Patent Application
20230407359
The present invention relates to synthetic biology and microelectromechanical systems (MEMS), particularly to a high-throughput automated gene synthesis device using cluster arrays.
Oligonucleotide synthesis and gene assembly, as powerful tools of synthetic biology, are widely used in molecular biology (including library construction, sequencing, gene editing, etc.), protein engineering, metabolic engineering, biomedical engineering and genetic testing and other fields.
In the traditional commercial solid-phase oligonucleotide synthesis method, each oligonucleotide is synthesized in a separate synthesis tube or in a well in a synthesis plate, and the yield of each nucleic acid is high, which is usually in nanomole level. However, such synthesis method consumes large amounts of reagents and thus the cost is high. Besides, during gene synthesis, mixing oligonucleotides into an oligonucleotide pool is an essential step for performing subsequent gene assembly. Poor quality of manual mixed oligonucleotides pools may cause the pooling problem in subsequent gene assembly process. The microarray-based high-throughput synthesis method has been widely studied, which has the advantages of high throughput (up to millions of different oligonucleotide sequences can be synthesized on a single chip) and low cost. However, the yield of single oligonucleotide synthesis is relatively low, generally in the femtomole level (generally from 105 to 1012 molecules/sequence, even not enough to trigger a PCR reaction). Therefore, multiple PCR amplifications of oligonucleotide pool are required prior to subsequent gene assembly. When all the synthesized nucleic acid sequences on a chip need to be cut down into a mixture, to avoid interactions between different sequences in the mixture, the sequence and the amount of each nucleic acid need to be carefully designed. And during gene synthesis, the oligonucleotide mixture needs to be divided into several oligonucleotide sub-pools using universal primers and other methods, and then further gene assembly is performed. The mixing operation is complex which may cause the depooling problem. The nucleic acid synthesis method based on microfluidic device has been reported that it has the benefits of no cross-contamination, saving reagents, and a high synthesis yield (100 pmol level, which can directly apply to gene assembly without amplification). However, the microfluidic device itself needs to introduce a micropump and microvalve and the like, which makes its structure relatively complex, operation troublesome and efficiency reduced. Therefore, microfluidic-device-based synthesis approach has not been widely applied and commercialized. In addition, some companies have reported a commercialized DNA synthesis platform based on semiconductor silicon chips. This unique honeycomb microwell design reduces the reaction volume by one million times. But this method requires relatively complex substrate processing technology, unique liquid processing technology and substrate fixing device, resulting in a high cost of synthesizing nucleic acid. Although no manual mixing or splitting process is required during semiconductor silicon chips-based synthesis, the yield is still not high enough to perform following gene assembly without PCR amplification. Therefore, it is necessary to continue to develop nucleic acid synthesis technologies that can produce suitable amounts of synthetic oligonucleotides and has the advantages of simplicity, low cost, high-throughput and potential of commercializing automated gene assembly.
The purpose of the present invention is to provide a high-throughput gene synthesis device based on cluster arrays, which can synthesize oligonucleotides of various lengths on the same plate. This method is more conducive to the PCA splicing of oligonucleotides or ligase-mediated assembly using a combination of short primers and long oligonucleotides.
The high-throughput gene synthesis device based on cluster arrays provided by the present invention includes a substrate and a microwell plate;
In the gene synthesis device mentioned above, the micropore is a funnel-shaped micropore or a cylindrical micropore;
In the gene synthesis device mentioned above, the substrate can be a silicon wafer, and the micropore can be prepared by the MEMS micro-nano processing method.
In the above-mentioned gene synthesis device, the substrate can be a polymer plastic plate, and the micropore can be prepared by 3D printing or injection molding.
In the above-mentioned gene synthesis device, the solid phase carriers can be glass microspheres or polystyrene microspheres;
In the above-mentioned gene synthesis device, each cluster of the micropores includes 4 to 68 micropores;
When using the gene synthesis device of the present invention to synthesize oligonucleotides, the following steps can be performed:
The device of the present invention can synthesize oligonucleotides of various lengths on the same plate and is more conducive to the polymerase-mediated gene assembly splicing of oligonucleotides or ligase-mediated assembly using a combination of short primers and long oligonucleotides. Compared with the traditional multi-step splicing method using short oligonucleotides, the one-step splicing method can be adopted to accomplish gene assembly using ultra-long oligonucleotides as initial splicing elements, which is easier to automate.
When synthesizing oligonucleotides using the device of the present invention, the synthesized oligonucleotides are automatically recovered into the corresponding size of standard SBS plates (96-well plate, 384-well plate, 1536-well plate, etc.) under the device to form an oligonucleotide pool for each gene. The yield of oligonucleotides is at the picomole level, which can meet the needs of subsequent gene assembly through polymerase chain reaction (PCR) or ligation chain reaction (LCR) without amplification. After error correction, the oligonucleotides can be used to complete the full-length assembly of genes, realizing high-throughput automated gene synthesis.
The experimental methods in the following examples are conventional methods unless otherwise specified.
The materials, reagents, and etcetera used in the following examples can be obtained from commercial sources unless otherwise specified.
The process flow of processing funnel-shaped micropores arranged in clusters on a silicon wafer is shown in
As shown in
As shown in
As shown in
Nucleic acid synthesis reactions were carried out on the solid phase carriers in funnel-shaped micropores on silicon wafers or in micropores in polymer plastic plates: standard chemical synthesis methods (including the steps of deprotection, coupling, capping and oxidation) may be used.
Specific chemical synthesis implementation method is as follows: using different micro-nano dispensing heads for four or more different (deoxygenated/modified) nucleotide monomer solutions and activators and/or auxiliary reagents, according to the sequence information to be synthesized in each synthesis pore. The liquid type, position and liquid amount of the dispensing head were automatically controlled to complete the chemical synthesis of nucleic acid.
The liquid dispensing device used in the synthesis process is a micro-nanoliquid dispensing head, as shown in
A purified 150 nt oligonucleotide product was detected on the 2100 Bioanalyzer using the capillary electrophoresis kit, RNA Pico 6000 Kit (Agilent, Cat. No. 5067-1513).
The flow chart of the overall process of gene synthesis is shown in
The oligonucleotides synthesized in each cluster of funnel-shaped micropores are recovered into one well of the corresponding multi-well plate (96 wells, 384 wells, 1536 wells), and gene splicing and assembly are performed directly in the corresponding wells to achieve automated parallel synthesis of 96 or 384 or 1536 genes.
For example, to synthesize the 1546-base CDS sequence (with a 27-base tag sequence at the N-terminus for protein purification) of the methylcytosine dioxygenase (Tet1, mouse) gene, the sequence is as follows (SEQ ID NO: 1 in the Sequence Listing):
The process of gene synthesis is as follows:
(1) Design of Oligonucleotide Sequences According to the DNA Sequence of the Target Gene
By using DNAWorks, the target DNA sequence was codon-optimized and split into 12 sequence fragments connected end to end. And each segment was about 150 nt in length, the average number of bases in the overlapping region was about 20 bp, and the Tm value was 62° C. The head and tail primers for amplifying the 1546 nt fragment, Pa and Pb, were designed. The sequences of the 12 fragments and the head and tail primers are shown in Table 1:
(2) Synthesis of Oligonucleotides
The designed oligonucleotides were synthesized on the solid phase carriers in the funnel-shaped micropores in cluster arrays, and each cluster of oligonucleotides after ammonolysis was recovered into one well of the corresponding 96-well plate/384-well plate. The recovered oligonucleotide pools (Seq1-Seq12) were directly used for gene assembly without further purification steps. The oligonucleotide pools (Seq1-Seq12) were detected by capillary electrophoresis on Agilent 2100 Bioanalyzer, and the results are shown in
(3) One-Step Gene Assembly Using the Polymerase Approach
The polymerase-based assembly method comprises two steps. The first step was Polymerase Cycling Assembly (PCA). 12 oligonucleotide fragments were used as primers and templates for each other to perform one-step splicing. PCR amplification of the spliced target fragments was carried out using the head and tail primers, Pa and Pb, and the product was tested by capillary electrophoresis on Agilent 2100 Bioanalyzer.
The PCA reaction system: 2×HiFi HotStart ReadyMix (Roche, Cat. No. KK2602), oligomix (4 pmoL each), and nuclease-free water to bring the volume to 4 μL (minimum reaction volume: 2 μL, maximum volume: 50 μL)
The following reaction program was executed:
The head and tail primers, Pa and Pb, were used to amplify the spliced target fragment by PCR. The reaction system:
The following PCR reaction program was executed:
After the above PCA reaction, the Smear product after the fragment fusion was obtained, and then the PCA product was subjected to a PCR reaction to carry out the full-length fragment synthesis of the gene to obtain the 1546 bp target fragment using bilateral primers. CorrectASE enzyme (Thermo Fisher, Cat. No. A14972) was used for the error correction of the PCR product to obtain the final product for downstream cloning. The PCA, PCR, and error-corrected products were tested on 2100 bioanalyzer using a capillary electrophoresis kit, High Sensitivity DNA Kit (Agilent, Cat. No. 5067-4626). The fragment analysis results are shown in
On the basis of one-step assembly, the PCA system can be reduced to 2-5 μL, and the components of PCR reaction system can be directly added into the PCA reaction tube for one-tube assembly.
(4) Clone Sequencing
After ligating the PCR product obtained in step (3) and the error-corrected product with the T vector, the plasmids were transferred into Escherichia coli DH5α competent cells, and every 10 to 16 positive clones were picked for first-generation sequencing. The sequencer used was ABI 3730 XL, and it was found that all the sequence results showed that the fragments of the target length had been successfully synthesized, and it was ensured that at least one strain contained completely correct sequences, while other sequences contained from 1 to 2 mutation sites. The sequencing results are shown in
Then two rounds of error correction were performed using CorrectASE. After the first and second rounds of correction, on the 2100 bioanalyzer, a sharper peak was detected, indicating a lower error rate. From the error-corrected products obtained in step (3), 2 to 4 colonies were picked for sequencing to obtain completely correct gene clones. Sequencing results showed that after error correction, the sequencing showed an error rate of about 1/3000-1/10000.
The high-throughput automated gene synthesis system based on cluster arrays completes the high-throughput oligonucleotide synthesis through the funnel-shaped pores in cluster structure. Then these cluster arrays are one-to-one automatically recovered into the wells of standard SBS plates to form oligonucleotide pools for subsequent gene assembly. The yield of oligonucleotides reaches picomole level, which can just meet the needs of gene splicing without amplification.
Compared with the traditional gene synthesis method, it avoids both a large number of the manual operations of mixing oligonucleotides, and waste caused by the nanomole level products of traditional oligonucleotide synthesis approach. Compared with oligonucleotides synthesized based on microarray chips, the yield of a single oligonucleotide is higher which can be directly used for subsequent gene assembly without amplification. There is no need for a PCR splitting step in high-throughput oligonucleotide sub-pools. At the same time, errors caused by amplification can be effectively reduced, thereby reducing the error rate.
At the same time, ultra-long oligonucleotides can realize one-step splicing and simplify the operation steps. The synthesis amount of each oligonucleotide just meets the picomole level of gene splicing, which reduces the synthesis cost. At the same time, the cluster synthesis of unique oligonucleotides is innovatively connected with the standard microwell plate for downstream gene splicing, which achieves a higher automation level than traditional multi-step splicing.
The present invention solves the current bottlenecks in the field of gene synthesis, such as low-throughput, and cumbersome manual operation, provides a commercialized and low-cost high-throughput automated gene synthesis method.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110344493.0 | Mar 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/094310 | 5/18/2021 | WO |
