Gene synthesis process, gene chip and kit

Abstract

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 gene chip formed by immobilizing oligonucleotide probes onto the surface of a solid substrate, wherein the oligonucleotide has at the 3′-end a linker sequence of 15-150 bases in length and is anchored onto the surface of the chip via a nicking endonuclease recognition site within the linker sequence. 
     
     
         2 . The gene chip according to  claim 1 , wherein the microarray is divided into sub-arrays by using a method of physical segmentation, each sub-array contains oligonucleotide sequences for synthesizing more than 0.2 kb in length. 
     
     
         3 . The gene chip according to  claim 1 , wherein the solid substrate is selected from the group consisting of nitrocellulose membrane, nylon membrane, glass slide, silicon wafer and plastic sheet. 
     
     
         4 . The gene chip according to  claim 2 , wherein the solid substrate is selected from the group consisting of nitrocellulose membrane, nylon membrane, glass slide, silicon wafer and plastic sheet. 
     
     
         5 . A gene synthesis kit, comprising the gene chip according to  claim 1 , a nicking endonuclease, a strand displacement DNA polymerase, a high-fidelity DNA polymerase and a mismatch-specific endonuclease. 
     
     
         6 . A gene synthesis kit, comprising the gene chip according to  claim 2 , a nicking endonuclease, a strand displacement DNA polymerase, a high-fidelity DNA polymerase and a mismatch-specific endonuclease. 
     
     
         7 . A gene synthesis kit, comprising the gene chip according to  claim 3 , a nicking endonuclease, a strand displacement DNA polymerase, a high-fidelity DNA polymerase and a mismatch-specific endonuclease. 
     
     
         8 . A gene synthesis kit, comprising the gene chip according to  claim 4 , a nicking endonuclease, a strand displacement DNA polymerase, a high-fidelity DNA polymerase and a mismatch-specific endonuclease. 
     
     
         9 . The gene synthesis kit according to  claim 5 , wherein the kit further comprises dNTP, BSA, Thermopol II buffer and oligonucleotide primer, and the Thermopol II buffer consists of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , and 0.1% Triton X-100, pH 8.8 at 25° C. 
     
     
         10 . The gene synthesis kit according to  claim 6 , wherein the kit further comprises dNTP, BSA, Thermopol II buffer and oligonucleotide primer, and the Thermopol II buffer consists of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , and 0.1% Triton X-100, pH 8.8 at 25° C. 
     
     
         11 . The gene synthesis kit according to  claim 7 , wherein the kit further comprises dNTP, BSA, Thermopol II buffer and oligonucleotide primer, and the Thermopol II buffer consists of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , and 0.1% Triton X-100, pH 8.8 at 25° C. 
     
     
         12 . The gene synthesis kit according to  claim 8 , wherein the kit further comprises dNTP, BSA, Thermopol II buffer and oligonucleotide primer, and the Thermopol II buffer consists of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , and 0.1% Triton X-100, pH 8.8 at 25° C.