Super Long Fragment Rapid Gene Synthesis Technology
Rapid gene synthesis of ultra long fragments is one of the most difficult technologies in the field of gene synthesis. As a public service platform for large segment gene synthesis and precise sequencing, recently, the ultra long segment rapid gene synthesis technology developed by Tsingke biology has successfully broken through the core technology of yeast assembly of multiple segments and large segments, and can industrially produce 50kb large segment DNA, with a maximum synthetic length of 160kb.
The industrialized production of large DNA fragments is a key link in the development and application of synthetic biology. To develop high fidelity synthesis and assembly technology of large DNA fragments and chromosomes, the first is to synthesize short fragments through PCR reaction, which are often less than 1KB in length. Several small 1KB fragments are assembled into a large DNA molecule. Gibson recombination is the most commonly used assembly technology at present.
Gibson assembly technology was first proposed by Dr.Daniel and his colleagues in 2009. It is famous for its easy assembly of multiple linear DNA fragments. It can also be used to insert a target fragment into a selected vector to complete basic cloning. In Gibson ligation, DNA fragments with homologous regions at the ends need to be obtained first, which are generally obtained by PCR, and then these fragments are incubated with an enzyme master mix to complete the ligation.
The ultra long fragment rapid gene synthesis technology of Tsingke biology can assemble multiple linear DNA fragments. At the same time, it has also developed a yeast platform for large fragment assembly. The yeast assembly platform refers to the homologous recombination mechanism in yeast cells to construct yeast artificial synthetic chromosomes (YACs). Some studies have shown that yeast cells can ingest multiple DNA fragments and assemble four or five overlapping DNA fragments to connect to carrier DNA[1]. However, the existing yeast recombination technologies need to select yeast artificial single colony culture and screening one by one, and the linearized yeast carrier needs to be obtained by PCR[2], which will correspondingly prolong the overall time, reduce the fidelity, and increase the complexity and workload of operation. Therefore, how to screen and clone correctly at low cost and high throughput has become the key technology for yeast to assemble large DNA molecules.
The ultra long fragment rapid synthesis technology used by Tsingke biology is fast, simple and feasible, with high success rate, low cost, high efficiency and easy operation. It can expand the scale of industrialization and has a wide range of uses. At present, the application of synthetic ultra long DNA molecules has been extended to many aspects of biotechnology. With the development of synthetic technology, scientists have made further achievements in breeding transformation, medical health, pollution prevention and data storage; It also provides more solutions for human beings to improve food problems, develop new drugs, optimize disease treatment methods, solve environmental problems, and explore more ideal data storage methods.
In practical operation, Tsingke biology also carried out a series of optimization, and finally developed the super long fragment rapid gene synthesis technology, which can produce fragments up to 160kb in length, ensuring that it can provide customers with high-quality gene synthesis services in the shortest time. After long-term verification, this is indeed an effective method for the synthesis of long DNA fragments.
At the same time, in order to ensure the accuracy of synthetic fragments, the service process also includes sequence optimization, primer design, small fragment synthesis, large fragment recombination and other processes, as well as two sequencing verification before and after large fragment assembly. The second generation sequencing technology was used to sequence and verify the large fragments, and the accurate gene detection technology was used to ensure the 100% accuracy of the synthetic products. At the same time, Tsingke biological hasa established 21 branches / subsidiaries all over the country to quickly respond to customer needs with a strong service network system and logistics system.
Reference:
[1]Raymond,C.K.et.al.Biotechniques(1999)26:134-138
[2]Gibson,D G .et .al .Science(2008)5867:1215‐1220
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