DPO (dual priming oligonucleotide) primer combination and detection method for multi-PCR detection of transgenic crops
A DPO primer and primer combination technology, which is applied in the direction of recombinant DNA technology, DNA / RNA fragments, etc., can solve the problems of low resolution, easy primer competition, and time-consuming operation, and achieve high throughput, good specificity, and improved detection. efficiency effect
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Embodiment 1
[0033] Embodiment 1 is used for the acquisition of the DPO primer combination of seven multiplex PCR detection transgenic crops
[0034] Taking FMV35S, Cry1Ab, NPTII, NOS, CaMV35S, Pat, and Bar genes as target genes, design and artificially synthesize the following seven-fold DPO-PCR detection DPO primer combination (I is hypoxanthine):
[0035] FMV35S-F: 5'-CAGTCCAAAAGCCTCAACAAGGTCIIIIIIACAGAGTC-3';
[0036] FMV35S-R: 5'-ATTAGTGAGTGGGCTGTCAGGACAIIIIITTTCCACG-3';
[0037] Cry1Ab-F: 5'-GGCCAGGGAGTGTACCGCAIIIIIIAGCAGCAC-3';
[0038] Cry1Ab-R: 5'-GCTCACGCTGCTGTTGCTGAAIIIIITGCGGAAC-3';
[0039] NPTII-F: 5'-TCACCTTGCTCCTGCCGAGAAIIIIICCATCATG-3';
[0040] NPTII-R: 5'-GGGCATGCGCGCGCCTTGAIIIIIIGCGAACAG-3';
[0041] NOS-F: 5'-CTTAAGATTGAATCCTGTTGCCGIIIIIIGCGATGATTATC-3';
[0042] NOS-R: 5'-AGGTTGCGCGCTATATTTTGTTTIIIIICGCGTATTAAATG-3';
[0043]CaMV35S-F: 5'-TCCTACAAATGCCATCATTGCGIIIIIGGAAAGGC-3';
[0044] CaMV35S-R: 5'-ATCACATCAATCCACTTGCTTTGAIIIIITGGTTGGA-3';
[0045] Pat-F: 5'...
Embodiment 2 7
[0050] Embodiment 2 Seven-fold DPO-PCR detection method
[0051] 1. Extraction of DNA
[0052] Genomic DNA of transgenic maize TC1507, transgenic maize MON863, transgenic maize BT176, and transgenic maize MON89034 were extracted with reference to the DNA extraction kit (Plant Genomic DNA Extraction Kit, Cat. No. DP305-02, Tiangen Biotechnology Co., Ltd.).
[0053] 2. Seven-fold DPO-PCR amplification
[0054] Using FMV35S-F, FMV35S-R, Cry1Ab-F, Cry1Ab-R, NPTII-F, NPTII-R, NOS-F, NOS-R, CaMV35S-F, CaMV35S-R, Pat-F, Pat-R, Bar -F, Bar-R, a total of 14 seven-fold DPO-PCR primers, the reaction system was prepared as shown in Table 1, the DNA templates were the genomes of four transgenic maize lines including transgenic maize TC1507, transgenic maize MON863, transgenic maize BT176, and transgenic maize MON89034 DNA mixture. Among them, a multiplex PCR amplification kit was used, which contained 10×MultiHotStart reaction buffer, dNTPs (2.5mmol / L), and MultiHotStart DNA polymerase,...
Embodiment 3 7
[0061] The specificity analysis of embodiment 3 seven-fold DPO-PCR detection method
[0062] 1. Extraction of DNA
[0063] Refer to the DNA extraction kit (Plant Genomic DNA Extraction Kit, Cat. No. DP305-02, Tiangen Biotechnology Co., Ltd.) to extract transgenic corn TC1507, genetically modified corn MON863, genetically modified corn BT176, genetically modified corn MON89034, and genetically modified soybean GTS40-3 -2. Genomic DNA of transgenic rice TT51-1 and transgenic rapeseed GT73.
[0064] 2. Seven-fold DPO-PCR amplification
[0065] Using the method in Example 2, seven-fold DPO-PCR amplification was performed using the genomic DNAs of the 10 transgenic crop lines extracted in step 1 as templates to obtain seven-fold DPO-PCR amplification products.
[0066] 3. Capillary electrophoresis detection of seven-fold DPO-PCR amplification products
[0067] After the seven-fold DPO-PCR reaction was completed, 1 μL of the seven-fold DPO-PCR amplification products were taken an...
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