Gene synthesis process, gene chip and kit
Provided is a chip process of gene synthesis, and the process comprises incorporating the whole procedure, which comprises amplifying oligonucleotides and assembling the oligonucleotides into a gene in parallel, onto a single chip. A specific mismatch endonuclease is also used in the process to establish an error repair system in gene synthesis, and the error rate is decreased to about 0.19 mismatched bases/kb. The high-throughput, high-fidelity and low-cost chip process of gene synthesis provided in the present invention can meet the requirements of gene synthesis and the optimization and screening of protein expression on a large scale at the frontier of life sciences such as synthetic biology, genomics, and systems biology.
1. A method of gene synthesis, comprising the steps of:
(1) immobilizing a pool of oligonucleotides to the surface of a solid substrate to form a gene chip, wherein the oligonucleotides are anchored to the surface of the chip via a linker sequence that is 15-150 bases in length, located at the 3′ end, and contains a nicking endonuclease recognition site;
(2) carrying out oligonucleotide pool amplification on the gene chip by performing an isothermal nicking and strand displacement amplification reaction, which comprises performing the following steps under isothermal conditions: (i) hybridizing a universal primer to the linker at the 3′ end of the chip-immobilized oligonucleotides, (ii) extending the universal primer using a strand displacement polymerase to form a newly amplified oligonucleotide chain, (iii) nicking the newly amplified oligonucleotide chain using a nicking endonuclease to re-free the 3′ end of the universal primer for a new extension reaction, and (iv) using the strand displacement polymerase to extend the universal primer of step (iii) and displace the newly amplified strand generated in step (ii), and
(3) carrying out parallel gene assembly on the gene chip by performing a polymerase cycling assembly reaction using the newly amplified oligonucleotide chains generated in the oligonucleotide pool amplification step.
2. The method of gene synthesis according to claim 1 , wherein the length of the synthesized gene is greater than or equal to 200 base pairs.
3. The method of gene synthesis according to claim 1 , wherein the method further comprises a step of (4) carrying out gene synthesis error repair using a mismatch-specific endonuclease.
4. The method of gene synthesis according to claim 3 , wherein the oligonucleotide pool amplification, the parallel gene assembly, and the gene synthesis error repair are carried out successively or stepwise in the same system.
5. The method of gene synthesis according to claim 3 , wherein the gene synthesis error repair is carried out on-chip.
6. The method of gene synthesis according to claim 3 , wherein the gene synthesis error repair is carried out by heat denaturation and re-annealing the synthesized gene to expose a mismatch site(s); recognizing and cleaving the mismatch site(s) using the mismatch-specific endonuclease and a 3′→5′ exonuclease; and assembling the resulting gene fragments into a complete gene by overlap-extension PCR.
7. The method of gene synthesis according to claim 3 , wherein the gene synthesis error repair is carried out separately off-chip.
8. The method of gene synthesis according to claim 1 , wherein the gene chip is divided into one or more sub-areas, and the oligonucleotide pool amplification and the parallel gene assembly are simultaneously carried out in the one or more sub-areas.