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Gallinaceous visfatin gene 9bp indel polymorphism detection method and application thereof

A polymorphism detection and lipoprotein technology, applied in the field of chicken genetics and breeding, can solve the problems of difficult insertion/deletion type identification, high cost of direct sequencing technology, cumbersome SSCP operation, etc., so as to shorten the generation interval and save the time of enzyme digestion. , the effect of reducing the cost of feeding

Inactive Publication Date: 2010-08-18
HENAN AGRICULTURAL UNIVERSITY +1
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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 PCR-RFLP method requires that the polymorphic site to be detected is a specific enzyme cutting site point, the scope of application is limited; the cost of direct sequencing technology is relatively high
Moreover, the above method is not suitable for the detection of insertion / deletion polymorphisms of several 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 agarose detection. - The 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 agarose detection can detect the insertion / deletion of a large fragment sequence of the gene, but it is difficult to judge the insertion / deletion of a few bp

Method used

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  • Gallinaceous visfatin gene 9bp indel polymorphism detection method and application thereof
  • Gallinaceous visfatin gene 9bp indel polymorphism detection method and application thereof
  • Gallinaceous visfatin gene 9bp indel polymorphism detection method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0027] Detection of chicken visfatin gene 9bp indel polymorphism by the method of the present invention

[0028] (1) According to the position of the chicken visfatin gene 9bp indel site in the genome sequence, a pair of primers were designed with the chicken visfatin gene sequence, and the primer sequence was:

[0029] PF: 5′-GTGGTAGTGAGAGATAGCAGAAGG-3

[0030] PR: 5'-AGTGAGACGCCGTTTCTAGTTC-3';

[0031] (2) Perform PCR amplification with genomic DNA from wing vein blood samples of 964 chickens in the Gushi chicken-Anka chicken resource group. The reaction system for PCR amplification is 25 μL, and the specific sample volume is shown in Table 1. Taq DNA polymerase, 10×Buffer and MgCl 2 Both were purchased from MBI Fermentas.

[0032] Table 1 PCR reaction system

[0033]

[0034] The PCR reaction program is: pre-deformation at 95°C for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 72°C for 60 seconds, 30-35 cycles; extension at 72°C for 10 minutes; st...

Embodiment 2

[0037] The method of the present invention detects the chicken visfatin gene 9bp indel polymorphism, and the primers used are the same as those in Example 1, with Gushi chicken-Anka chicken F 2 Genomic DNA of the wing vein blood sample of 964 chickens of the generation resource group was carried out by PCR amplification, and the method adopted was the same as that of Example 1, except that what was used for electrophoresis detection was 12% polyacrylamide gel for electrophoresis analysis, which was the same as that of Example 1. Methods Record the genotype of the gene corresponding to each individual chicken sample.

[0038] Verification example

[0039] ABI 3730 sequencer was used to select 9 PCR products of representative individuals with different genotypes (3 replicates for each of the 3 genotypes) for sequencing respectively. The sequencing results of the 9bp insertion of chicken visfatin gene are as follows: image 3 As shown in (a), the sequencing results of the 9bp de...

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Abstract

The invention relates to a gallinaceous visfatin gene 9bp indel polymorphism detection method and application thereof. The detection method comprises the following steps of: designing a primer in the position of a gallinaceous visfatin gene 9bp indel locus; then carrying out PCR amplification; and detecting the polymorphism of the gene 9bp indel by the polyacrylamide gel electrophoresis, wherein the gene is of a DD genotype when 9bp is deleted, the gene is of an II genotype when 9bp is inserted, and the heterozygous individual at the locus is of an ID genotype. The correlation analysis combining the detection results and economic characters of chickens is used for the assistant selection and the molecular breeding of chickens. The detection method does not need be subject to the enzyme digestion, thereby saving the cost and the time; the detection is carried out by adopting the polyacrylamide gel electrophoresis, thereby overcoming the limitation of the agarose electrophoresis to the small-fragment nucleotide difference detection. The method has the advantages of high resolution, high detection sensitivity and accurate genotype judgment; and the gel has on requirements for the staining method, and the ethidium bromide (EB) staining and the silver staining are both applicable.

Description

technical field [0001] The invention relates to a chicken visfatin (visfatin) gene 9bp indel polymorphism detection method, and also relates to the application of the detection method, which belongs to the technical field of chicken genetic breeding. Background technique [0002] In the genetic breeding of livestock and poultry, the application of modern breeding technology of molecular markers is an effective method to speed up the breeding of fine breeds and improve the genetic quality of the population, thereby increasing the advanced and effective method of livestock and poultry production performance. The application of molecular marker breeding is first to screen and detect genetic markers closely related to chicken economic traits at the DNA level; secondly, to establish a rapid detection method for its gene polymorphism; and then to realize genetic marker-assisted selection and early diagnostic selection. [0003] Single Nucleotide Polymorphism (SNP) is considered to...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 韩瑞丽康相涛陈宏李建群李转见蓝贤勇
Owner HENAN AGRICULTURAL UNIVERSITY
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