Scalable De Novo Synthesis Of Long DNA And Scalable Rapid Preparation Of Individual Oligonucleotides

Although several traditional de novo DNA synthesis methods have been developed,the cost is still too high for large scale synthesis,and the length of synthesized DNA is also limited.De novo synthesis of long DNA is traditional starting from pre-synthesized oligonucleotides which ordered from Controlled Pore Glass(CPG)synthesis platform.These oligonucleotides in liquid were mixed and assembled to longer DNA through DNA ligase-based method or DNA polymerase-based method.Due to the limitation of oligonucleotide chemical synthesis method,the throughputs of these traditional de novo gene synthesis technologies are limited and the prices of synthesized long DNAs are too high.The development of microchip technologies can partly solve the problems rising from oligonucleotides synthesis.However,the oligonucleotides synthesized from microchips have several disadvantages,such as high background sequences,low quantity of each sequence and low fidelity compared to traditional CPG oligonucleotides.Here,we optimized the assembly method for the long DNA using microchip-synthesized oligonucleotides,to establish a scalable,accurate,time-and cost-efficient de novo long DNA synthesis protocol.This de novo long DNA synthesis method consisted of several steps,including oligonucleotides design,oligonucleotides synthesis,subpools amplification,fragment(-330 base pair(bp))assembly,full-length(1-2 kilobase pairs(kb))gene assembly,large gene cluster assembly(?12 kb).Firstly,the microchip-synthesized oligonucleotides mixture were divided into several subpools through adding common primer sequences,providing decreased complexity of background sequences during gene assembly and increased amount of each oligonucleotide sequence.Secondly,the oligonucleotide subpools were assembled into gene fragments(-330 bp)through DNA ligase-dependent method or DNA polymerase-dependent method.We optimized the reaction conditions of key steps,to ensure the success for DNA synthesis and fidelity of assembled DNA products.In addition,a MutS-immobilized cellulose column(MICC)error removal method which can remove errors in oligonucleotides and assembled products with low cost and high efficiency,was brought into the de novo long gene synthesis protocol to generate high-fidelity DNA products.The established long DNA synthesis approach were tested by assembling 78 gene fragments coding for 11 genes(total gene length?21 kb),including a gene cluster(soluble methane monooxygenase gene cluster)and 3 long gene(epothilone A,B and C genes),from 893 unpurified microchip-synthesized oligos(total oligo length 75,618 nt).The de novo synthesis of genes(>1 kb)using unpurified microchip synthesized oligonucleotides can be finished in 2 days with high fidelity(error frequency:0.65 error/kb)which is better than gene assembled using commercially provided CPG oligonucleotides.And a gene-size DNA(?1.0 kb)can be obtained at a cost as low as $0.015/kb,which is much cheaper than the currently commercial provided gene-size DNA.Besides,the synthetic genes within the soluble methane monooxygenase(sMMO)gene cluster were heterologous expressed in Escherichia coli BL21 star(DE3),but it is a pity that the sMMO X and Y can not form soluble proteins.The chemical synthesized oligonucleotides can also be directly applied into a number of research fields without assembly process,such as metabolic engineering,drug development,genome function research.The traditional CPG synthetic oligonucleotides can only be used as primers for PCR or site-directed mutagenesis due to the limited throughput and high cost.With the development of microchip-synthesis technology,hundreds of thousands of oligonucleotides can be parallel synthesized,increasing the synthesis throughput and decreasing the cost of each oligonucleotide.However,the microchip-synthesized oligonucleotides will form a complex pool containing various sequences,the quantity of each oligonucleotide is very low.So the oligonucleotides can not be easily separated and only can be used as a mixture for downstream application like gene assembly and library construction.The overall developed oligonucleotides synthesis technology still can not provide a large number of separated oligonucleotides sequences even if the synthesis capacity of microchip were improved,limiting the potential applications of oligonucleotides.To meet the simultaneous requirement of a large number of individual oligonucleotides and expand the utilization of oligonucleotides in wider range of applications,like biomolecular engineering,DNA-dependent gene therapy,non-coding RNA research,we established a high-throughput,rapid,accurate and cost-efficient approach for preparing individual oligonucleotides(length up to 96 nt),which combines two platform including microchip synthesis and PAGE separation.The total oligonucleotides synthesized on one chip can be divided into several subpools which contain 5-11 distinct sequences in each subpool.The oligonucleotides within one subpool can be separated by virtue of different length(at least 4 nt).This approach was test by preparing 4,125 oligonucleotides(total length 394,199 nt),coding for 5 genes(total length 6,441 bp)and 1,723 kinds of microRNA precursor template DNAs(total length 261,705 bp).Combined with MICC error removal system,we can obtain individual oligonucleotides in 2 days with reasonable error rate(3.1 error/kb)in quantity of nmols and in purity of electrophoresis purification with low cost(as low as $0.004/nt).The cost can be further reduced with the improvement of the total number of oligonucleotides synthesized on one chip.In conclusion,we established two meaningful DNA synthesis approaches,including scalable long DNA synthesis and scalable rapid preparation of individual oligonucleotides.Here,long DNA can be scalable de novo synthesized using unpurified microchip-synthesized oligonucleotides with improved throughput,low cost and high fidelity in 2 days,overcoming the high cost and low throughput nature of conventional gene synthesis.On the other hand,we can easily obtain individual oligonucleotide of any sequence.The ability to generate individual oligonucleotide products with features of high-throughput,cost-efficiency,high yield and fidelity are competitive compared with commercially oligonucleotides(>60 nt)synthesis.