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Gene synthesis methods

Update time : 2020-08-25

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Gene synthesis methods

Gene synthesis methods mainly include assembly PCR, overlap extension PCR, TBIO method, and PTDS method.


Before gene synthesis, in order to allow genes to be well expressed in different biological expression systems, researchers usually perform codon optimization on genes. 

There are 64 genetic codes, but most organisms tend to use some of these codons. Those that are used most frequently are called optimal codons, and those that are not used frequently are called rare or low-utilized codons. 

Actually every organism used for protein expression or production (including E. coli, yeast, mammalian cells, plant cells, and insect cells) exhibits a certain degree of codon preference. 

Therefore, the expression of recombinant proteins may be affected by codons. The impact of child utilization (especially in heterologous expression systems).

Without changing its coding protein, the process of changing the coding gene of a protein, removing rare codons, and reasonably optimizing its mRNA secondary structure, GC content, etc. is called codon optimization. 

The principle of codon optimization is to select different codons for each amino acid in the target protein sequence according to the codon preference of the expression system to be used to perform the combination of the entire sequence and find the best one.

1. Primer synthesis
After the researchers determine the synthetic gene sequence according to their own wishes, since there is no template, they first need to design and synthesize primers based on the double-stranded DNA to be synthesized.
At present, the primer synthesis basically adopts the solid phase phosphoramidite triester method. The phosphoramidite triester method to synthesize DNA fragments has the characteristics of high efficiency, rapid coupling and relatively stable initial reactants.
2. PCR amplification with primers as templates
After the primers are synthesized, the primers serve as templates for PCR amplification. The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity relies on oligonucleotide primers complementary to both ends of the target sequence.
3. Connection transformation, bacteria inspection and testing
Through the PCR reaction, the double-stranded DNA we need can be amplified. But the stability of the PCR product is low, we need to ligate it to the plasmid, transform it into a competent state, and spread it on a plate overnight. The positive clones can be picked from the plate the next morning. The picked clones need to be verified by colony PCR again to verify whether there is a full-length gene insertion. PCR is performed with the head and tail primers. If there is a product, it indicates that there is a target gene. The verified clones are transformed into test tube culture shake bacteria, after which the plasmids are extracted, purified and sent for sequencing.
4. First-generation sequencing
Currently, we are using first-generation sequencing, also called sanger sequencing. Sanger sequencing uses a DNA polymerase to extend a primer bound to a template of a pending sequence until a chain terminating nucleotide is incorporated. Each sequence determination consists of a set of four separate reactions, each containing all four deoxynucleotide triphosphates (dNTPs), mixed with a limited amount of a different dideoxynucleotide triphosphate (ddNTP).
5. QC verification

The sequencing result is compared with the target gene, and QC enzyme digestion verification is performed at the same time. If the sequencing result and the digestion verification result are correct, the synthesis of the target gene is completed. According to the above steps, a gene sequence of about 800 bp can be synthesized in 5 days under smooth conditions.