Multiple DPO-PCR primer combination for detecting transgenic maize MON810 and MIR604 and method
A DPO-PCR, MON810-F technology, applied in the field of multiplex DPO-PCR primer combinations, can solve the problems of complex system, low stability, limited use, etc., and achieve high throughput, good specificity, and improved detection efficiency. Effect
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Embodiment 1
[0029] Example 1 Multiplex DPO-PCR method for detecting transgenic maize MON810 and MIR604
[0030] 1. Design of Multiplex DPO-PCR Primer Combinations
[0031] According to the flanking sequences of transgenic maize MON810 and MON810, the following multiple DPO-PCR primer combinations for detecting transgenic maize MON810 and MIR604 were designed (I in the primer sequence is hypoxanthine):
[0032] MON810-F: 5'-CTAACGTTTAACATCCTTTGCCATTIIIIIGCTATCTGTC-3' (SeqIDNo.1)
[0033] MON810-R: 5′-TAGTGGGATTGTGCGTCATCCCTIIIIIIICAGTGGAGA-3′ (SeqIDNo.2)
[0034] MIR604-F: 5′-GCACGCAATTCAACAGAAGGCGIIIIICGACAATC-3′ (SeqIDNo.3)
[0035] MIR604-R: 5'-TGCGGTTCTGTCAGTTCCAAACGTIIIIIIGGCTTGTC-3' (SeqIDNo.4)
[0036] Among them, MON810-F and MON810-R are primers for detecting transgenic maize MON810, and the product size is 266bp; MIR604-F and MIR604-R are primers for detecting transgenic maize MIR604, and the product size is 127bp.
[0037] 2. Multiplex DPO-PCR method for detection of transge...
Embodiment 2
[0052] The specific detection of embodiment 2 multiple DPO-PCR primers
[0053] 1. Extraction of DNA
[0054] Refer to the kit instructions to operate (Plant Genomic DNA Extraction Kit, Cat. No. DP305-02, Tiangen Biotechnology Co., Ltd.) to extract transgenic corn MON810, genetically modified corn MIR604, genetically modified corn TC1507, genetically modified corn MON863, genetically modified corn BT176, and genetically modified corn Genomic DNA of MON89034, transgenic soybean GTS40-3-2, transgenic rice TT51-1, and transgenic rapeseed GT73.
[0055] 2. Multiplex DPO-PCR amplification
[0056] Using the method for detecting transgenic maize MON810 and MIR604 in step 2 of Example 1, the genomic DNA of the nine transgenic lines extracted in step 1 was used as a template for multiplex DPO-PCR amplification to obtain multiplex DPO-PCR amplification products.
[0057] 3. Electrophoretic detection of multiplex DPO-PCR amplification products
[0058] After the multiplex DPO-PCR rea...
Embodiment 3
[0063] The sensitivity detection of embodiment 3 multiplex DPO-PCR primer combination
[0064] 1. Extraction of DNA
[0065] Genomic DNA of transgenic corn MON810 and MIR604 were extracted according to the instructions of the kit (Plant Genomic DNA Extraction Kit, Cat. No. DP305-02, Tiangen Biotechnology Co., Ltd.). And the obtained genomic DNA was diluted 10 times, and the genomic DNA of the transgenic maize 810 with the concentration of 50ng / μL, 5ng / μL, 0.5ng / μL, 0.05ng / μL and 0.005ng / μL and the concentration of 50ng / μL were prepared respectively. μL, 5ng / μL, 0.5ng / μL, 0.05ng / μL and 0.005ng / μL of genomic DNA of transgenic maize MIR604.
[0066] 2. Multiplex DPO-PCR amplification
[0067] Using the method for detecting transgenic maize MON810 and MIR604 in step 2 of Example 1, the following 5 groups of genomic DNA were used as templates to perform multiple DPO-PCR amplification respectively to obtain multiple DPO-PCR amplification products:
[0068] Group 1: 50 ng / μL of tr...
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