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Fast detection method for chicken Pax7 gene 31 bp indel polymorphism and application thereof

A detection method and polymorphism technology, applied in the field of molecular genetics, can solve the problems of high cost of direct sequencing technology, cumbersome SSCP operation, and complicated experimental operation, so as to save the time of enzyme digestion, overcome the limitations of complicated operation, Simple operation effect

Active Publication Date: 2014-03-26
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the SSCP operation is cumbersome, time-consuming, has many influencing factors, and there are false negative problems in the experimental process, so it is not an ideal SNP detection method; the ordinary PCR-RFLP method requires that the polymorphic site to be tested is a specific enzyme Cutting site, limited scope of application; direct sequencing technology is costly
Moreover, the above method is not suitable for the detection of insertion / deletion polymorphisms of tens of bases in genomic DNA. For the detection of insertion / deletion of genomic DNA sequences, many studies have used the PCR-RFLP method or the combination of PCR and polyacrylamide electrophoresis detection. method, the PCR-RFLP method requires a special endonuclease for the mutation site, and the digested product needs to be detected by agarose or polyacrylamide electrophoresis, which is costly and time-consuming
The method of PCR and polyacrylamide detection can detect the insertion / deletion of a few nucleotide sequences of the gene, but for the insertion / deletion of dozens of bp, the experimental operation is complicated and time-consuming

Method used

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  • Fast detection method for chicken Pax7 gene 31 bp indel polymorphism and application thereof
  • Fast detection method for chicken Pax7 gene 31 bp indel polymorphism and application thereof
  • Fast detection method for chicken Pax7 gene 31 bp indel polymorphism and application thereof

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Experimental program
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Effect test

Embodiment 1

[0033] Example 1, the extraction and purification of chicken genomic DNA

[0034] 1. Extraction of Chicken Genomic DNA

[0035] Take Gushi chicken-Anka chicken resource group F 2 A total of 772 chickens were used as materials, and 5 mL of jugular venous blood was extracted from each chicken, put into a centrifuge tube, placed at an angle at room temperature for 30 minutes, and put into a centrifuge after precipitation of serum, and centrifuged at a speed of 3000r / min for 30 minutes After the serum was separated, it was stored in a -80°C refrigerator for later use.

[0036] from F by phenol-chloroform extraction 2 Genomic DNA was extracted from the blood samples of the generation resource group, dissolved in TE, and stored at 4°C for later use. The specific method is as follows:

[0037]

[0038]

[0039] 2. Purification of genomic DNA

[0040] 1) Add 10% SDS to 500 μL DNA solution to make the final concentration 0.1%, add proteinase K until the final concentration re...

Embodiment 2

[0057] Embodiment 2, PCR amplification

[0058] 1. PCR amplification primers

[0059] Taking the genome sequence of chicken Pax7 gene in Genbank (accession number NC006108) as a reference, Primer5.0 was used to design PCR primers for the third exon and third intron fragment of chicken Pax7 gene. The primer sequences are as follows:

[0060] Upstream primer: 5'-CTTTTTTCTCTCCCCCTTCC-3';

[0061] Downstream primer: 5'-CAGACCCTCAGCACAACTCA-3'.

[0062] 2. PCR amplification reaction system

[0063] Take Gushi chicken-Anka chicken resource group F 2 A total of 772 chicken genomic DNAs were used as templates. In the presence of Taq PCR Master Mix, PCR amplification was performed using the designed PCR amplification primers. The PCR reaction system is shown in Table 1:

[0064] Table 1 PCR reaction system

[0065] components

[0066] 3. PCR amplification reaction program

[0067] The PCR amplification reaction program is shown in Table 2:

[0068] Table 2PCR reaction...

Embodiment 3

[0071] Embodiment 3, agarose gel electrophoresis

[0072] Agarose gel electrophoresis detection:

[0073] 1) Make 2.0% agarose gel: Weigh 2g of agarose, transfer it into a conical flask, add 1×TAE100mL to suspend it, heat it in a microwave oven on medium heat, take it out after boiling twice, and quickly transfer it to another conical flask Add the EB solution to a final concentration of 5 μg / mL, then pour the agarose solution quickly and shake slightly to prevent air bubbles.

[0074] 2) After the gel is completely cooled and solidified, unplug the comb, remove the tape paper at both ends, and move the gel into the electrophoresis tank.

[0075] 3) Take 3 μL of DNA sample, add 2 μL of loading buffer, mix well, load the sample uniformly, electrophoresis at 100V for 60 min, and stain with EB.

[0076] 4) Imaging on the GBOX gel imaging system, see figure 2 .

[0077] from figure 2 It can be seen that lanes 5 and 6 are PCR amplification products, and the fragment size is ...

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Abstract

The invention discloses a fast detection method for chicken Pax7 gene 31 bp indel polymorphism and the application thereof, wherein the detection method comprises the following steps: designing a pair of primers based on gene sequences of a third exon and a third intron of a chicken Pax7 gene; then performing PCR amplification; and finally detecting the 31 bp indel polymorphism of the gene through agarose gel electrophoresis, when the third intron 31 bp is inserted, the gene is an II genotype, when the third intron 31 bp is in deletion, the gene is a DD genotype, and when the locus is a heterozygous individual, the gene is an ID genotype. Meanwhile, the method utilizes detection result to perform relevance analyze with chicken economical characters, which is used for chicken assistant selection and molecular breeding. According to the invention, during big DNA fragment detection, the method not only has high resolution, sensitive detection and accurate type judgment, but also has the characteristics of simplicity in operation, time conservation and low cost, and can be widely popularized.

Description

technical field [0001] The invention relates to a rapid detection method of chicken Pax7 gene 31bp indel polymorphism, and also relates to the application of the rapid detection method, belonging to the field of molecular genetics. Background technique [0002] Animals provide humans with a stable source of protein, which is the material basis necessary for human life. With the improvement of the growth rate of poultry in the poultry industry and the selection of feed efficiency, the production performance of poultry has been greatly improved, but at the same time, it has also produced negative effects that reduce the quality of the meat, such as excessive deposition of abdominal fat and flavor loss. Decreased, thicker muscle fiber diameter, decreased tenderness, etc. my country is a big chicken raising and chicken consumption country. With the improvement of people's living standards, people's demand for chicken is not only the output, but also its flavor and palatability....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 康相涛张锁韩瑞丽王乐乐田亚东孙桂荣李国喜蒋瑞瑞
Owner HENAN AGRICULTURAL UNIVERSITY
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