restriction enzyme: multiple restriction enzyme technology (Multiple enzyme digestion technology) is to adopt a plurality of enzymes to cut simultaneously a fragment or cut simultaneously a plurality of fragments, and the reaction conditions of these several enzymes, damping fluid and reaction times etc. all are the same.
The main meat gene of pig of the present invention refers to these three genes of halothane, sour meat gene and heart fat acid gene.
The present invention is described further below in conjunction with accompanying drawing and concrete embodiment:
The invention provides a kind of method of using multiple PCR method and multiple restriction enzyme method that the main meat gene of pig halothane, sour meat gene and heart fat acid gene polymorphism are detected, technological line is characterized in that as shown in Figure 6:
At first for fluothane, the mutational site of acid meat and these three meat genes of heart fat acid is selected primer and is cut the corresponding restriction endonuclease of pleomorphism site with enzyme, search the enzyme of these restriction endonucleases under various enzyme cutting buffering liquid conditions and cut efficient, and list accordingly all enzymes and cut the more much higher heavy enzymes combinations of efficient, according to the selected primer of experiment and all multiple restriction enzyme combinations, before using each enzymes combinations of primer5 computed in software for corresponding DNA cloning fragment sudden change and the endonuclease bamhi length after the sudden change, analyze and whether can recognize that the electrophoresis result that causes because of sudden change changes.Filter out the enzymes combinations that to recognize sudden change, select in these combinations severally, and make up Multiplex PCR amplification system and the multiple restriction enzyme system of corresponding with it dna fragmentation.
The design of primer: concrete primer information is as follows:
Halothane: upstream primer: 5 '-TCCAGTTTGCCACAGGTCCTACCA-3 '
Downstream primer: 5 '-ATTCACCGGAGTGGAGTCTCTGAG-3 '
Fragment length: 659bp
Annealing temperature: 61 ℃
Acid meat gene: upstream primer: 5 '-GAGGCCCAAATAAGTCAATGTA-3 '
Downstream primer: 5 '-ACCGGGGTCAAATGCTC-3 '
Fragment length: 616bp
Annealing temperature: 62 ℃
Heart fat acid gene: upstream primer: 5 '-GGACCCAAGATGCCTACGCCG-3 '
(5 '-upstream) downstream primer: 5 '-CTGCAGCTTTGACCAAGAGG-3 '
Fragment length: 693bp
Annealing temperature: 59 ℃
Heart fat acid gene: upstream primer: 5 '-TTGCTTCGGTGTGTTTGAG-3 '
(intron 2 district) downstream primer: 5 '-CAGGAATGGGAGTTATTGG-3 '
Fragment length: 816bp
Annealing temperature: 63 ℃.
The selection of restriction endonuclease: search the enzyme of the used restriction endonuclease of the present invention in the situation of using various different buffer and cut efficient, as shown in Table 1:
Table one: each restriction endonuclease is using the enzyme in the different buffer situations to cut efficient (%)
According to form, select the enzymes combinations that enzyme is cut efficient higher (more than 50%).
Make up as follows:
Enzyme buffer
Alw21l＋
Hinfl： O、R、T2
Alw21l＋
Haelll： R、T2
Alw21l＋
Mspl： T2
BsrBrl＋
Hinfl： T1
BsrBrl＋
Haelll： T1
BsrBrl＋
Mspl： G、T1
Hinfl＋
Haelll： R、T1、T2
Hinfl＋
Mspl： T1、T2
Haelll＋
Mspl： T1、T2
Alw21l＋
Hinfl＋
Haelll： R、T2
Alw21l＋
Haelll＋
Mspl： T2
Alw21l＋
Hinfl＋
Mspl： T2
BsrBrl＋
Haelll＋
Mspl： T1
BsrBrl＋
Hinfl＋
Mspl： T1
Hinfl＋
Haelll＋
Mspl： T1、T2
BsrBrl＋
Hinfl＋
Haelll＋
Mspl： T1
Alw21l＋
Hinfl＋
Haelll＋
Mspl： T2。
According to this combination, each enzymes combinations of use primer5 computed in software is the front and rear fragment length that is cut into of sudden change for corresponding DNA cloning fragment sudden change, analyzes and whether can recognize the electrophoresis result change that causes because of sudden change.
According to analysis, filter out the three pairs of enzymes combinations that can tell sudden change, as follows:
Alw21l＋
Hinfl：
Use buffer:O, R, T2,
Fragment length (bp) before the halothane sudden change: 140,495,
Fragment length (bp) after the sudden change: 635,
Fragment length (bp) before the sudden change of heart fat acid gene 5 '-upstream: 339,172,98,59,
Fragment length (bp) after the sudden change: 339,231,98.
Hinfl＋
Mspl：
Use buffer:T1, T2,
Fragment length (bp) before the sudden change of heart fat acid gene 5 '-upstream: 172,144,111,58,55,50,
Fragment length (bp) after the sudden change: 222,144,111,58,55,
Fragment length (bp) before the heart fat acid gene intron 2 region mutation: 520,264
Fragment length (bp) after the sudden change: 454,264,66.
BsrBrl＋
Mspl：
Use buffer:G, T1,
Fragment length (bp) before the transgenation of acid meat: 298,187,70,46,
Fragment length (bp) after the sudden change: 298,233,70,
Fragment length (bp) before the heart fat acid gene intron 2 region mutation: 816,
Fragment length (bp) after the sudden change: 750,66.
In order to guarantee that the pcr amplification product on each gene can both be digested, the present invention adopts
AlwThe 21l restriction endonuclease and
HinThe fl restriction endonuclease carries out double enzyme to the double PCR amplified production of halothane and heart fat acid gene 5 '-upstream and cuts; Adopt
BsrThe Brl restriction endonuclease and
MspThe l restriction endonuclease carries out double digestion to the double PCR amplified production in sour meat gene and heart fat acid gene intron 2 district, uses at last
HaeThe lll restriction endonuclease carries out single endonuclease digestion to the double PCR amplified production in sour meat gene and heart fat acid gene intron 2 district.
The structure of Multiplex PCR method:
Multiplex PCR reaction system following (μ L):
1. reaction system: 50;
2.ddH
2O： 25；
3.buffer： 8；
4.dNTP： 8；
5. upstream primer 1:1;
6. upstream primer 2:1;
7. downstream primer 1:1;
8. downstream primer 2:1;
9.
TaqEnzyme: 2;
10.DNA template: 3;
Reaction conditions is: 94 ℃/10min denaturation; 94 ℃/35sec-61 ℃/35sec-72 ℃/35 circulations of 35sec; 72 ℃/10min extends.
Detected result is used 140V voltage, and 2.5% agarose gel electrophoresis, electrophoresis time are 35 minutes, and electrophoresis result is as shown in Fig. 1 and Fig. 2.
The structure of multiple restriction enzyme method:
The endonuclease reaction system is 10 μ l, PCR product 5 μ l wherein, and two kinds of each 0.5 μ l of restriction enzyme, distilled water 3 μ l, enzyme Qie Wendu are that 37 ℃ of enzymes are cut time 12h.Detected result is used 140V voltage, and 3% agarose gel electrophoresis, electrophoresis time are 22 minutes.Enzyme is cut the result as shown in Fig. 3, Fig. 4 and Fig. 5.
On the halothane pcr amplified fragment except there being one
AlwOutside the 21l restriction enzyme site, also have two
HinThe fl restriction enzyme site, gene is divided into size is respectively 636,7,3 fragments of 16bp, but because length to be two fragments of 7 and 16 less, can ignore when carrying out the electrophoresis comparison, so can think when halothane is not undergone mutation, the halothane pcr amplified fragment can quilt
Alw21l+
HinFl double digestion system is cut into two fragments that size is respectively 493bp and 143bp, and genotype is
NN, when halothane is undergone mutation,
AlwThe 21l restriction enzyme site can't be identified, and only can detect size and be the fragment of 636bp, and genotype is
Nn, when if halothane only has a DNA chain to undergo mutation, can obtain 636 bp, 493 bp and 143 bp3 bands, genotype is
Nn
Heart fat acid gene 5 '-upstream pcr amplified fragment is not subjected to restriction endonuclease
AlwThe 21l impact, so when not undergoing mutation, each restriction enzyme site is digested opening fully, common property is given birth to 5 fragments of 339,172,98,59,25 bp, genotype is
H, during sudden change, endonuclease bamhi is 339 bp, 231 bp, and 98 bp and 25 bp, genotype is
h(Fig. 3).The restriction enzyme digestion and electrophoresis result of two sections extension increasing sequences is independent of each other, and can normally detect so double enzyme is cut.As shown in Figure 3, swimming lane 1,3, heart fat acid gene 5 '-upstream genotype of 4,6 is
Hh, the genotype of swimming lane 2,5 is
HhThe halothane genotype of all swimming lanes is
NN.
When acid meat gene was not undergone mutation, amplified fragments can quilt
BsrBrl+
MspL double digestion system is cut into that size is respectively 298,233, the fragment of 70bp, and genotype is
Rn, after undergoing mutation, can be cut into that size is respectively 298,187,70, the fragment of 46bp, genotype is
RNHeart fat acid gene intron 2 district amplified fragments is not subjected to restriction endonuclease
BsrThe Brl impact,
MspThe I enzyme is cut and is respectively
AType and
aType,
AThe type endonuclease bamhi has 750 bp and 66bp,
aThe type endonuclease bamhi is 816 bp(Fig. 4).The restriction enzyme digestion and electrophoresis result of two sections extension increasing sequences is independent of each other, and can normally detect so double enzyme is cut.As shown in Figure 4, the sour meat gene genotype of all swimming lanes is
Rn/rnThe heart fat acid gene intron 2 district of all swimming lanes
MspThe I enzyme is cut genotype and is
AA
Heart fat acid gene intron 2 district amplified fragments
HaeThe III enzyme is cut allelotrope and is respectively D type and d type, and D type endonuclease bamhi size is 683,117 and 16bp; D type endonuclease bamhi is 405,278,117 and 16bp; The amplified fragments of acid meat gene can quilt
HaeIt is 190,124,103 and the fragment of 102bp that the III restriction endonuclease is cut into size, and these fragments can not affect heart fat acid gene intron 2 district amplified fragments
HaeThe III enzyme is cut polymorphic detection.As shown in Figure 5, swimming lane 1,2,5,7,8,9 heart fat acid gene intron 2 district
HaeThe III enzyme is cut genotype
Dd, swimming lane 3,4,6 genotype is
Dd
Claims (5)
1. a multiplex PCR and multiple restriction enzyme detect the method for the main meat gene pleiomorphism of pig, it is characterized in that: carry out according to the following steps:
(1), at first selects primer and cut the corresponding restriction endonuclease of pleomorphism site with enzyme for the mutational site of fluothane, sour meat and these three meat genes of heart fat acid, search the enzyme of restriction endonuclease under various enzyme cutting buffering liquid conditions and cut efficient, and list accordingly enzyme and cut the more much higher heavy enzymes combinations of efficient;
(2), according to selected primer in the step (1) and all multiple restriction enzyme combinations, calculate each enzymes combinations for the sudden change of corresponding DNA cloning fragment before and the endonuclease bamhi length after the sudden change, analyze and whether can recognize that the electrophoresis result that causes because of sudden change changes;
(3), filter out the enzymes combinations that can recognize sudden change, select in these combinations severally, and make up Multiplex PCR amplification system and the multiple restriction enzyme system of corresponding with it dna fragmentation.
2. multiplex PCR according to claim 1 and multiple restriction enzyme detect the method for the main meat gene pleiomorphism of pig, it is characterized in that: adopt in the step (3)
AlwThe 21l restriction endonuclease and
HinThe fl restriction endonuclease carries out double enzyme to the double PCR amplified production of halothane and heart fat acid gene 5 '-upstream and cuts; Adopt
BsrThe Brl restriction endonuclease and
MspThe l restriction endonuclease carries out double digestion to the double PCR amplified production in sour meat gene and heart fat acid gene intron 2 district; Use at last
HaeThe lll restriction endonuclease carries out single endonuclease digestion to the double PCR amplified production in sour meat gene and heart fat acid gene intron 2 district.
3. multiplex PCR according to claim 1 and multiple restriction enzyme detect the method for the main meat gene pleiomorphism of pig, it is characterized in that: at first the double PCR amplification is carried out in the specific fragment of halothane and heart fat acid gene 5 '-upstream, then the double PCR amplification is carried out in the specific fragment in sour meat gene and heart fat acid gene intron 2 district;
Multiplex PCR reaction system following (μ L):
1. reaction system: 50;
2.ddH
2O： 25；
3.buffer： 8；
4.dNTP： 8；
5. upstream primer 1:1;
6. upstream primer 2:1;
7. downstream primer 1:1;
8. downstream primer 2:1;
9.
TaqEnzyme: 2;
10.DNA template: 3;
Reaction conditions is: 94 ℃/10min denaturation; 94 ℃/35sec-61 ℃/35sec-72 ℃/35 circulations of 35sec; 72 ℃/10min extends; Detected result is used 140V voltage, and 2.5% agarose gel electrophoresis, electrophoresis time are 35 minutes.
4. multiplex PCR according to claim 1 and multiple restriction enzyme detect the method for the main meat gene pleiomorphism of pig, it is characterized in that: described multiple restriction enzyme total reaction system is 10 μ l, PCR product 5 μ l wherein, two kinds of each 0.5 μ l of restriction enzyme, distilled water 3 μ l, enzyme Qie Wendu is 37 ℃, and enzyme is cut time 12h; Detected result is used 140V voltage, and 3% agarose gel electrophoresis, electrophoresis time are 22 minutes.
5. multiplex PCR according to claim 4 and multiple restriction enzyme detect the method for the main meat gene pleiomorphism of pig, and it is characterized in that: described two kinds of restriction enzymes are
Alw21l+
HinFl or
BsrBrl+
MspL.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611349A (en) * | 2014-11-24 | 2015-05-13 | 吉林大学 | FDFT1 gene key loci affecting Chinese simmental cattle fat deposition |
| CN117778589A (en) * | 2023-12-25 | 2024-03-29 | 中国农业科学院深圳农业基因组研究所(岭南现代农业科学与技术广东省实验室深圳分中心) | Pig economic character associated mutation site detection reagent, kit and detection method |
-
2011
- 2011-09-29 CN CN 201110298964 patent/CN103031363A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104611349A (en) * | 2014-11-24 | 2015-05-13 | 吉林大学 | FDFT1 gene key loci affecting Chinese simmental cattle fat deposition |
| CN104611349B (en) * | 2014-11-24 | 2017-10-03 | 吉林大学 | Influence the FDFT1 gene critical sites of Chinese Simmental fat deposition |
| CN117778589A (en) * | 2023-12-25 | 2024-03-29 | 中国农业科学院深圳农业基因组研究所(岭南现代农业科学与技术广东省实验室深圳分中心) | Pig economic character associated mutation site detection reagent, kit and detection method |
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