Primer sequence for cauliflower mosaic virus 35s promoter-containing transgenic crop nucleic acid amplification

A cauliflower flower leaf and primer sequence technology, applied to the determination/testing of microorganisms, biochemical equipment and methods, etc.

Inactive Publication Date: 2004-01-28
QIAGEN SHENZHEN CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Primer sequence for cauliflower mosaic virus 35s promoter-containing transgenic crop nucleic acid amplification
  • Primer sequence for cauliflower mosaic virus 35s promoter-containing transgenic crop nucleic acid amplification
  • Primer sequence for cauliflower mosaic virus 35s promoter-containing transgenic crop nucleic acid amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Qualitative detection: Select genetically modified and non-genetically modified agricultural products such as soybeans, corn, rape, tomato, and potatoes, and extract genomic DNA with the Promega magnetic bead method, specifically: weigh 100mg of plant material, place it in a 2ml centrifuge tube; add 500ul Lysis Buffer A and 5ul RNase A, mix with the material; add 250ulLysis Buffer B, mix well, and let stand at room temperature for 10 minutes; add 750ul precipitation buffer, mix well and centrifuge at high speed (13000×g) for 10 minutes; pipette the supernatant into a clean 2ml Add 50ul magnetic powder solution to the centrifuge tube, mix well; add 0.8 volume of isopropanol to the mixture, mix well, and let it stand at room temperature for 5 minutes; put the centrifuge tube on the magnetic rack for 1 minute, remove the clarified liquid; take out the centrifuge tube, and then Add 250ul Lysis Buffer B, mix well and place on the magnetic rack for 1 minute to remove the clari...

Embodiment 2

[0071] Quantitative detection: Take the Fluka soybean standard product that meets the international standard, the genomic DNA extraction method is the same as before, and use the 35s7240 / 35s2R primers for quantitative detection of fluorescent PCR, and the fluorescent PCR instrument uses RocheLight-Cycler. The results are shown in Table 4

[0072] sample

[0073] The regression curve that obtains from above-mentioned data is y=-0.346x+7.9383; Correlation r2=0.99; Then use 1.0% and 2.0% standard substance as tested sample, the GMO content that measures is respectively 0.94% and 2.26%, The quantitative deviation is 5%-15% (the quantitative deviation of GENESCAN company in the European Union is ≤20%), suggesting that the detection efficiency of the primers used has reached the international level of this project in the field of nucleic acid amplification detection.

Embodiment 3

[0075] Electrophoresis detection: take the Fluka transgenic soybean standard product that meets international standards, use the same method for genomic DNA extraction as before, and use 35s7240 / 35s2R primers for PCR amplification. The PCR amplification conditions are as follows:

[0076] 93°C: 3min 1 cycle

[0077] 93°C: 15 sec; 60°C: 30 sec; 72°C 1min 40 cycles

[0078] Take the above-mentioned amplification products, use 1% agarose, 2V / CM electrophoresis for one hour, soak in EB working solution for 15 minutes, and observe the results under ultraviolet light: a clear amplification band can be seen in positive samples, while negative samples have no amplification belt, see image 3 .

[0079] image 3 middle

[0080] 1) 0.0% Genetically Modified Soybean Standard 2) 0.1% Genetically Modified Soybean Standard

[0081] 3) 0.5% genetically modified soybean standard product 4) 2.0% genetically modified soybean standard product

[0082] M) 2000ladder

[0083] Advantages of th...

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Abstract

The present invention provides the primar sequences for nucleic acid amplification of the transgenic crop containing caulimovirus 35s promotor, they are contained in the extended regional range of four pairs of optimal primer sequences, and the extended regional range of every pair of primer sequence is: the primer pair is formed from upstream primer and downstream primer, in the upstream primer position of said primer pair 10 bases are forward extended and 10 bases are backward extended, and in the downstream primer position 10 bases are forward extended and 10 bases are backward extended, in the regional range of upstream and downstream extended positions of said primer pair the primer sequence can be obtained.

Description

technical field [0001] The invention relates to a primer sequence for nucleic acid amplification of transgenic crops containing cauliflower mosaic virus 35s promoter Background technique [0002] In the past three to four years, the detection of genetically modified products has become a major event at home and abroad, which has attracted widespread attention and attention from all walks of life. Although agencies affiliated to the United Nations (FAO, WHO, etc.) and some regional international organizations are convening to establish a unified worldwide testing technology and method standard, due to inconsistencies in the safety of genetically modified products, risk management measures and other interests, countries currently It is still difficult to reach a consensus. Based on the detection technology of GMO products in various countries in the world, it is mainly divided into the following three types according to the detection target: detection of inserted foreign gene...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 朱文斯黄茜华朱水芳
Owner QIAGEN SHENZHEN CO LTD
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