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Plasmid reference molecule of quantitative determination of nucleic acids from transgenic soybean GTS40-3-2
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A technology of genetically modified soybeans and standard molecules, applied in the field of bioengineering, can solve the problem of lack of positive standard product configuration for positive products, and achieve the effect of good traceability
Inactive Publication Date: 2012-06-27
SHANGHAI INST OF MEASUREMENT & TESTING TECH
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[0008] The technical problem to be solved by the present invention is to provide a plasmid standard for transgenic soybean GTS40-3-2 in order to overcome the lack of positive products and the configuration of positive standard products in the existing nucleic acid quantitative PCR detection of transgenic soybean GTS40-3-2 molecular
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Embodiment 1
[0073] The construction of embodiment 1 plasmid standard molecule
[0074] 1. Experimental reagents
[0075] Restriction endonucleases KpnI, HindIII, XbaI, and restriction enzyme buffers were purchased from Shanghai Haojia Technology Development Co., Ltd.
[0076] pUC19 vector, Taq DNA polymerase, T4 DNA ligase, dNTP, DL2000 Marker
[0077] Purchased from Shanghai Haojia Technology Development Co., Ltd.;
[0078] DNA primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.
[0079] Other biochemical reagents are imported subpackages or domestic analytical pure.
[0083] DNA electrophoresis analysis system, including dark box, digital camera, computer, scanner, inkjet printer, photosensitive printer, Tanon UV-2000 ultraviolet analyzer, Gis gel image analysis software (Sha...
Embodiment 3
[0121] Application of the plasmid standard molecule pXL02 constructed in embodiment 3 in actual detection
[0122] 1. Experimental reagents
[0123] Plant genomic DNA was extracted and purified using the Plant Genomic DNA Extraction Kit from OMEGA.
[0124] Plasmid DNA was extracted and purified using a plasmid DNA extraction kit from OMEGA.
[0125] The pUC19 vector, T4 DNA ligase and its buffer, dNTPs, Taq DNA polymerase and its buffer, and DNA Marker were purchased from Shanghai Haojia Technology Development Co., Ltd.
[0126] Primers and probes were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
[0127] Other biochemical reagents are imported subpackages or domestic analytical pure.
[0130] Opticon-2 Quantitative PCR Amplifier (MJ Research Inc.).
[0131] DNA electrophoresis analysis system, including dark box, dig...
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Abstract
The invention discloses a plasmid reference molecule which is suitable for the quantitative determination of nucleic acids from a transgenic soybean GTS40-3-2, comprising a structure-specific sequence and a strain-specific sequence of the transgenic soybean GTS40-3-2 and a specific fragment of a soybean endogenous reference gene Lectin. The invention is characterized by designing primers to obtain the structure-specific sequence and the strain-specific sequence of the transgenic soybean GTS40-3-2 and the specific fragment of the soybean endogenous reference gene Lectin by PCR amplification through the analysis of inserting the exogenous source of the transgenic soybean GTS40-3-2 in the gene sequence; constructing by molecular cloning to form a artificial recombinant plasmid molecule pXL02 in a plasmid molecule; detecting the phosphorus content of the plasmid reference molecule by inductively coupled plasma mass spectrometry (ICP-MS) to calculate the concentration of the plasmid reference molecule. According to the invention, the plasmid reference molecule constructed in the invention can completely replace a transgenic soybean GTS40-3-2 positive standard sample, and the plasmid reference molecule is completely suitable for the quantitative PCR analysis and detection of structure specificity and strain specificity of the transgenic soybean GTS40-3-2 sample.
Description
technical field [0001] The invention relates to a plasmid molecule in the technical field of bioengineering, in particular to a plasmid standard molecule for quantitative detection of genetically modified soybean GTS40-3-2 and its construction and quantitative method. Background technique [0002] Genetically modified biotechnology is known as "the most rapidly applied biotechnology". With the continuous development of biotechnology, more and more genetically modified plants are used in the agricultural field to promote agricultural production, help farmers increase income, and solve the problems caused by global population growth. The food problem brought about opened up new avenues. The so-called transgenic plant (Genetically modified organism, GMO) refers to the use of biotechnology to recombine genes isolated or artificially synthesized from different organisms in vitro, and then introduce them into recipient plant cells, so that the new genes can be integrated in the re...
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