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A method for cloning the complete sequence of goat mc4r gene coding region

A technology of gene coding and full sequence is applied in the field of cloning the full sequence of the gene coding region of goat melanocortin receptor-4 gene, which can solve the problems of high test cost, high experimental technical requirements and heavy workload.

Inactive Publication Date: 2011-12-07
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

1. The gene fragment length of the target PCR product is not clear
2. High test cost
3. Heavy workload and high requirements for experimental technology
The cost is high and the workload is heavy. A gene needs to be cloned several times if it is divided into several segments.

Method used

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  • A method for cloning the complete sequence of goat mc4r gene coding region
  • A method for cloning the complete sequence of goat mc4r gene coding region
  • A method for cloning the complete sequence of goat mc4r gene coding region

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Embodiment Construction

[0048] The present invention will be described in detail below in conjunction with specific embodiments.

[0049] Step 1 extracts the total RNA (ribonucleic acid) in the goat eye muscle tissue, and then reverse-transcribes the total RNA into cDNA;

[0050] 1. Collection of Tissue

[0051] Immediately after bleeding from the carotid artery of one-year-old Tianfu mutton sheep, about 1 g of eye muscle was put into a liquid nitrogen tank for total RNA extraction.

[0052] 2. RNA preparation and cDNA synthesis

[0053] In the laboratory, the eye muscle was ground into powder with liquid nitrogen and put into a 1.5ml EP tube. Put it in an ultra-low temperature refrigerator at minus 80°C for later use.

[0054] Take a new 1.5ml EP tube, add 1ml of Trizol solution, and then add about 80mg of eye muscle tissue powder. After mixing by hand shaking, shake for about 30 seconds with a vibrator and then place at room temperature for 5 minutes.

[0055] Centrifuge at 13000rpm for 3 minu...

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Abstract

The invention discloses a method for cloning the complete sequence of goat MC4R gene coding region, comprising the following steps: A1, extracting total RNA in goat eye muscle tissue, and then reverse-transcribing the total RNA into cDNA; A2, designing primers and PCR Reaction: The upstream primer is designed in the upstream region of the start codon, and the downstream primer is designed in the downstream region of the stop codon; the designed primers are: upstream primer P1: 5′-CTCTCCGCCGCCTAACTTTCGTTT-3′ downstream primer P2: 5′-GTGCCTATAACCTGTGATGTGGAT- 3'. A3, recovery and purification of PCR products; A4, cloning. The invention provides a simple and fast method for obtaining the complete coding region sequence of goat MC4R gene, fills in the blank of the gene in goat, and lays the foundation for further research on the gene of goat.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for cloning the complete sequence of the coding region of goat melanocortin-4 receptor (MC4R) gene. Background technique [0002] Melanocortin receptor-4 (MC4R) is one of the five members of the melanocortin receptor family, which is the smallest G protein-coupled receptor subfamily known so far. Belongs to class A of seven transmembrane alpha-helical G protein receptors and is the product of a series of minigenes. The MC4R gene is encoded by a single exon and is a peptide substance secreted by the ventromedial hypothalamus (VMH). MC4R is coupled with G-protein, and finally affects the transcription and translation of intracellular genes through the cAMP signaling pathway and the phosphatidylinositol signaling pathway. Plays an important role in the regulation of body weight, energy homeostasis and feed intake in humans and mice. MC4R can regulate Leptin, which is an impo...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/10
Inventor 郑程莉徐刚毅陈浩林汪代华徐洪刚
Owner SICHUAN AGRI UNIV
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