Probe primer combination for detecting high-oleic acid transgenic soybeans, kit and method thereof

A technology of genetically modified soybeans and primer combinations, which is applied in the field of genetic engineering and can solve problems such as detection

Inactive Publication Date: 2020-03-24
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] After searching and analyzing existing patents and literature, there are currently no articles or patents related to MLPA detection of high oleic acid transgenic soybean E2D8037-3. How to detect high oleic acid transgenic soybean is an urgent problem to be solved

Method used

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  • Probe primer combination for detecting high-oleic acid transgenic soybeans, kit and method thereof
  • Probe primer combination for detecting high-oleic acid transgenic soybeans, kit and method thereof
  • Probe primer combination for detecting high-oleic acid transgenic soybeans, kit and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1. MLPA Ligation-Dependent Probe and Universal Primer Design for Transgenic Soybean

[0047] Use primer design software such as Primer Premier to design junction-dependent probes for the exogenous gene (nucleotide sequence shown in SEQ ID NO: 1) and the junction region of the soybean genome for the left border flanking sequence of E2D8037-3 (for the design principle, see figure 1 ). The hybridization sequence in the ligation-dependent probe can be designed in one of the following three ways:

[0048] 1. The hybridization sequences of the upstream and downstream connection-dependent probes are exactly located on both sides of the insertion site, that is, the hybridization sequences of the upstream connection-dependent probes are designed based on exogenous gene sequences, and the hybridization sequences of the downstream connection-dependent probes are designed based on the soybean genome;

[0049] 2. The hybridization sequence of the upstream connection-depende...

Embodiment 2

[0058] Example 2. Transgenic soybean event E2D8037-3 specific MLPA PCR detection method

[0059] Using the CTAB method to extract the genome of transgenic soybean E2D8037-3 seeds, first crush the seed material into powder with a grinder; weigh 60±10 mg powder and add it to a 1.5 mL centrifuge tube; add 1200 μL (preheated at 65°C) CTAB lysis buffer , and incubated in a water bath at 65°C for 40 min, during which time the mixture was mixed by inverting 5 times. Centrifuge at 12000g for 10min at room temperature, transfer the supernatant to another new centrifuge tube; add an equal volume of Tris saturated phenol / chloroform / isoamyl alcohol (V / V / V=25 / 24 / 1) to extract two Once, extract once with chloroform / isoamyl alcohol (V / V=24 / 1), centrifuge at 12000g for 10min; carefully absorb the supernatant, add 2 / 3 volume of isopropanol, and mix carefully; precipitate at -20°C for 30min Centrifuge at 12000g for 10min, discard the supernatant, and suck up the liquid as much as possible; add...

Embodiment 3

[0066] Example 3 Transgenic soybean event E2D8037-3 specific MLPA detection sensitivity

[0067] Five samples with different concentrations of E2D8037-3 genome (5ng / μL, 0.5ng / μL, 0.05ng / μL, 0.05ng / μL, 0.005ng / μL) were prepared and amplified using these samples as templates. MLPA products were detected by 2% agarose gel electrophoresis, and the electrophoresis bands were observed under ultraviolet light. The detection sensitivity of this method can be as low as 0.5ng.

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Abstract

The invention discloses a probe primer combination for detecting high-oleic acid transgenic soybeans. The probe primer combination comprises a pair of ligation-dependent probes and a pair of universalprimers, the ligation-dependent probe is designed and obtained for the exogenous gene of the high-oleic acid transgenic soybean based on a certain principle, and the universal primer only has specificity for the ligation-dependent probe and has no specificity for a soybean genome sequence. The invention also provides a detection method of the high-oleic acid transgenic soybean and a kit. By utilizing the exogenous insertion element left boundary flanking sequence and the specificity detection method provided by the invention, specificity detection is carried out on a transgenic soybean eventE2D8037-3, parents, other transgenic soybean events and common crops, so that effective supervision and management on transgenic soybeans and products thereof are realized.

Description

technical field [0001] The application relates to the technical field of genetic engineering, in particular to a probe primer combination, method and kit for detecting the MLPA of the high oleic acid transgenic soybean event E2D8037-3. Background technique [0002] The detection of genetically modified products can be carried out from several aspects such as the DNA level, the mRNA transcription level of the new gene, the expressed protein level, and the metabolite level. At present, the research on the detection technology of genetically modified products at home and abroad is mainly concentrated on the two levels of nucleic acid and protein. In the actual detection work, the detection technology based on DNA has been widely used because of its high sensitivity and good specificity. [0003] RNAi interference (RNA interference, RNAi) refers to the phenomenon that double-stranded RNA (double-stranded RNA, dsRNA) induces the efficient and specific degradation of homologous mR...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2600/13C12Q2600/16C12Q2537/143C12Q2531/113C12Q2521/501C12Q2533/107
Inventor 商颖许文涛黄昆仑李飞武罗云波张晨雷展
Owner KUNMING UNIV OF SCI & TECH
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