Multiplex PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize
A technology of PCR-DHPLC and transgenic corn, which is applied in the direction of recombinant DNA technology, measuring devices, DNA/RNA fragments, etc., to achieve the effect of high-throughput detection method, strong specificity and reliable flux detection method
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Embodiment 1D
[0027] Example 1DNA Extraction
[0028] Sample DNA was extracted by CTAB method, as follows:
[0029] a) Weigh 5 g of the sample, add liquid nitrogen to the mortar and grind until the sample is a powder with a size of about 0.5 mm;
[0030] b) Weigh 300 mg of the ground sample, quickly transfer it to a 2 mL centrifuge tube, add 700 μL of CTAB extract solution preheated at 65 °C, mix well, and put it in a water bath at 65 °C for 30 min;
[0031] c) Add 5 μL RNase (10 mg / mL), and bathe in water at 37°C for 30 minutes;
[0032] d) Add an equal volume of Tris saturated phenol, mix thoroughly, and centrifuge at 12000r / min for 15min;
[0033] e) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;
[0034] f) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;
[0035] g) Add an equal volume of pre-cooled isopropanol, shake gently, ...
Embodiment 2D
[0038] Example 2 DNA concentration determination
[0039] The concentration and purity of the extracted sample DNA were measured; the absorbance values at 260nm and 280nm were measured by an ultraviolet spectrophotometer, and the purity and concentration of nucleic acid were calculated respectively. The calculation formula is as follows:
[0040] DNA purity = OD260 / OD280
[0041] DNA concentration=50×OD260mg / mL
[0042] The purity ratio of DNA was between 1.7 and 1.9, and the concentration was greater than 10ng / μL.
Embodiment 3
[0043] Embodiment 3PCR amplification
[0044] Design specific detection primer binding sites according to transgenic maize lines MON863, NK603, MON810, T25, MON88017, MIR604, 59122, and add at the 5' end of the specific detection primer binding sites of MON810, T25, MON88017, MIR604 lines Regulatory sequence, synthetic detection primers containing regulatory sequences (Table 1), synthetic primer pair 1 upstream / downstream primer specific binding site, primer pair 2 upstream / downstream primer specific binding site, primer pair 7 upstream / downstream primer specificity The binding site and the primer pair 3 up / downstream primers adding regulatory sequences, the primer pair 4 upper / downstream primers adding regulatory sequences, the primer pair 5 upper / downstream primers adding regulatory sequences, the primer pair 6 upper / downstream primers adding regulatory sequences The downstream primers were used as primers for PCR amplification, and the PCR sample settings included the mixed...
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