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Qualitative PCR detection method for transgenic rice kefeng No. 6

A technology of transgenic rice and detection method, applied in the field of qualitative PCR detection of transgenic rice Kefeng 6

Inactive Publication Date: 2011-08-24
CHINA NAT RICE RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

my country has not yet approved the commercial production of genetically modified rice, but some varieties (strains) have been approved for environmental release and productive experiments

Method used

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  • Qualitative PCR detection method for transgenic rice kefeng No. 6
  • Qualitative PCR detection method for transgenic rice kefeng No. 6
  • Qualitative PCR detection method for transgenic rice kefeng No. 6

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Cloning of flanking sequences of transgenic rice line Kefeng No. 6 exogenous insertion vector

[0029] 1. Experimental materials

[0030] 1. Plant material: transgenic rice Kefeng 6 and its receptor variety Minghui 86, provided by Fujian Academy of Agricultural Sciences;

[0031] 2. Reagents: Taq DNA polymerase, 10×PCR Buffer (100mM Tris-HCl pH8.3, 500mMKCl, 15mM MgCl 2 ), dNTP were purchased from Treasure Bioengineering Dalian Co., Ltd.; 100bp ladder DNA Marker was purchased from Beijing Quanshijin Biotechnology Co., Ltd. PCR product purification kit (Cat.NO.SK1141) and PCR product cloning kit (Cat.NO.SK2213) were purchased from Shanghai Sangon Bioengineering Co., Ltd. Primer synthesis and clone sequencing were completed by Invitrogen. Other biochemical reagents are imported subpackages or domestic analytical pure.

[0032] 3. Experimental instrument: PCR amplification instrument: PTC-200 (produced by Bio-Rad)

[0033] Biological spectrophotometer 6131 (m...

Embodiment 2

[0054] Example 2: Line-specific qualitative PCR detection based on two exogenous insertion flanking sequences of the transgenic rice line Kefeng 6

[0055] 1. Experimental materials: transgenic rice line Kefeng 6 and its derivative variety II Youkefeng 6 were provided by Fujian Academy of Agricultural Sciences; other transgenic rice Huahui 1 and Bt63 were provided by Huazhong Agricultural University; The non-transgenic rice pairs Zhaominghui 86 and II Youming 86 were provided by Fujian Academy of Agricultural Sciences, Xianyou 63 was provided by Huazhong Agricultural University, and Xianyou 10 was provided by Zhejiang University. The enzymes and reagents used in the experiment are the same as in Example 1.

[0056] 2. Experimental process and methods:

[0057] 1. Extraction and detection of DNA: same as Example 1.

[0058] 2. Specific detection based on two different insertion sites: According to the two sequences determined in Example 1, two pairs of PCR primers were design...

Embodiment 3

[0064] Example 3: Sensitive detection of strain-specific detection

[0065] 1. Experimental materials: transgenic rice line Kefeng 6 and control recipient variety Minghui 86. The enzymes and reagents used in the experiment are the same as in Example 1.

[0066] 2. Experimental process and methods:

[0067] 1. Extraction and detection of DNA. After grinding the seeds of Kefeng 6 and its non-transgenic control recipient Minghui 86 into powder, a series of mixed samples were prepared. The content of Kefeng 6 in each sample was 10% and 5.0% respectively. , 2.5%, 1.25%, 0.5%, 0.1%, 0.05% and 0.01% (W / W), after mixing, the DNA extraction method is the same as in Example 1.

[0068] 2. Sensitivity detection based on two different insertion sites: the primers used are shown in Table 4, and the PCR reaction system is 1×PCR buffer, 200umol / L dNTP, 0.2umol / L specific primer, and 0.025U / uLTaq enzyme. The reaction program was 95°C, 5min; 35 cycles of 94°C for 30s, 59°C for 45s, and 72°C...

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Abstract

The invention provides a qualitative polymerase chain reaction (PCR) detection method for flanking sequences of transgenic rice kefeng No. 6. The method comprises a step of amplifying deoxyribonucleic acid (DNA) of a sample to be tested by using specific primers, namely KF6-2-F and KF6-2-R, wherein the sample to be tested is the transgenic rice kefeng No. 6 if a target fragment is obtained through amplification; and the sample to be tested is not the transgenic rice kefeng No. 6 if no target fragment is obtained through amplification. The invention provides two other copy flanking sequences of the kefeng No. 6 and an event-specific detection method based on the two copy flanking sequences; and important information is provided for molecular characteristics of the transgenic rice kefeng No. 6.

Description

(1) Technical field [0001] The invention relates to a qualitative PCR detection method for transgenic rice Kefeng 6. (2) Background technology [0002] Rice (Oryza sativa) is a major food crop, and it is also an early successful application of transgenic technology for genetic improvement. Transgenic rice varieties containing different traits have been bred so far. my country has not yet approved the commercial production of genetically modified rice, but some varieties (strains) have been approved for environmental release and productive experiments. Effective supervision of genetically modified crops is the prerequisite and guarantee for the safe use of genetically modified crops. The flanking sequence of the exogenous insert in a transgenic plant is one of the most important molecular characteristics of a transgenic plant line. Therefore, the flanking sequence of the exogenous insert is an important technical data for establishing a specific detection method for a trans...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 王渭霞
Owner CHINA NAT RICE RES INST
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