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Method for cloning intron-containing partial sequence of GAPDH (glyceraldehyde-phosphate dehydrogenase) gene of Cyprinus carpio var. Jian and real time PCR method thereof

A cloning method and intron technology, applied in the field of molecular biology, can solve problems such as DNA residues, reduce RNA yield, and undetectable genes, and achieve the effect of eliminating pollution and increasing efficiency

Active Publication Date: 2013-06-05
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0003] In the RNA sample extracted with Trizol, there will inevitably be a small amount of DNA residue. After reverse transcription, the cDNA still contains DNA, and the DNA will also be amplified together with the cDNA as a template, which will inevitably affect the amplification efficiency. , thus affecting the accuracy of the experimental results
In order to eliminate DNA contamination, it is usually necessary to treat the RNA sample with DNase enzyme, but this will increase the probability of sample contamination, and also reduce the yield of RNA, resulting in genes with very low expression levels that cannot even be detected

Method used

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  • Method for cloning intron-containing partial sequence of GAPDH (glyceraldehyde-phosphate dehydrogenase) gene of Cyprinus carpio var. Jian and real time PCR method thereof
  • Method for cloning intron-containing partial sequence of GAPDH (glyceraldehyde-phosphate dehydrogenase) gene of Cyprinus carpio var. Jian and real time PCR method thereof
  • Method for cloning intron-containing partial sequence of GAPDH (glyceraldehyde-phosphate dehydrogenase) gene of Cyprinus carpio var. Jian and real time PCR method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Amplification of Partial Sequence Containing Introns of the Gene Carp GAPDH Gene

[0023] Jian carp comes from the Yixing Breeding Base of the Freshwater Fishery Research Center of the Chinese Academy of Fishery Sciences. According to the cDNA sequence of the carp GAPDH gene published on GenBank and the DNA sequence of the zebrafish GAPDH gene, a pair of primers are designed to amplify the intron of the Jian carp GAPDH gene. See Table 1.

[0024] Table 1 Primers for amplifying Jian carp GAPDH

[0025]

[0026] PCR was carried out with 50ng genomic DNA, 15mmol / L Tris-HCl, 50mmol / L KCl (pH8.0), 2mmol / L MgCl 2 , 200umol / L dNTPs, 10μmol / L primer and 0.5U Taq DNA polymerase in a 25μl reaction system, the reaction conditions were 95°C for 3min, 30 cycles (94°C for 30s, 57°C for 30s, 72°C for 1min), 72°C extension 8min, stored at 4°C. The amplified PCR product was purified and connected to the pMD18-T vector, transformed into competent cells, screened by blue a...

experiment example 2

[0027] Experimental Example 2 Establishment of a real time PCR method for GAPDH in carps based on SYBR Green I dye technology

[0028] About 50 mg of Jian carp adult liver was taken, and total RNA was extracted by Trizol lysis method according to the RNA extraction manual of Takara Company. Use 1 μg of total RNA, use Oigo dTPrimer and Random 6 mers as primers, carry out RT reaction according to the instructions of M-MLV, the total reaction volume is 10 μl, and then carry out real time PCR with this RT solution as template, the fluorescent dye is SYBR Green I, See Table 2 for primers. The reaction conditions are: 94°C for 3min, then 40 cycles of 94°C for 5sec, 62°C for 20sec, and finally 72°C for 3min, and store at 4°C. To test the specificity of the reaction, a melting curve analysis was performed after PCR to determine whether the obtained product was the target product. The amplification temperature was slowly increased from 65°C to 95°C with an increment of 0.2°C, and the...

experiment example 3

[0031] Experimental example 3 establishment of standard curve

[0032] Taking the reverse-transcribed Jian carp liver cDNA as a standard, use the diluent provided by Takara to perform multiple dilutions at a 10-fold dilution, and dilute to 1 / 10, 1 / 100, 1 / 1000, 1 / 10000, 1 / 100000. Then standard products with 6 concentrations (100~10 -5 ) for real time PCR reaction, the reaction conditions are: 94°C for 3min, then 40 cycles of 94°C for 5sec, 62°C for 20sec, and finally 72°C for 3min, and store at 4°C. The standard curve equation is Y=-0.3705x+7.48, and its correlation coefficient is r 2 = 0.991, see image 3 .

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Abstract

The invention relates to a method, which is useful for research on functional genes of Cyprinus carpio var. Jian by real-time polymerase chain reaction and using GAPDH (glyceraldehyde-phosphate dehydrogenase) as an internal control gene. The method includes cloning an intron-containing partial sequence of the GAPDH of Cyprinus carpio var. Jian, designing an intron-spanning primer, optimizing conditions for polymerase chain reaction to eliminate DNA contamination, thereby improving the amplification efficiency.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for cloning a partial sequence of an intron-containing gene of the carp housekeeping gene GAPDH gene and a real time PCR method thereof. Background technique [0002] In real-time quantitative PCR (real time PCR), the relative expression of the target gene is determined by the normalization of the internal reference gene. An ideal internal reference gene should have a constant expression level in different tissues and not be affected by experimental treatment conditions. A large number of studies have shown that there is currently no universal internal reference gene that meets this condition, and the best choice is that the expression level of the internal reference gene has little change in the samples studied. Glyceraldehyde-3-phosphate dehydrogenase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) is an enzyme in the glycolysis reaction, consi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10C12N15/11
Inventor 唐永凯李建林俞菊华李红霞董在杰
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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