Synthesis method of target gene segment

Abstract

The invention discloses a synthesis method of a target gene segment. The method comprises the following steps: obtaining a plurality of upstream primers and a plurality of downstream primers of a target gene segment; uniformly mixing the plurality of upstream primers, the plurality of downstream primers and double distilled water to obtain a primer pair solution; uniformly mixing the primer pair solution, PCR Buffer and ddH2O to obtain an annealing reaction solution; putting the annealing reaction solution into hot water with a temperature of 95-105 DEG C, and then naturally cooling to room temperature to obtain an annealing product; carrying out double enzyme digestion on a vector, and then treating with T4 DNA polymerase to obtain a linear vector with a second cohesive end; and incubating the annealed product and the linear vector with the longer cohesive end, and then transforming into competent cells to obtain a transformant with the target gene segment. The method is high in synthesis efficiency and high in accuracy, and is suitable for synthesis of target gene segments with special structure sequences such as highly repeated sequences, high GC sequences, hairpin sequences andthe like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for synthesizing an objective gene fragment. Background technique [0002] At present, the traditional gene synthesis methods all rely on PCR technology, which uses the annealing and extension of primers to gradually generate larger DNA fragments. However, the primer-dependent PCR synthesis technology has the following disadvantages: ① Currently, the chemical synthesis of oligonucleotides mostly adopts the solid-phase phosphoramidite synthesis method (phosphoramidite chemistry), which uses controlled pore glass (CPG) ) or polystyrene (polystyrene, PS) as a solid-phase support carrier, the deoxygenated nuclei are deoxygenated by a cycle reaction of four consecutive chemical reactions of activation (deprotection), coupling (coupling), capping (capping) and oxidation (oxidation). The nucleotides are connected sequentially and gradually extended, and the primer itself is prone ...

AI Technical Summary

Problems solved by technology

However, the primer-dependent PCR synthesis technology has the following disadvantages: ① Currently, the chemical synthesis of oligonucleotides mostly uses the solid-phase phosphoramidite synthesis method (phosphoramidite chemistry), which uses controlled pore glass (CPG) ) or polystyrene (polystyrene, PS) as a solid-phase support carrier, the deoxygenated nuclei are deoxygenated by a cycle reaction of four consecutive chemical reactions of activation (deprotection), coupling (coupling), capping (capping) and oxidation (oxidation). The nucleotides are connected sequentially and gradually extended, and the primer itself is easy to accumulate errors in the synthesis reaction, that is, mutation

During the PCR process, the mutation of the total primers will be amplified exponentially, resulting in a high error rate of the synthesized target fragment. This is the main reason for the mutation rate of the current gene synthesis method relying on PCR, and it cannot be completely avoided; at the same time, the error rate of the synthesized target fragment is high. On the one hand, it increases the workload of synthesis, on the other hand, it also increases the cost of sequencing

②Because the synthesis method relies on PCR technology, when there are special structures such as high GC, high AT, hairpin, and high repetition in the gene sequence to be synthesized, the designed primers will not be able to anneal and extend each other, or the primers will have multiple annealing extensions site, so that it would be known what caused the failure of the synthesis

③The synthesis method relying on PCR technology has cumbersome operation steps such as preparation of PCR system, detection of PCR products, recovery, enzyme digestion, connection of target gene fragment and carrier, etc., which reduces the throughput of synthesis to a certain extent and improves the efficiency of synthesis. cost

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