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

A technology of expression level and real-time fluorescence quantification, applied in the field of genetic engineering, can solve problems such as affecting the accuracy of quantification, and achieve the effects of good stability, low experimental cost, and convenient and rapid quantitative detection.

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

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Problems solved by technology

However, since SYRB Green binds to all double-stranded DNA, false positives caused by primer-dimers, single-stranded secondary structures, and erroneous amplification products will affect the accuracy of quantification

Method used

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

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Embodiment 1

[0031] The fluorescence quantitative PCR detection kit of MSTN 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 MSTN 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 upstream primer sequence is: 5'-AACCAGGAGAAGATGGAC-3';

[0039] The downstream primer sequence is: 5'-TTAGAGGGTAACGACAGC-3'.

[0040]Standard MSTN gene template: the concentration is 0.8 μmol / L, and the MSTN gene template sequence is 5’-AACCAGGAGAAGATGGACTGACTCCTTTTTAGAAGTCAAGGTAACAGACACACCAAAAAGATCTAGGAGAGATTTTGGGCTTGATTGTGATGAACACTCCAGAATCTCGA...

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Abstract

The invention provides a specific primer for detecting the mRNA expression levels of MSTN genes of cows. The specific primer is characterized by having the nucleotide sequence as follows: an upstream primer has the sequence: 5'-AACCAGGAGAAGATGGAC-3'; and a downstream primer has the sequence: 5'-TTAGAGGGTAACGACAGC-3'. The invention also provides a corresponding fluorescent quantitative PCR detecting kit. The specific primer can be used for achieving the aims of conveniently, rapidly and quantitatively detecting the mRNA transcriptional levels of MSTN genes, monitoring the muscle growth and development states of different cows (including milk cows, cattle and yaks), different tissues and different periods and also identifying diseases related to cow muscle dysplasia. 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 MSTN 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
CPCC12Q1/6851C12Q1/6888C12Q2600/124C12Q2600/158C12Q2531/113C12Q2545/113C12Q2563/107
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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