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Molecular maker for detecting resistant DNA (Deoxyribose Nucleic Acid) methylation of dairy cow mastitis

A molecular marker and methylation technology, applied in recombinant DNA technology, DNA/RNA fragments, microbial determination/inspection, etc., to achieve strong specificity

Active Publication Date: 2013-09-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in traditional dairy cow mastitis resistance studies, DNA methylation studies of candidate genes are rarely seen

Method used

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  • Molecular maker for detecting resistant DNA (Deoxyribose Nucleic Acid) methylation of dairy cow mastitis
  • Molecular maker for detecting resistant DNA (Deoxyribose Nucleic Acid) methylation of dairy cow mastitis
  • Molecular maker for detecting resistant DNA (Deoxyribose Nucleic Acid) methylation of dairy cow mastitis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 optimizes primer design

[0036] 1. Using Oligo software to design PCR-bisulfite amplification primers

[0037] For the bovine CD4 gene, the sequence rich in CpG dinucleotides within 0-1000bp upstream of the transcription start point is selected as the target sequence, and its nucleotide sequence is shown in SEQ ID No.1. First replace the "CG" locus of the target sequence with "YG", and then replace all "C" with "T" to obtain the bisulfite-converted sequence of the CD4 gene. The nucleotide sequence is as shown in SEQ ID No. .2 shown. Design primers for the bisulfite-converted sequences. The design principles are: the length of the primers is generally between 18 and 25 bases; the G+C content is between 40% and 60%, and the Tm value is between 55 and 65°C. The difference between the Tm value of the upstream and downstream primers should not exceed 4°C; the energy should be less than 5kcal as much as possible, and the maximum should be 7kcal / mol; no hairpin...

Embodiment 2

[0044] Embodiment 2 optimizes PCR reaction

[0045] 1. Bisulfite treatment of blood genomic DNA

[0046] Quantitative DNA was optimized with bisulfite using the EZ DNA Methylation-Gold Kit (ZYMO, agent of Beijing Tianmo Technology). The specific steps are as follows: mix 130 μL CT Conversion Reagent and 20 μL blood genomic DNA (1 μg), divide them into two PCR tubes, each tube has 65 μL CT Conversion Reagent and 10 μL DNA (500 ng) after PCR thermal cycle, and combine the two PCR tubes into Add 600μL M-Binding buffer to the column, mix by inversion, centrifuge at 12000rpm for 30s, discard the waste liquid; put 100μL M-Wash buffer into the column, centrifuge at 12000rpm for 30s, discard the waste liquid; 200μL M-Desulphonation buffer, put into the column, room temperature Place for 20min, centrifuge at 12000rpm for 30s, discard the waste liquid; put 200μL M-Wash buffer into the column, centrifuge at 12000rpm for 30s, discard the waste liquid; put 20μL M-Elution buffer into the c...

Embodiment 3

[0055] Embodiment 3 bisulfite direct sequencing

[0056] 1. Bisulfite mixed pool sequencing and individual direct sequencing

[0057] The 1:5 diluted bisulfite-treated DNA was mixed in equal amounts, and the pooled DNA was selected as a template, and the temperature gradient hot-start PCR amplification was performed to obtain a suitable PCR product, and the PCR product was used after gel electrophoresis detection was qualified. ABI 3730XL direct sequencing detection. Individual sequencing is to directly sequence the individual DNA after amplifying it at a suitable temperature in the previous step. The sequencing work was completed by Beijing Sanbo Polygala Biotechnology Co., Ltd.

[0058] 2. Bisulfite Cloning and Sequencing

[0059] (1) Carry out gel cutting and recovery of the second-step amplification product of Example 2

[0060] (2) connection

[0061] Add the recovered target fragment and the pMD18-T vector into the reaction tube according to the components in Table ...

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Abstract

The invention provides a molecular marker for detecting resistant DNA (Deoxyribose Nucleic Acid) methylation of dairy cow mastitis and a primer pair of the molecular marker. According to an amplification primer pair and a sequencing primer pair designed by the molecular marker, the resistant DNA methylation of the dairy cow mastitis can be detected. The detecting method comprises the following steps of: treating genome DNA of a to-be-detected dairy cow by bisulfate; carrying out PCR (Polymerase Chain Reaction) amplification onto a CD4 gene by utilizing the amplification primer pair; sequencing by utilizing the sequencing primer pair to match with a pyrophosphoric acid apparatus, so that the methylation degree of a CD4 gene promoter region of the to-be-detected dairy cow can be detected. The molecular marker for detecting resistant DNA methylation of dairy cow mastitis disclosed by the invention can be used for providing theoretical basis and hereditary basis for molecular breeding of the dairy cow with the mastitis.

Description

technical field [0001] The invention relates to a molecular marker, in particular to a molecular marker for detecting DNA methylation. Background technique [0002] The QTL (quantitative trait locus) research on functional traits of dairy cows has only been carried out since the mid-1990s, and the most studied are mastitis and fecundity traits. Mastitis will reduce milk production, affect milk quality, and increase the abortion rate and culling rate of cows. In dairy cattle breeding for mastitis resistance, due to the low heritability of mastitis resistance, traditional genetic improvement methods are difficult to improve its disease resistance. Researchers are trying to find stable DNA methyl groups related to mastitis resistance Genetic markers and target genes were selected to select individuals with high resistance to mastitis, laying the foundation for breeding new lines of dairy cows resistant to mastitis. [0003] Leukocytes are a type of nucleated cells, and the co...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68C12N15/85A01K67/027
Inventor 俞英王晓铄王雅春孙东晓张毅张胜利张沅
Owner CHINA AGRI UNIV
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