Single nucleotide polymorphic locus of milk goat PITX1 gene, and detection method and application of single nucleotide polymorphic locus

A single nucleotide polymorphism and milk goat technology, applied in the field of animal molecular genetics and breeding, can solve problems such as no related reports, and achieve low cost effects

Inactive Publication Date: 2012-08-29
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Up to now, there is no relevant report on the SNP study of dairy goat PITX1 gene

Method used

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  • Single nucleotide polymorphic locus of milk goat PITX1 gene, and detection method and application of single nucleotide polymorphic locus
  • Single nucleotide polymorphic locus of milk goat PITX1 gene, and detection method and application of single nucleotide polymorphic locus
  • Single nucleotide polymorphic locus of milk goat PITX1 gene, and detection method and application of single nucleotide polymorphic locus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Cloning and DNA Sequencing of Partial DNA Sequence of PITX1 Gene in Dairy Goats

[0063] 1. Collection of milk goat samples

[0064] The blood samples (venous blood) and ear tissue samples used for DNA extraction were collected from 658 healthy and unrelated Guanzhong dairy goats aged 2-4 years old, from the dairy goat breeding farm in Sanyuan County, Shaanxi Province.

[0065] 2. Extraction and isolation of genomic DNA from blood samples

[0066] (1) Thaw frozen blood samples (mainly blood cells) at room temperature, transfer 500 μL to a 1.5 mL Eppendorf tube, add an equal volume of PBS, mix well, centrifuge at 12,000 r / min for 10 min (4°C), discard the supernatant, and repeat the above Step until the supernatant is transparent and the precipitate is pale yellow.

[0067] (2) Add 500 μL of DNA extraction buffer to the centrifuge tube, shake it to make the blood cell pellet detach from the wall of the centrifuge tube, and bathe at 37° C. for 1 hour.

[0...

Embodiment 2

[0111] Embodiment 2: MspI PCR-RFLP Analysis of G>A Polymorphism at Position 201 of PITX1 Gene in Dairy Goats

[0112] 1. Analysis of polymorphic loci

[0113] When G>A mutation occurs at position 201 of PITX1 gene of dairy goat, that is, G mutation becomes A, the original CCG The sequence accordingly becomes CCG As a result, the MspI restriction endonuclease recognition sequence is lost, and it cannot be cleaved by the restriction endonuclease MspI.

[0114] 2. PCR reaction conditions

[0115] The PCR amplification system and reaction conditions were as described above, wherein the template was dairy goat individual genomic DNA, the PCR amplification product was detected by 1.0% agarose gel electrophoresis, and a 230 bp band could be clearly seen.

[0116] 3. PCR product digestion (MspI) and RFLP detection

[0117] The PCR amplification products were first digested with MspI, and then the single nucleotide polymorphism of the site was determined according to the result...

Embodiment 3

[0124] Embodiment three: The diagnostic application of the DNA marker prepared by the invention in the population polymorphism of Guanzhong dairy goats

[0125] 1. Diagnosis in Population Polymorphism:

[0126] Using the above SNP detection method, genotype identification by PCR-RFLP was performed on the DNA samples of all the relevant middle-dairy goats respectively.

[0127] 2. Statistical analysis of the frequency of SNP loci:

[0128] Genotype frequency refers to the ratio between various genotypes for a trait in a population. P AA =N AA / N, where P AA Represents the AA genotype frequency at a locus; N AA Indicates the number of individuals with AA genotype in the population; N is the total number of the detected population.

[0129] Gene frequency refers to the relative ratio of a gene to its alleles in a population. The formula for the calculation can be written as: P A =(2N AA +N Aa1 +N Aa2 +N Aa3 +N Aa4 +...+N Aan ) / 2N

[0130] In the formula, P A rep...

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Abstract

The invention relates to a single nucleotide polymorphic locus of a milk goat PITX1 gene, and a detection method and application of the single nucleotide polymorphic locus, and belongs to the technical field of animal molecular genetic breeding. The gene single nucleotide polymorphic locus comprises the single nucleotide polymorphic locus of the milk goat PITX1 gene with the 201st position is G or A, wherein the genotype is GG, GA and AA. The detection method comprises the following steps: performing polymerase chain reaction (PCR) amplification on the milk PITX1 gene by using the DNA sequence of the PITX1 gene as a template and a primer pair P as a primer; performing enzyme digestion on the PCR product by using restriction endonuclease MspI; and detecting the enzyme-digested product through agarose gel electrophoresis to identify the single nucleotide polymorphic locus of the milk goat PITX1 gene. Through the adoption of the method, the milk goat species with excellent genetic resources can be established quickly in marker assisted selection (MAS) breeding of the milk goat. The method has the advantages of simplicity and quickness in operation, low cost and high detection precision.

Description

technical field [0001] The invention belongs to the technical field of animal molecular genetics and breeding, relates to the detection of gene single nucleotide polymorphism (SNP) and its application, and in particular relates to dairy goat PITX1 (paired homeotype domain protein transcription factor 1) gene (with sequence Accession No. NW_003104033 for reference) single nucleotide polymorphism sites and detection methods and applications thereof. Background technique [0002] Compared with cows, dairy goats are more suitable for rearing in harsh environmental conditions because of their strong resistance to stress; compared with cows, goat milk has higher nutritional value because of its small fat globules and is free of allergies Original, low acid value, easier to be absorbed by the human body than milk, known as "the king of milk" in the international nutrition community. However, at present, the dairy goat industry in my country is relatively lagging behind. The quanti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 蓝贤勇赵海谕李转见潘传英陈宏雷初朝
Owner NORTHWEST A & F UNIV
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