A method for detecting single nucleotide polymorphism of yak foxo1 gene and its kit

A technology of single nucleotide polymorphism and detection method, which is applied in the field of detection method and kit of single nucleotide polymorphism of yak FOXO1 gene, can solve the problem of reducing the tightness of the binding of probe and target sequence, and the problem of reducing probe and target sequence. Base mismatch with the target sequence, affecting the fluorescence release of the probe, etc., to achieve the effects of good specificity, high sensitivity and low cost

Active Publication Date: 2018-10-19
GANSU AGRI UNIV
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Problems solved by technology

[0007] At present, there are few studies on the genetic variation of the FOXO1 gene in the existing technology, and the research on the function of the gene locus and the research on the relationship between genetic variation and economic traits are still blank, and the existing research on the single nucleotide polymorphism of the FOXO1 gene There is a problem of base mismatch between the probe and the target sequence in the detection, which will greatly reduce the binding tightness of the probe and the target sequence, affect the fluorescence release of the probe, and lead to inaccurate detection results

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  • A method for detecting single nucleotide polymorphism of yak foxo1 gene and its kit
  • A method for detecting single nucleotide polymorphism of yak foxo1 gene and its kit
  • A method for detecting single nucleotide polymorphism of yak foxo1 gene and its kit

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Embodiment 1

[0045] Embodiment one: a kind of detection method of single nucleotide polymorphism of yak FOXO1 gene, comprises the following steps:

[0046] Preparation of yak FOXO1 gene PCR amplification product: first extract yak blood genomic DNA, after dilution, use the FOXO1 gene of the yak whole genome DNA to be tested as a template, use specific primer pair A and specific primer B as primers, and use The yak FOXO1 gene was amplified by PCR, followed by polyacrylamide gel electrophoresis, and then the PCR amplified product was purified with a purification kit, sequenced, and the single nucleotide polymorphism at position 720 of the yak FOXO1 gene (occurrence C-T mutation).

[0047] Specific steps are as follows:

[0048] Step 1: Preparation of yak FOXO1 gene amplification product: first extract yak blood genomic DNA, dilute it, use 1% agarose gel electrophoresis and spectrophotometer to double detect the quality of DNA, use the detected and extracted DNA and extract 20 μL was dilute...

Embodiment 2

[0071] Embodiment 2: Data statistics

[0072] The genotype and allele frequency were calculated according to the theory of population genetics, and the homozygosity (Ho), heterozygosity (He), effective allele number (Ne) and polymorphism information of the FOXO1 gene polymorphic sites in the yak population were calculated content (PIC). Hardy-Weinberg balance law was used for balance test, and SHEsis software was used for paired linkage disequilibrium analysis. The chi-square test was performed on the genotype distribution of the FOXO1 gene polymorphism site in the yak population by using SPSS17.0 software, and the correlation between different genotypes and body size traits was analyzed.

Embodiment 3

[0073] Embodiment three: result analysis

[0074] PCR amplification product: using yak blood genomic DNA as a template, and using the designed primers for PCR specific amplification, the results are shown in figure 1 , figure 1 middle: figure 1 Among them, 1 and 2 represent primers FOXO1 and FOXO2 respectively; M represents Mark. Depend on figure 1 It can be seen that the PCR amplification products are all single-specific bands, which are basically consistent with the expected size. The above-mentioned specific primer pair A and specific primer pair B are sequenced, and the results show as follows figure 2 As shown, there are 720(C / T)1 polymorphic sites in the partial CDS region sequence of FOXO1 gene.

[0075] Typing of yak FOXO1 gene by HRM method: After sequencing the FOXO1 gene of yak through the constructed DNA pool, the sequence of the spliced ​​CDS region was obtained by analyzing and splicing with Chromas software. The results of genotype analysis of 1 polymorph...

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Abstract

The invention relates to the technical field of gene polymorphism detection, in particular to a detection method of yak FOXO1 gene single nucleotide polymorphism and a kit thereof. The detection method comprises the following steps: 1) preparing a yak FOXO1 gene amplification product; 2) synthesizing a high and low temperature interior label; 3) preparing an HRM-PCR (High Resolution Melt-Polymerase Chain Reaction) amplification product; 4) collecting a fluorescence signal. Compared with an HRM method used for detecting polymorphic sites in the prior art, a gene probe designed by the detection method is more accurate and flexible in positioning, is accurate in parting, does not need PCR post-processing, has high working efficiency and good repeatability and is low in sample quality and quantity requirements, especially, the concentration requirement of a DNA template can be lowered to 0.1ng / mu L, and time and labor are saved when a sample size is large. Meanwhile, the detection kit has the advantages of high sensitiveness, high detection speed and good stability.

Description

technical field [0001] The invention relates to the technical field of gene polymorphism detection, in particular to a method for detecting the single nucleotide polymorphism of yak FOXO1 gene and a kit thereof. Background technique [0002] FOXO1 is a member of the FOXO family. As a transcription factor, it regulates the transcription of downstream target genes through post-translational modification, and participates in biological processes such as oxidative stress, apoptosis, DNA damage repair, and cell cycle arrest. At present, the mechanism of action of FOXO1 gene has not been fully elucidated, and research on FOXO1 gene has become a research hotspot today. Qualitative analysis of FOXO1 gene is helpful for further research on FOXO1 gene. [0003] Single nucleotide polymorphisms (SNPs) are the most frequently occurring variants in the genome. Studying SNP can promote people's understanding of genome structure, gene evolution history and disease origin. SNP is an impor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q1/6888C12Q2600/124C12Q2600/156C12Q2545/101C12Q2527/107C12Q2563/107
Inventor 张全伟赵兴绪王琪张勇马友记
Owner GANSU AGRI UNIV
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