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Method for detecting linked mutation of AMPD1 gene of Qinchuan cattle

A technology of gene linkage, Qinchuan cattle, applied in the field of detection of Qinchuan cattle AMPD1 gene linkage mutation, can solve problems such as lack of research

Inactive Publication Date: 2012-02-29
NORTHWEST A & F UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

At present, there is a lack of research on the genetic variation of the AMPD1 gene in local yellow cattle in China, especially Qinchuan cattle. The functional research of this gene and its genetic variation and growth and carcass traits (such as: body height, body oblique length, waist angle width, fasting for 24 hours before slaughter Live weight, carcass weight, slaughter rate and other traits) research is still blank

Method used

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  • Method for detecting linked mutation of AMPD1 gene of Qinchuan cattle
  • Method for detecting linked mutation of AMPD1 gene of Qinchuan cattle
  • Method for detecting linked mutation of AMPD1 gene of Qinchuan cattle

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Experimental program
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Effect test

Embodiment 1

[0065] 1. Collection of experimental animals

[0066] From September to November 2008, at Qinbao Animal Husbandry Development Co., Ltd. in Shaanxi Province, 30±2-month-old bulls were randomly selected from the Qinchuan cattle population and slaughtered, and a total of 215 Qinchuan cattle blood samples were collected. Add 0.5mL of ACD anticoagulant to 15mL of whole blood, mix upside down and put it in an ice box and bring it back to the laboratory. Store the sample at -80°C for later use.

[0067] 2. Extraction process of genomic DNA from bovine blood samples

[0068] Extraction of genomic DNA from blood samples, the specific operation is as follows:

[0069] (1) After taking out the blood sample from -80°C, put it at room temperature and let it thaw slowly; in the semi-frozen-thawed state, draw 800 μL of blood into a 2 mL sterilized centrifuge tube;

[0070] (2) Draw 800 μL from the PBS buffer to make the total volume reach 1600 μL, and shake gently for 10 min;

[0071] (3)...

Embodiment 2

[0106] 1. Design of PCR primers for the 11th intron of Qinchuan cattle AMPD1 gene

[0107] Taking the bovine (accession number: NC_007301) sequence published by GenBank in the NCBI database as a reference, the PCR primers (fragment size: 299bp) of the 11th intron of the AMPD1 gene of Qinchuan cattle were designed using Premier 5.0 software combined with Oligo 6.0 software. The primer sequence as follows:

[0108] Upstream primers:

[0109] 5'-AACCCTCAGGCTCACCCA-3'18bp;

[0110] Downstream primers:

[0111] 5'-GGGCTTAGGGCTCTTGGA-3' 18bp.

[0112] The primer pair amplifies the 11th intron region of Qinchuan cattle AMPD1 gene.

[0113] 2. PCR amplification

[0114] (1) PCR reaction system

[0115] The PCR reaction system adopts the mixed sample addition method. In the experiment, we usually calculate the amount required for each component first, then calculate the total amount of sample addition, and then divide it into each PCR tube equally, then add template DNA, and the...

Embodiment 3

[0137] According to the electropherogram results after sequencing of individual and mixed DNA templates, using the bovine NC_007301 sequence published by GenBank in the NCBI database as a reference, the sequenced sequence and the published reference sequence were sequenced using BioXM (Version 2.6) software and DNA Star 7.10 software. Comparison and mutation site analysis showed that the base mutation site was quickly screened. In order to further verify the variation of the base mutation site, the next experiment was carried out in the entire Qinchuan cattle population.

[0138] Example 4: Polyacrylamide gel electrophoresis analysis of Qinchuan cattle AMPD1 gene

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Abstract

The invention discloses a method for detecting the linked mutation of an AMPD1 gene of Qinchuan cattle. The method is characterized in that: a complete genomic DNA sequence of Qinchuan cattle to be measured and containing the AMPD1 gene is used as a template; polymerase chain reaction primers are designed under the presence of 2*Reaction Mix, ddH2O, a Forward primer, a Reverse primer, a Golden DNA polymerase, and a DNA template and are amplified under a PCR reaction program; PCR products are degenerated after the amplification; and amplified fragments are subjected to polyacrylamide gel electrophoresis and are analyzed through a gel imaging system to determine single nucleotide polymorphisms (SNPs) of the AMPD1 gene. The detection method has the advantages of simple operation, rapidness, low cost, high detection accuracy and convenient popularization and application.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to the detection of gene single nucleotide polymorphisms (SNPs), in particular to a rapid detection of Qinchuan cattle adenosine monophosphate deaminase 1 (AMPD1) gene intron 11 region The PCR-SSCP (PCR-single-strand conformation polymorphism) method of the 19416th and 19421st single nucleotide polymorphism, the method utilizes polyacrylamide gel electrophoresis to include this single nucleotide polymorphic position The gene sequence of the point is detected, and the fragments are separated, and the polymorphism of the fragment is analyzed by the gel imaging system, and then combined with the sequencing technology, so as to accurately determine the SNPs. Background technique [0002] Through the analysis of SNP, we can well explain the differences caused by individual phenotypes from the perspective of genetics. Compared with other molecular markers, SNP has the highest and most ab...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 刘小林贺花张慧林
Owner NORTHWEST A & F UNIV
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