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Specific primer for detecting mRNA expression levels of MYF5 genes of cows and fluorescent quantitative detecting kit

A technology of expression level and specificity, applied in the field of genetic engineering, can solve the problems of abnormal development of myogenic cells, and achieve the effects of high sensitivity, low experimental cost and good stability.

Inactive Publication Date: 2015-09-09
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lack of expression of the MYF5 gene prevents myoblasts from developing normally during the decisive period of myogenesis

Method used

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  • Specific primer for detecting mRNA expression levels of MYF5 genes of cows and fluorescent quantitative detecting kit
  • Specific primer for detecting mRNA expression levels of MYF5 genes of cows and fluorescent quantitative detecting kit

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Embodiment 1

[0031] The fluorescence quantitative PCR detection kit of MYF5 gene mRNA expression level of the present invention is made up of following components:

[0032] 2×SYBR GREEN MIX 5mL;

[0033] Positive control: 500 μL of standard MYF5 gene template;

[0034] Primer mixture 500μL;

[0035] Ultrapure water 5mL.

[0036] Among them, 2×SYBR GREEN MIX consists of the following components: Taq DNA polymerase 0.1 U / μL, dNTPs substrate 0.4 mmol / L, Mg 2+ 5.0 mmol / L, KCl 100 mmol / L, Tris·HCl 20 mmol / L at pH 8.3, 0.02% gelatin and SYBR Green dye.

[0037] Primer mixture: upstream primer 8 μmol / L, downstream primer 8 μmol / L. in,

[0038]The sequence of the upstream primer is: 5'-CCTGAATGTAACAGCCCTAT -3';

[0039] The sequence of the downstream primer is: 5'-TGATCCGATCCACTATGC-3'.

[0040] Standard MYF5 gene template: the concentration is 0.8 μmol / L, the MYF5 gene template sequence is

[0041] 5'-CCTGAATGTAACAGCCCTATCTGGTCCAGAAAGAGCAGCAGTTTTGACAGCGTCTACTGTCCTGATGTACCAAATGTATATGCCACG...

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Abstract

The invention provides a specific primer for detecting the mRNA expression levels of MYF5 genes of cows. The specific primer is characterized by having the nucleotide sequence as follows: an upstream primer has the sequence: 5'-CCTGAATGTAACAGCCCTAT-3'; and a downstream primer has the sequence: 5'-TGATCCGATCCACTATGC-3'. The invention also provides a corresponding fluorescent quantitative PCR detecting kit. The aim of conveniently, rapidly and quantitatively detecting the mRNA transcriptional levels of MYF5 genes of different cows is achieved. The specific primer can be used for monitoring the mRNA expression states of the MYF5 genes of different cows, different tissues and different periods, detecting the expression status of the gene in a muscle development process and also identifying diseases related to muscle growth retardation. The specific primer has the advantages of high sensitivity, good stability and low experimental cost on the aspect of detecting the transcriptional levels of the genes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a specific primer and a fluorescence quantitative PCR detection kit for detecting the expression level of bovine MYF5 gene mRNA. Background technique [0002] Polymerase chain reaction (Polymerase Chain Reaction, PCR) is the most commonly used technique in DNA manipulation technology in vitro. PCR technology puts template DNA, specific primers, dNTPs substrates, heat-resistant DNA polymerase, magnesium ions, etc. in the same buffer reaction system, and performs repeated thermal cycles of high-temperature denaturation, low-temperature annealing, and medium-temperature chain extension to achieve target DNA Fragments appear as 2 in the reaction solution n Double amplification (where n is the number of thermal cycles). [0003] Fluorescent quantitative PCR technology is based on ordinary PCR reaction, combined with real-time fluorescence detection technology...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q1/6851C12Q2600/124C12Q2600/158
Inventor 裴杰郭宪包鹏甲褚敏梁春年丁学智阎萍冯瑞林王宏博朱新书
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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