Gene synthesis process, gene chip and kit

Gene synthesis is an efficient and cost-effective alternative to molecular cloning for custom gene production, where the DNA is manufactured by assembling strings of oligos together.Gene synthesis is the process of chemically synthesizing double-stranded DNA molecules in vitro. The main concept of gene synthesis is to assemble custom oligos into long DNA molecules. Email: marketing@medicilon.com.cn web:www.medicilon.com

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.

Low-cost, high-throughput gene synthesis and precise control of protein expression are the critical technical problems of synthetic biology. Currently, the main method for artificially synthesizing gene is to obtain long chain DNA by splicing and assembling short chain oligonucleotides. The cost of the method for artificially synthesizing short chain oligonucleotide by chemical method is very high (RMB 0.6 per nucleotide), and the error rate of synthesis remains as high as one base deletion per 100 bases and one mismatch or insertion per 400 bases. As a result, synthesis of gene or genome from assembling oligonucleotide is expensive, and the accumulative error rate of synthesis is high. Repairing errors by clone sequencing and mutagenesis methods further increases the amount of labor and total cost.

Oligonucleotide synthesis by performing large scale parallel synthesis on microfluidic chip can significantly reduce the cost. Currently, the method for synthesizing oligonucleotide on the chip mainly includes ink-jet printing (Agilent), 5'-end modified photolabile protecting groups (Nimblegen/Affymetrix), photo-generated acid deprotection (Atactic/Xeotron) and electrochemical method (Oxamer/Combimatrix). However, due to the very small surface area of the microfluidic chip, the yield of the oligonucleotide synthesis is low, with the concentration of each sequence of oligonucleotide in the solution being 10-12 M or less. Thus, extensive amplification is required prior to assembling into gene. Currently, a feasible method is as follows: releasing the synthesized oligonucleotide from the microfluidic chip by chemical or enzymatic treatment, amplifying by PCR, digesting by restriction enzyme, and purifying, then assembling the obtained oligonucleotide into gene or genome, and the error repair method of the gene synthesis mainly involves polyacrylamide gel electrophoresis and HPLC. Since the current methods of gene synthesis and error repair are still compromised by cumbersome steps, new strategies are urgently required to increase integration and miniaturization, lower cost, and increase the efficiency in synthesis and error repair.

The present invention intends to provide a high-throughput, high-fidelity, low cost gene synthesis method, which integrates the steps of oligonucleotide pool amplification and parallel gene assembly on one microfluidic chip to perform simultaneously, and also employs mismatch-specific endonucleases to establish an efficient error-repair system for gene synthesis, which decreases the error rate of synthesis from about 1.9 error base per kb to less than 0.19 error base per kb. The method presents a cost that is one-tenth of the lowest reported cost.

The invention provides a method of gene synthesis, wherein isothermal nicking and strand displacement amplification, and polymerase cycling assembly reactions are performed on one gene chip to achieve oligonucleotide amplification and gene assembly, the gene chip is formed by immobilizing oligonucleotides to the surface of a solid substrate. The oligonucleotide has at 3'-end a linker sequence of 15-150 bases and is anchored onto the surface of the chip via a nicking endonuclease recognition site within the linker sequence.

Preferably, the length of the synthesized gene is greater or equal to 200 base pairs.

Preferably, the method further comprises and employs mismatch-specific endonuclease to perform the gene synthesis error-repair step.

Preferably, the three reactions of oligonucleotide amplification, assembly and gene synthesis error-repair are carried out successively or stepwise in the same system.

Preferably, the gene synthesis error-repair reaction is carried out on-chip or separately off-chip.

Preferably, the gene chip can be divided into one or more sub-areas, and oligonucleotide amplification and gene assembly are simultaneously carried out in one or more sub-areas.

Preferably, a universal primer is used to hybridize to the linker at 3'-end of the oligonucleotides in the isothermal nicking and strand displacement amplification and polymerase cycling assembly reactions. While the strand displacement polymerase extends and displaces the oligonucleotide, the nickning endonuclease separates the universal primer from the newly amplified oligonucleotide chain to re-free 3'-end of the universal primer for new extension reaction.

Preferably, the gene synthesis error-repair step is carried out by heat denaturing and re-annealing the synthesized gene to expose the mismatch site; recognizing and cleaving the mis-match site by the mismatch-specific endonuclease and 3'→5' exonuclease activities; assembling the resulting gene fragments into a complete gene by overlap-extension PCR reaction.

Another purpose of the present invention is to provide a gene chip formed by immobilizing oligonucleotide probes onto the surface of a solid substrate, wherein the oligonucleotides has at 3'-end a linker sequence of 15-150 bases and is anchored onto the surface of the chip via a nicking endonuclease recognition site within the linker sequence.

Preferably, for the gene chip, the microarray is divided into sub-arrays by using the method of physical segmentation, and each sub-array contains oligonucleotide sequence for synthesizing more than 0.2 kb in total length.

The solid substrate used in the gene chip of the present invention is selected from any material that is suitable for the preparation of a gene chip, including but not limited to nitrocellulose membrane, nylon membrane, glass slide, silicon wafer and plastic sheet. The gene chip of the present invention is prepared by immobilizing oligonucleotides onto the substrate through successively dotting the oligonucleotides onto the substrate and anchoring the oligonucleotides by means of a nicking endonuclease recognition site within the 3'-end linker sequence.

The present invention further provides a gene synthesis kit comprising any gene chip as described above, a nicking endonuclease, a strand displacement DNA polymerase, a high-fidelity DNA polymerase and a mismatch-specific endonuclease.