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Primer sequence for NOS terminator-containing transgenic crop mucleic acid amplification

A primer sequence and transgenic technology, applied in 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

AI Technical Summary

Problems solved by technology

None of this would be possible without proven GMO detection methods

Method used

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  • Primer sequence for NOS terminator-containing transgenic crop mucleic acid amplification
  • Primer sequence for NOS terminator-containing transgenic crop mucleic acid amplification
  • Primer sequence for NOS terminator-containing transgenic crop mucleic acid amplification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] 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 250ul Lysis BufferB, 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

[0076] Electrophoresis detection: take the transgenic rapeseed standard product that meets the international standard as the material, the genomic DNA extraction method is the same as before, and use nos-U / nos-R primers for PCR amplification. The PCR amplification conditions are as follows:

[0077] 93°C: 3min 1 cycle

[0078] 93℃: 15 sec; 60℃: 30 sec; 72℃1min 40 cycles

[0079] 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 .

[0080] image 3 middle,

[0081] 1) 0.0% transgenic canola standard 2) 0.1% transgenic canola standard

[0082] 3) 0.5% transgenic canola standard 4) 2.0% transgenic canola standard

[0083] M) 2000 ladder

[0084] Advantages of the present invention: the nos-U / nos-R ...

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Abstract

The present invention provides the primer sequences for nucleic acid amplification of the transgenic crop containing NOS terminator, these sequences are distributed in the extended regional range of two pairs of optimal primers, and the extended regional range of every pair of described two pairs of primers is that in the upstream primer position 10 bases are backward extended, and in the downstream primer position 10 bases are forward extended and 10 bases are backward extended, and in the regional range of upstream and down stream extended positions of said primer pair the primer sequence is obtained. The fluorescence PCR detection of said optimal primer pairs shows that their detection sensibility can be up to 0.1%, and non-transgenic crop product soybean and corn have no amplificationsignal.

Description

technical field [0001] The invention relates to a primer sequence for nucleic acid amplification of transgenic crops containing NOS terminator 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, detection of foreign ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 朱文斯黄茜华朱水芳
Owner QIAGEN SHENZHEN CO LTD