CN202658155U

CN202658155U - Single nucleotide polymorphism (SNP) parting kit for chicken quality character related gene Akirin2

Info

Publication number: CN202658155U
Application number: CN2012202225167U
Authority: CN
Inventors: 赵振华; 黎寿丰; 黄华云; 李春苗
Original assignee: Jiangsu Institute Poultry Sciences
Current assignee: Jiangsu Institute Poultry Sciences
Priority date: 2012-05-17
Filing date: 2012-05-17
Publication date: 2013-01-09
Anticipated expiration: 2022-05-17

Abstract

The utility model relates to a single nucleotide polymorphism (SNP) parting kit for a chicken quality character related gene Akirin2, and belongs to the technical field of biology. The SNP parting kit for the chicken quality character related gene Akirin2 is characterized by consisting of a kit body, a pad in the kit body, and a primer mixture P, a deoxyribonucleic acid (DNA) Tag enzyme, amplification buffer, deoxynucleoside triphosphate (dNTP), buffer and an incision enzyme which are filled in hole cavities of the pad. By designing specific primers, a polymerase chain reaction (PCR) amplification method and a restriction fragment length polymorphism (RFLP) method, a product has high specificity, and SNP parting accuracy is ensured; and the kit is reasonable in design, convenient to use and low in cost, and a result is reliable.

Description

Chicken meat quality trait related gene-Akirin2 gene SNP parting kit

Technical field

The utility model belongs to biological technical field, utilizes the PCR-RFLP technology for detection that the test kit of a kind of chicken meat quality trait related gene-Akirin2 gene SNP somatotype is provided.

Background technology

Akirin2 is the important muscle-derived factor, be subject to the regulation and control of MSTN gene, its expression can impel Growth of Cells and sarcoplast to merge, finally cause the myocyte loose, the Akirin2 gene can promote the secretion of interleukin 6 (IL-6), and IL-6 is the excreted factor in the skeletal muscle regeneration process, can not only regulate skeletal muscle regeneration, and its expression amount also there are differences in dissimilar myofibers.Akirin2 participates in the body growth metabolic process, especially plays an important role in the tenderization after sarcostyle upgrades and kills.Myofiber is the fundamental unit that consists of muscle tissue, dissimilar myofibers differs greatly at aspects such as contained protein type, shrinkage character, metabolic characteristics, therefore also there is larger difference between the muscle that is consisted of by dissimilar myofibers, and finally has influence on meat quality (Karlsson et al. 1999; Klont et al. 1998).The meat quality that Akirin2 gene pairs chicken is described plays important regulating and controlling effect.

Single nucleotide polymorphism (single nucleotide polymorphism, SNP), obtained develop rapidly as third generation genetic marker, at present multiple full-fledged detection technique has been arranged, such as technique means such as single strand conformation polymorphism (SSCP), Restriction fragment length polymorphism (RFLP), DNA chip and Taqman probes.Restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) technology is utilized the pcr amplification target DNA exactly, and amplified production cuts into different big or small fragments, directly somatotype in gel electrophoresis with the digestion of specificity restriction endonuclease again.This technology has the specificity height, detects fast, highly versatile, the characteristics such as applied widely, with low cost, and the SNP that has been successfully applied to human diseases and poultry diease genes involved molecule marker at present detects and the research and development of gene diagnosis kit.

Through the retrieval to existing domestic and foreign literature and patent, so far there are no the report of Akirin2 gene polymorphism sites and chicken meat quality proterties dependency.

The utility model content

The purpose of this utility model is to provide a kind of accuracy high, quick, cheap gene type test kit for the detection to chicken muscle quality trait genes involved-Akirin2 gene C 397T.

The purpose of this utility model is achieved through the following technical solutions: chicken meat quality trait related gene-Akirin2 gene SNP parting kit, it is characterized in that, described test kit is by box body, liner in the box body, the upstream and downstream primer mixture that is used for the amplification of Akirin2 gene PCR, DNA Tag enzyme, amplification buffer, dNTP, Buffer and the restriction endonuclease of inserting respectively in each vestibule of liner form.

Be used for the upstream and downstream primer mixture of Akirin2 gene PCR amplification, primer information is as follows:

Upstream primer: 5 '-ATG CAA AAGCTAGCA GTC TATGT-3 '

Downstream primer: 5 '-GGAGAGCCTGAGAAGGAGTTG-3 '.

This test kit using method:

1, place, pcr amplification SNP site fragment

1) the pcr amplification reaction system is prepared

The reaction system of PCR is 20 μ l, comprising:

Amplification buffer (10 * buffer) 2.5 μ l

2.5mM dNTP 　　 2.0μl

Primer (5uM) 1 μ l

Template DNA (about 50 ng) 1 μ l

5U/ul Taq 　　0.5μl

Adding water mends to 20 μ l

In the PCR of 200 ul thin-walled tube, add reagent according to above reaction system, fully of short duration centrifugal behind the mixing.

2) pcr amplification reaction programming

Arrange as described in Table 1ly at eppendorf PCR instrument, after programming is complete, the PCR reaction tubes is put into the PCR instrument react amplification.

Table 1 pcr amplification reaction program

Figure DEST_PATH_779598DEST_PATH_IMAGE001

3) after reaction finishes, get the 5ul reaction product at 2% agarose gel, electrophoresis among 1 * TBE, whether detection reaction is successful.

2, the RFLP enzyme is cut and the identified gene type

The first step gained PCR product is got 7ul, and adding 2ul 10 * buffer(restriction endonuclease is supporting), restriction endonuclease ( The Ear I) 0.5ul, ultrapure water 10.5 μ l, fully of short duration centrifugal behind the mixing, then put into 37 ℃ of water-bath water-baths 10 hours, to get at last 6 ul enzymes and cut product at 3% sepharose, electrophoresis among 1 * TBE is with the video picture of gel imaging analysis system, identified gene type.

The utility model is by the design Auele Specific Primer, and pcr amplification and RFLP method so that product has more specificity, have guaranteed the accuracy of SNP somatotype.The utility model is reasonable in design, easy to use, reliable results and with low cost.

Description of drawings

Fig. 1 is structural representation of the present utility model;

Fig. 2 is the utility model Akirin2 gene PCR product amplification figure;

Fig. 3 is the utility model Akirin2 gene PCR product RFLP somatotype figure;

Among the figure: 1, box body; 2, liner; 3, the upstream and downstream primer mixture that is used for the amplification of Akirin2 gene PCR; 4, DNA Tag enzyme; 5, amplification buffer; 6, dNTP; 7, Buffer; 8, restriction endonuclease.

Embodiment

The utility model with specific embodiments and the drawings is described further.Should be understood that these embodiment only are used for illustration purpose, and be not used in the restriction scope of the invention.

Chicken meat quality trait related gene-Akirin2 gene SNP parting kit, by box body, liner in the box body, the upstream and downstream primer mixture, DNA Tag enzyme, amplification buffer, dNTP, the Buffer(restriction endonuclease damping fluid that are used for the amplification of Akirin2 gene PCR of inserting respectively in each vestibule of liner) and the restriction endonuclease composition.

Use this test kit that somatotype is carried out in 282 uncle Shao chicken Akirin2 gene G195C, A234G SNP sites.

1, sample

Gather the blood of uncle 282 Shaos chickens, use traditional phenol/chloroform method extraction DNA, and with the DNA concentration dilution to 50ng/ ul.

2, pcr amplification reaction and result

Pcr amplification and detected result are carried out according to the test kit using method:

Place, pcr amplification SNP site fragment

1) the pcr amplification reaction system is prepared

The reaction system of PCR is 20 μ l, comprising:

Amplification buffer (10 * buffer) 2.5 μ l

2.5mM dNTP 　　 2.0μl

Primer (5uM) 1 μ l

Template DNA (about 50 ng) 1 μ l

5U/ul Taq 　　0.5μl

Adding water mends to 20 μ l

2) pcr amplification reaction programming

Table 1 pcr amplification reaction program

Figure DEST_PATH_696739DEST_PATH_IMAGE001

Akirin2 gene PCR product size is 391bp, and is consistent with the result who estimates, as shown in Figure 2.

3, the RFLP of Akirin2 gene PCR amplified production identifies

Akirin2 PCR product RFLP identifies and carries out according to the test kit using method:

Above-mentioned gained PCR product is got 7ul, and adding 2ul 10 * buffer(restriction endonuclease is supporting), restriction endonuclease ( The Ear I) 0.5ul, ultrapure water 10.5 ul, fully of short duration centrifugal behind the mixing, then put into 37 ℃ of water-bath water-baths 10 hours, to get at last the 6ul enzyme and cut product at 3% sepharose, electrophoresis among 1 * TBE is with the video picture of gel imaging analysis system, identified gene type.

The detected result basis for estimation as shown in Figure 3.

Claims

1. chicken meat quality trait related gene-Akirin2 gene SNP parting kit, it is characterized in that, described test kit is by box body, liner in the box body, the upstream and downstream primer mixture that is used for the amplification of Akirin2 gene PCR, DNA Tag enzyme, amplification buffer, dNTP, Buffer and the restriction endonuclease of inserting respectively in each vestibule of liner form.