Gene chip detection method of transgenic maize
A technology of transgenic corn and gene chips, applied in the field of detection, can solve problems such as false negatives, reduced detection sensitivity, and the inability to detect multiple samples at the same time
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Embodiment 1
[0033]For the sequences of 7 exogenous genes commonly used in transgenic maize and control genes (18SrRNA, Invertase endogenous gene), primers and probes were designed by primer design software primer5.0 and oligo6.0, and nucleotide Sequence alignment, from which primers and probes that meet the requirements are selected, and the information of the selected primers and probes is shown in Table 1 and Table 2.
[0034] Table 1
[0035] Primer number
Primer sequence
target DNA
1
TAA TAG AGC AAT GAA CAG TCG G
18SrRNA
2
CTT TCA ACA ACG GAT CTC TTG G
3
GGC CGG ATC GTC ATG CTC TAC A
Invertase endogenous gene
4
TTG GCG TCC GAC TTG ACC CAC T
5
GAT TCC ATT GCC CAG CTA TCT G
CaMV35S promoter
6
TAG AGG AAG GGT CTT GCG AAG G
7
GAA TCC TGT TGC CGG TCT TG
T-nos terminator
8
CGA TT CCC GAT CTA GTAAC
9
CGT CTT GAA GGT CGT GGTA
Modified ...
Embodiment 2
[0044] Using a fully automatic chip spotter, it is designed into a 6×9 array, including 18 detection target probes, two negative controls, 4 points for each pair of probes, and a gene for each row. The setting on the chip in Anhui A human gene that has no homology with the detection probe was used as a negative control to exclude the interference of non-specific hybridization during the hybridization process.
Embodiment 3
[0046] The established transgenic corn gene detection chip was used to repeatedly test and verify 20 standard samples. The results showed that only the corresponding internal reference gene (18SrRNA, invertase endogenous gene) hybridization detection signal appeared in the standard sample without the transformed gene, 0.1% The maize not only obtained the hybridization detection signal of the internal reference gene, but also obtained the hybridization detection signal of the specific exogenous gene. The verification results showed that the designed chip had very high sensitivity, and the accuracy of the experimental results was very high. The selected primers, probes Needle and reaction conditions are optimal.
[0047] The established transgenic corn gene detection chip was used to detect 10 unknown corn samples. The results showed that 18SrRNA, invertase, insect-resistant zSSIIb gene, insecticidal protein CryIAb gene, CaMV 35S promoter, T- The nos terminator and herbicide-res...
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