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Visualized detection method of BAR transgenic crops

A detection method and crop technology, applied in the field of genetic engineering, can solve the problem of high detection cost of fluorescent probes, achieve the effects of cheap reagents, lower detection costs, and convenient synthesis

Inactive Publication Date: 2012-11-21
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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Problems solved by technology

Among them, the first type of method is widely used. The detection method based on nucleic acid is mainly based on the detection method of fluorescent quantitative PCR. Fluorescent quantitative PCR has many advantages, including high sensitivity, high specificity, and can be both qualitative and quantitative. etc., but the required equipment and detection costs of fluorescent probes are much higher than ordinary PCR

Method used

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  • Visualized detection method of BAR transgenic crops
  • Visualized detection method of BAR transgenic crops
  • Visualized detection method of BAR transgenic crops

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: Detection of BAR gene in rice

[0021] 1. Extraction of rice genomic DNA;

[0022] Rice genomic DNA was extracted by the improved CTAB method. ①Grind an appropriate amount of rice leaves or seeds into a fine powder in a mortar under the protection of liquid nitrogen, put a small amount of the powder into a 1.5mL EP tube, add 700μl of CTAB extraction buffer preheated at 65°C, shake and mix well, and incubate at 65°C for 2h ; ② Add 600 μl of chloroform-isoamyl alcohol (24:1) after the sample is cooled to room temperature, and mix it upside down for 5 minutes; ③ Centrifuge at 7500r.min-1 for 10 minutes, and transfer the supernatant to a new EP tube; ④ Add etc. Volume -20 ℃ pre-cooled isopropanol, mix well, and place at 20 ℃ for 30 minutes; ⑤10,000r.min-1 centrifuge for 15 minutes, discard the supernatant; Centrifuge at 10,000r.min-1 for 3 minutes, discard the supernatant, evaporate ethanol at room temperature, dissolve DNA in sterile high-purity water, and ...

Embodiment 2

[0033] Embodiment 2: the detection of BAR gene in cotton

[0034] 1. Extraction of cotton genomic DNA;

[0035] Cotton genomic DNA was extracted by the improved CTAB method. ①Grind an appropriate amount of cotton leaves or seeds into a fine powder in a mortar under the protection of liquid nitrogen, take a small amount of powder into a 1.5mL EP tube, add 700μl of CTAB extraction buffer preheated at 65°C, shake and mix well, and incubate at 65°C for 2h ; ② Add 600 μl of chloroform-isoamyl alcohol (24:1) after the sample is cooled to room temperature, and mix it upside down for 5 minutes; ③ Centrifuge at 7500r.min-1 for 10 minutes, and transfer the supernatant to a new EP tube; ④ Add etc. Volume -20 ℃ pre-cooled isopropanol, mix well, and place at 20 ℃ for 30 minutes; ⑤10,000r.min-1 centrifuge for 15 minutes, discard the supernatant; Centrifuge at 10,000r.min-1 for 3 minutes, discard the supernatant, evaporate ethanol at room temperature, dissolve DNA in sterile high-purity wa...

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Abstract

The invention belongs to the technical field of genetic engineering and relates to a detection technology of transgenic crops, in particular to a visualized detection method of BAR transgenic crops. The method comprises steps as follows: adding DNAzyme-marked specific probes for detecting the BAR gene into a PCR (polymerase chain reaction) system, amplifying the BAR gene by forward and reverse primers, releasing the DNAzyme markers from the probes by exonuclease activity of nucleic acid polymerase 5'-3', and detecting the markers released from the PCR product to judge whether the sample is a transgenic sample or not. The method provided by the invention has the characteristics of being efficient and convenient and low in cost and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a detection technology of transgenic crops, in particular to a visual detection method of transgenic BAR gene crops. Background technique [0002] Genetically modified crops are bred by introducing genes from other organisms or species by using tissue culture technology and genetic recombination technology. Such crops often have characteristics such as resistance to diseases and insect pests, resistance to herbicides, or higher nutrition than non-genetically modified crops. At present, there are more than 20 kinds of genetically modified plants in China, among which genetically modified soybeans, potatoes, corn, tobacco, peanuts, spinach, sweet peppers, wheat, etc. have undergone field tests, genetically modified cotton has been used on a large scale, and foreign genetically modified soybeans have also entered my country in large quantities. Although genetically modifie...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 唐卓余懋群陈蓉龙海
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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