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Method for cloning complete sequence of coding region in goat PID1 (phosphotyrosine interactiondomain containing 1) gene

A technology of gene encoding and complete sequence is applied in the field of cloning the complete sequence of the coding region of goat phosphotyrosine interaction domain 1 gene, which can solve the problems of high experimental technical requirements, high experimental cost, and unclear length of gene fragments

Inactive Publication Date: 2012-08-01
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

Method used

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  • Method for cloning complete sequence of coding region in goat PID1 (phosphotyrosine interactiondomain containing 1) gene
  • Method for cloning complete sequence of coding region in goat PID1 (phosphotyrosine interactiondomain containing 1) gene
  • Method for cloning complete sequence of coding region in goat PID1 (phosphotyrosine interactiondomain containing 1) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Step 1 extracts the total RNA (ribonucleic acid) in the goat eye muscle tissue, and then reverse-transcribes the total RNA into cDNA;

[0052] 1. Collection of Tissue

[0053] Immediately after bloodletting from the carotid artery of one-year-old Tianfu mutton sheep, about 1 g of abdominal muscle was taken and placed in a liquid nitrogen tank for total RNA extraction.

[0054] 2. RNA preparation and cDNA synthesis

[0055] In the laboratory, the abdominal muscle was ground into powder with liquid nitrogen and loaded into a 1.5ml EP tube. Put it in an ultra-low temperature refrigerator at minus 80°C for later use.

[0056] 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.

[0057] Centrifuge at 13000rpm for 3 minutes at 4°C, transfer the liquid to another new 1.5ml EP tube with a pipette gu...

Embodiment 2

[0100] real-time fluorescent quantitative PCR

[0101] (1) The reaction system and reaction procedure of PID1 and GAPDH gene real-time fluorescent quantitative PCR are as follows:

[0102] Table 2-5PCR reaction system Table.2-5Reaction system of PCR

[0103]

[0104] The reaction conditions of PID1 and GAPDH gene real-time fluorescence quantitative PCR: pre-denaturation at 95°C for 10 sec → 45 cycles (denaturation at 95°C for 10 s → annealing at 61°C for 30 s).

[0105] The reaction system was carried out on the Bio-Rad iq5 fluorescent quantitative PCR instrument. During the PCR reaction, a blank tube without cDNA samples was set as a negative control.

[0106] (2) Drawing of standard curve

[0107] take 10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、10 10 Add 1 μl of each standard substance to the same reaction system as above, and carry out PCR amplification under the same reaction conditions. According to the PCR kinetic curve drawn by the fluorescent quantitative...

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Abstract

The invention discloses a method for cloning a complete sequence of the coding region in goat PID1 (phosphotyrosine interactiondomain containing 1) gene. The method comprises the following steps of: A1. extracting the total RNA (ribonucleic acid) in goat longissimus dorsi tissue, then reversely transcribing the total RNA into cDNA (complementary desoxyribonucleic acid); A2. designing primers and carrying out PCR (polymerase chain reaction): taking a conserved region of the gene in a mammal as a template, and designing conserved primers in the upstream of an initiation codon and in the downstream of a termination codon: the upstream primer P1: 5'-CCCCGCGGCTGGAAGATGTG-3', and the downstream primer P2: 5'-TCCACACTCCCACCCTCCTCA-3'; A3. recovering and purifying a PCR product; and A4. carrying out cloning. The invention provides a simple and quick method for obtaining a complete coding region sequence of the goat PID1gene, and fills in the gap of the gene in goats, thus laying a foundation for further study of the gene in goats.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for cloning the complete sequence of a goat phosphotyrosine interaction domain containing 1 (PID1) gene coding region. Background technique [0002] The phosphotyrosine interaction domain (PID1) was screened for differentially expressed genes in retroperitoneal adipose tissue between obese and normal people by Suppression subtractive hybridization (SSH) technology. The first English letter named the gene NYGGF4 gene, and after submitting to NCBI, it was uniformly named PID1 gene internationally according to the structural characteristics of the gene. The gene has a PTB (phosphotyrosine interaction site) domain, which is similar to the PTB domain on the signaling protein Shc, and is involved in downstream effects such as cell proliferation stimulated by growth factors. The overexpression of PID1 gene in 3T3-L1 adipocytes can significantly down-regulate the expression of gluc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/10C12N15/70C12R1/19
Inventor 徐洪刚徐刚毅汪代华郑程莉马基斯万璐
Owner SICHUAN AGRI UNIV
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