Method for separating unknown flanking sequences of multiple known sequence DNA fragments at one time
A flanking sequence and sequence technology, which is applied in the field of separating multiple unknown flanking sequences of DNA fragments with known sequences at one time, can solve the problems of cumbersome experimental procedures, low success rate, and long experimental period, achieving flexible throughput and reliable results , the effect of simple operation
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Embodiment 1
[0032] Example 1 Using Q-PCR to verify that the flanking sequence of the rice GAPDH fragment can be enriched by GPS-seq
[0033] In order to verify whether the designed GPS-seq method is feasible, take the 1655-1704bp fragment of the GAPDH gene in the rice genome as an example, try to isolate its upstream DNA region, and use Q-PCR to test whether the upstream region of the fragment is enriched. Proceed as follows:
[0034] 1.1 The transposase complex interrupts the rice genome and connects GPS-seq adapters at DNA fragment breakpoints
[0035] Use the Tn5 complex to interrupt the rice genomic DNA, configure the reaction system in a sterilized PCR tube according to the components in Table 1, then place the reaction tube in a PCR instrument, and run the following reaction program: 105°C hot lid, 55°C for 10 minutes, 10°C hold.
[0036] Table 1. Transposase complex reaction system
[0037]
[0038] 1.2 Purification of the disrupted product of the transposase complex by the m...
Embodiment 2
[0064] Example 2 Verification that the flanking sequence of rice Tos17 can be separated by GPS-seq by Sanger sequencing
[0065] 2.1 Transposase breaks rice genome (reaction system and reaction procedure are the same as Example 1.1)
example 12
[0067] 2.3 Using GPS-seq to amplify the flanking sequence of Tos17
[0068] According to the experimental requirements, design specific primers for amplifying the flanking sequences of Tos17, and the primer sequences and primer binding position information required for PCR amplification are shown in Table 8:
[0069] Table 8. Primer sequences and primer binding position information required for PCR amplification of Tos17
[0070]
[0071] Note: The bases in bold italics are the base sequences of the 3' end of the Illumina sequencing adapter P5.
[0072] Configure a reaction system for two rounds of PCR in a sterilized PCR tube according to the components in Table 9 and Table 10, and run the reaction program set in Table 11 in the PCR instrument to amplify the flanking sequence of Tos17.
[0073] Table 9. The first round of PCR reaction system
[0074]
[0075] Note: Template1 is the product of rice genomic DNA fragmented by transposase, adapter added, and purified.
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