TWI845557B - A high-throughput gene synthesis method based on chip primer surface extraction - Google Patents

Abstract

The present invention provides a high-throughput gene synthesis method based on chip primer surface extraction. The method classifies primers into clusters and synthesizes them in a fixed area of the chip and cuts them. Then, a sampler is used to dissolve the primers of different clusters and transfer them to a 96-well plate for gene synthesis.

Description

A high-throughput gene synthesis method based on chip primer surface extraction

The present invention relates to the field of gene synthesis, specifically to chip primer synthesis and a high-throughput automated gene synthesis method based on the chip primer.

Gene synthesis is usually based on polymerase chain assembly (PCA) and polymerase chain reaction (PCR), and pre-designed primer sequences are spliced into the target gene sequence according to the method of head-to-tail complementary pairing. Primers in conventional gene synthesis are synthesized by column synthesis, and the primers are mixed one by one. Since the synthesis scale of primers (25nmol) is much larger than the amount of primers required for gene synthesis (50pmol), conventional gene synthesis has the disadvantages of low throughput, high cost, and complex operation. In order to overcome the above shortcomings, a high-throughput gene synthesis method has been developed. This method relies on a high-throughput primer synthesis platform, which can synthesize tens of thousands or even millions of primers on a chip with an area of only a few square centimeters to dozens of square centimeters. The synthesis scale of each primer is at the fmol level, which greatly reduces the cost of primer synthesis. The primers are then divided into several libraries using methods such as specific PCR, each library containing all the primer sequences required to synthesize a target gene. After removing the tag sequences on the primers using methods such as enzyme cutting, the primer libraries are assembled into the corresponding target genes one by one (Nature, 2004, 1050-1054). Although this high-throughput gene synthesis method greatly improves throughput and reduces costs, it still has the following problems: 1) The overall operation process is relatively complicated, requiring the primers to be merged and then grouped, and undergoing tedious enzyme cutting, purification and other operations, and the degree of automation is low. 2) The primer utilization rate is low and the mutation rate is high. Since PCR binding sites and enzyme cleavage sites need to be added to both ends of each primer, the primer sequence used for gene synthesis usually only accounts for 50% or even less of the total length of the primer. On the other hand, the longer the length of the primer synthesis, the higher the mutation rate, resulting in a higher mutation rate in the final gene synthesis. 3) Low specificity and cross-contamination risk. All primers are mixed together after cutting. The specificity differences between different PCR primer sequences will greatly affect the PCR efficiency, so there is a greater possibility of cross-contamination.

Although the aforementioned high-throughput gene synthesis method has greatly improved throughput and reduced costs, the following problems still exist: 1) The overall operation process is relatively complicated, requiring primers to be merged and then grouped, and undergoing tedious enzyme cutting, purification and other operations, and the degree of automation is low. 2) The primer utilization rate is low and the mutation rate is high. Since sequences such as PCR binding sites and enzyme cutting sites need to be added to both ends of each primer, the primer sequence used for gene synthesis usually only accounts for 50% or even less of the total length of the primer. On the other hand, the longer the primer synthesis length, the higher the mutation rate, resulting in a higher mutation rate in the final gene synthesis. 3) Low specificity and cross-contamination risk. All primers are mixed together after cutting. The specific differences between different PCR primer sequences will greatly affect the PCR efficiency, so there is a greater possibility of cross-contamination.

On one hand, the present invention provides a high-throughput gene synthesis method based on chip primer surface extraction, the aforementioned method comprises classifying primers into clusters and synthesizing them in different areas of the chip, cutting the primer clusters on the chip by aminolysis, then using a liquid transfer workstation to dissolve the primers of different clusters, and transferring each dissolved primer cluster to an independent reaction container suitable for gene synthesis, performing a gene synthesis reaction under conditions suitable for gene synthesis, and harvesting the synthesized target gene fragment.

In some embodiments, the primers classified by cluster are primer clusters encoding multiple target gene fragments, wherein each primer cluster contains all primers encoding the same target gene fragment. In some embodiments, the different primer clusters are synthesized in different regions of the chip, and blank regions are left between the different regions. In some embodiments, multiple hydrophobic groups are synthesized on the blank regions.

In some embodiments, at least one linker that can be cleaved by aminolysis and at least three dTs are additionally added to the 3' end of each primer as a protective base when the primers are synthesized on the chip. In some embodiments, 2, 3, 4 or 5 linkers that can be cleaved by aminolysis and 3, 4, 5 or 6 dTs are additionally added to the 3' end of the primer when the primers are synthesized on the chip. In some more desirable embodiments, 2 linkers that can be cleaved by aminolysis and 5 dTs are additionally added to the 3' end of the primer when the primers are synthesized on the chip as a protective base. The aforementioned linker arm is selected from any structure that can generate a 3' free hydrocarbon group in the primer after aminolysis, preferably a structure based on a phosphoramidite structure that can provide a 3' free hydroxyl group, and more preferably a succinyl hexylamine phosphoramidite. In some embodiments, the primer cut by the aforementioned aminolysis method contains a 3' free hydroxyl group.

In some embodiments, the aforementioned aminolysis method is aminolysis with a non-aqueous aminating agent; ideally, the aforementioned aminating agent is selected from ammonia, monomethylamine, and more ideally monomethylamine. In some embodiments, the aforementioned aminolysis is carried out in a high-pressure reaction vessel filled with an aminating agent. In some embodiments, the aforementioned aminolysis is carried out at 25°C to 120°C and 20 to 120 psi for 15 minutes to 4 hours; ideally, the aforementioned aminolysis is carried out at 60°C to 90°C and 20 to 60 psi for 1 to 4 hours; more ideally, the aforementioned aminolysis is carried out at 80°C and 40 psi for 3 hours.

In some embodiments, the liquid transfer workstation in the aforementioned high-throughput gene synthesis method is a sampler. In some embodiments, the aforementioned liquid transfer workstation is a Biodot AD 1500 sampling platform. In one embodiment, the aminolyzed chip is placed on the sampling platform, and after position correction, the solvent for dissolving the primer is transferred.

In some embodiments, the solvent used for primer dissolution is selected from solvents that can be used for PCR reaction, preferably TE solution or distilled water, more preferably distilled water. In some embodiments, the primer dissolution includes using a sampler to drop 10~1000nL of solvent such as distilled water into each primer cluster, standing for 0.5~3 minutes, and then using a needle to blow and aspirate to dissolve it. Ideally, the primer dissolution includes using a sampler to drop 50nL of distilled water into each primer cluster, standing for 1 minute, and then using a needle to blow and aspirate to dissolve it.

In some embodiments, the aforementioned independent reaction container suitable for gene synthesis is a well of a multi-well plate, which can be selected from a 96-well plate, a 24-well plate, and a 384-well plate. In some embodiments, the aforementioned step of transferring to an independent reaction container suitable for gene synthesis includes adjusting the height of the needle of the liquid transfer workstation to the surface of the chip and aspirating the dissolved liquid in the chip and transferring it to the multi-well plate. In some embodiments, after aspirating the dissolved liquid in the chip and transferring it to the multi-well plate, the sampling needle is further cleaned by a cleaning procedure, and then the primer dissolution and transfer steps are repeated until all primer clusters are completely transferred.

In some embodiments, the aforementioned gene synthesis step includes adding a gene synthesis reaction solution and/or F/R primers of the target gene fragment to each independent reaction container to perform a synthesis reaction. In some embodiments, the aforementioned gene synthesis reaction solution is selected from a PCA reaction solution and a PCR reaction solution. In some embodiments, the aforementioned gene synthesis step includes adding a PCA reaction solution to each reaction container to cluster the primers for assembly reaction, and then adding a PCR reaction solution and F/R primers of the target gene fragment to perform a PCR reaction.

Another aspect of the present invention provides a high-throughput gene synthesis method based on chip primer surface extraction, comprising the following steps:

1) Synthesize primer clusters encoding multiple target gene fragments on a chip, wherein each primer cluster contains all primers encoding the same target gene fragment, and primer clusters encoding different target gene fragments are synthesized in different regions of the chip;

2) After the synthesis is completed, the chip is subjected to aminolysis to cut the primer from the chip, and the cut primer contains a 3' free hydroxyl group;

3) Add solvent to each primer cluster to fully dissolve the primers in the primer cluster, collect each dissolved primer cluster and transfer them to independent reaction containers respectively;

4) Add gene synthesis reaction solution to the reaction container to perform gene synthesis;

5) Recover the reaction product to obtain multiple target gene fragments.

In some embodiments, a blank area is left between the aforementioned different areas in the aforementioned step 1). In another embodiment, a plurality of hydrophobic groups are synthesized on the aforementioned blank area.

In some embodiments, when the primers are synthesized on the chip in the aforementioned step 1), at least one linker that can be cleaved by aminolysis and at least three dTs are additionally added to the 3' end of each primer as a protective base. In some embodiments, 2, 3, 4 or 5 linkers that can be cleaved by aminolysis and 3, 4, 5 or 6 dTs are additionally added to the 3' end of each primer as a protective base. In some embodiments, when the primers are synthesized on the aforementioned chip, 2 linkers that can be cleaved by aminolysis and 5 dTs are additionally added to the 3' end of the primer as a protective base. The aforementioned linker is selected from any group that can generate a 3' free hydroxyl group in the primer, and is preferably a structure based on a phosphoramidite structure and capable of providing a 3' end free hydroxyl group, and more preferably a succinyl hexamethylene phosphoramidite. In some embodiments, the primer cleaved by the aforementioned aminolysis method contains a 3' free hydroxyl group.

In some embodiments, the aforementioned step 2) comprises aminolysis with a non-aqueous aminating agent; ideally, the aforementioned aminating agent is selected from ammonia, monomethylamine; more ideally, monomethylamine. In some embodiments, the aforementioned aminolysis is carried out in a high-pressure reaction vessel filled with an aminating agent. In some embodiments, the aforementioned aminolysis is carried out at 25°C to 120°C and 20 to 120 psi for 15 minutes to 4 hours; ideally, the aforementioned aminolysis is carried out at 60°C to 90°C and 20 to 60 psi for 1 to 4 hours; more ideally, the aforementioned aminolysis is carried out at 80°C and 40 psi for 3 hours.

In some embodiments, the solvent for dissolving the primers in the aforementioned step 3) is selected from solvents that can be used for PCR reactions; ideally, it is TE solution or distilled water; more ideally, it is distilled water. In some specific embodiments, the aforementioned primer dissolution includes using a sampler to drop 10~1000nL of distilled water into each primer cluster, standing for 0.5~3 minutes, and then using a needle to blow and aspirate to dissolve it. Ideally, the aforementioned primer dissolution includes using a sampler to drop 50nL of distilled water into each primer cluster, standing for 1 minute, and then using a needle to blow and aspirate to dissolve it.

In some embodiments, the independent reaction container in the above step 3) is a hole of a porous plate, and the porous plate can be selected from a 96-well plate, a 24-well plate, and a 384-well plate. In some embodiments, the above step of transferring to an independent reaction container includes adjusting the needle height of a liquid transfer workstation such as a sampler to the surface of the chip and sucking the dissolved liquid in the chip and transferring it to the porous plate. In some embodiments, after sucking the dissolved liquid in the chip and transferring it to the porous plate, it further includes using a cleaning procedure to clean the sampling needle and then repeating step 3) until all primer clusters are completely transferred.

In some embodiments, the gene synthesis reaction solution in the aforementioned step 4) is selected from PCA reaction solution and PCR reaction solution. In some embodiments, optionally, the aforementioned step 4) drains the solvent in the reaction container before adding the gene reaction solution. In some embodiments, the aforementioned gene synthesis includes draining the solvent in the reaction container, adding PCA reaction solution to each reaction container, allowing the primers to cluster for assembly reaction, and then adding PCR reaction solution and F/R primers of the target gene fragment to perform PCR reaction.

On the other hand, the present invention provides a method for synthesizing primer clusters for high-throughput gene synthesis. The method comprises classifying primers by clusters, synthesizing different primer clusters in different areas of a chip, cutting the primer clusters on the chip by an aminolysis method, and then using a liquid transfer workstation to dissolve the primers of different clusters and collect each dissolved primer cluster.

In some embodiments, each of the aforementioned primer clusters includes all primers encoding the same target gene fragment, the aforementioned chip includes primer clusters synthesized to encode multiple target gene fragments, and primer clusters encoding different target gene fragments are synthesized in different regions of the chip, respectively, with blank regions left between the aforementioned different regions. Ideally, multiple hydrophobic groups are synthesized in the aforementioned blank regions.

In some embodiments, at least one linker that can be cleaved by aminolysis and at least three dTs are added to the 3' end of each primer as a protective base when the primers are synthesized on the chip. In some embodiments, two, three, four or five linkers that can be cleaved by aminolysis and three, four, five or six dTs are added to the 3' end of each primer as a protective base. In some embodiments, two linkers that can be cleaved by aminolysis and five dTs are added to the 3' end of the primer as a protective base when the primers are synthesized on the chip. The linker is selected from any structure that can generate a 3' free hydroxyl group in the primer, preferably a structure based on a phosphoramidite structure and capable of providing a 3' end free hydroxyl group, and more preferably a succinyl hexamethylene phosphoramidite. In some embodiments, the primer cleaved by the aforementioned aminolysis method contains a 3' free hydroxyl group.

In some embodiments, the aminolysis method comprises aminolysis with a non-aqueous aminating agent; ideally, the aforementioned aminating agent is selected from ammonia, monomethylamine; more ideally, monomethylamine. In some embodiments, the aforementioned aminolysis is carried out in a high-pressure reaction vessel filled with the aminating agent. In some embodiments, the aforementioned aminolysis is carried out at 25°C to 120°C and 20 to 120 psi for 15 minutes to 4 hours; ideally, the aforementioned aminolysis is carried out at 60°C to 90°C and 20 to 60 psi for 1 to 4 hours; more ideally, the aforementioned aminolysis is carried out at 80°C and 40 psi for 3 hours.

In some embodiments, the liquid transfer workstation in the aforementioned method of synthesizing primer clusters is a sampler. In some embodiments, the liquid transfer workstation is a Biodot AD 1500 sample station. In some embodiments, the aminolyzed chip is placed on the Biodot AD 1500 platform, and after position correction, the solvent for dissolving the primers is transferred.

In some embodiments, in the method for synthesizing primer clusters, the solvent for dissolving the primers is selected from solvents that can perform PCR reactions; ideally, it is TE solution or distilled water; more ideally, it is distilled water. In some specific embodiments, the primer dissolution includes using a sampler to drop 10-1000nL of distilled water into each primer cluster, standing for 0.5-3 minutes, and then using a needle to blow and aspirate to dissolve it. Ideally, the primer dissolution includes using a sampler to drop 50nL of distilled water into each primer cluster, standing for 1 minute, and then using a needle to blow and aspirate to dissolve it.

In some embodiments, the method for synthesizing primer clusters further comprises transferring each dissolved primer cluster to an independent reaction container, wherein the independent reaction container is a hole of a multi-well plate, and the multi-well plate can be selected from a 96-well plate, a 24-well plate, and a 384-well plate. In some embodiments, the step of transferring to an independent reaction container comprises adjusting the height of the sampler needle to the surface of the chip and transferring the dissolved liquid in the chip to the multi-well plate. In some embodiments, the step of transferring to an independent reaction container further comprises using a cleaning procedure to clean the sampler needle and then repeating the primer dissolution and transfer operations until all primer clusters are completely transferred.

The present invention provides a method for synthesizing a primer cluster for high-throughput gene synthesis, comprising the following steps:

1) Synthesize primer clusters encoding multiple target gene fragments on a chip, wherein the primer cluster encoding each target gene fragment includes all primers encoding the target gene fragment, and the primer clusters encoding different target gene fragments are synthesized in different fixed areas of the chip, leaving blank areas between the aforementioned different areas;

2) After the synthesis is completed, the chip is subjected to aminolysis to cut the primer from the chip, and the cut primer contains a 3' free hydroxyl group;

3) Add solvent to each primer cluster to fully dissolve the primers in the primer cluster, and collect each dissolved primer cluster.

In some embodiments, in the aforementioned step 1), at least one linker cleavable by aminolysis and at least three dTs are additionally added to the 3' end of the primer as a protective base when the primer is synthesized on the chip. In some embodiments, two, three, four or five linkers cleavable by aminolysis and three, four, five or six dTs are additionally added to the 3' end of each primer as a protective base. In some embodiments, two linkers cleavable by aminolysis and five dTs are additionally added to the 3' end of the primer as a protective base when the primer is synthesized on the chip. The aforementioned linker arm can be selected from any structure that can generate a 3' free hydroxyl group in the primer, preferably a structure based on a phosphoramidite structure that can provide a 3' end free hydroxyl group; more preferably, succinylated phosphoramidite.

In some embodiments, the aforementioned step 2) comprises aminolysis with a non-aqueous aminating agent; ideally, the aforementioned aminating agent is selected from ammonia, monomethylamine; more ideally, monomethylamine. In some embodiments, the aforementioned aminolysis is carried out in a high-pressure reaction vessel filled with an aminating agent. In some embodiments, the aforementioned aminolysis is carried out at 25°C to 120°C and 20 to 120 psi for 15 minutes to 4 hours; ideally, the aforementioned aminolysis is carried out at 60°C to 90°C and 20 to 60 psi for 1 to 4 hours; more ideally, the aforementioned aminolysis is carried out at 80°C and 40 psi for 3 hours.

In some embodiments, the solvent in the aforementioned step 3) is selected from solvents that can perform PCR reactions; ideally, it is TE solution or distilled water; more ideally, it is distilled water. In some specific embodiments, the aforementioned primer dissolution includes using a sampler to drop 10~1000nL of distilled water into each primer cluster, standing for 0.5~3 minutes, and then using a needle to blow and aspirate to dissolve it. Ideally, the aforementioned primer dissolution includes using a sampler to drop 50nL of distilled water into each primer cluster, standing for 1 minute, and then using a needle to blow and aspirate to dissolve it.

In some embodiments, the method for synthesizing primer clusters further comprises transferring each dissolved primer cluster to an independent reaction container, wherein the independent reaction container is a hole of a multi-well plate, and the multi-well plate can be selected from a 96-well plate, a 24-well plate, and a 384-well plate. In some embodiments, the step of transferring to an independent reaction container comprises adjusting the height of the sampler needle to the surface of the chip and transferring the dissolved liquid in the chip to the multi-well plate. In some embodiments, the step of transferring to an independent reaction container further comprises using a cleaning procedure to clean the sampler needle and then repeating the dissolution and transfer operations until all primer clusters are completely transferred.

The primer cluster synthesized by the above method can be used for high-throughput gene synthesis, including transferring the synthesized primer cluster to an independent reaction container, performing gene synthesis under conditions suitable for gene synthesis, and obtaining synthesized gene fragments.

The effects of this invention are:

First, due to the improvement of the overall strategy, the present invention can greatly shorten the length of the chip primers required for synthesis, which shortens the cycle of gene synthesis on the one hand, and reduces the mutation rate of gene synthesis on the other hand. Secondly, the present invention completely changes the scheme of mixing first and then grouping in the high-throughput gene synthesis based on chip primers, simplifies the process and improves the feasibility of automation. Finally, by avoiding the use of PCR for grouping, the present invention improves specificity and reduces the possibility of cross contamination.

The present invention will be more fully understood from the following detailed description in conjunction with the drawings, in which like elements are numbered in a similar manner.

【Figure 1】A schematic diagram showing the layout of primers in the chip synthesis area.

【Figure 2】Schematic diagram of primer extraction on chip surface.

【Figure 3】Chip layout and its enlarged view.

【Figure 4】Gene_Frag 4 sequencing results.

【Figure 5】Gene_Frag 13 sequencing results.

【Figure 6】Shows the electrophoresis gel of the target gene after gene synthesis.

The present invention adds a cleavable linker arm and a protective base to the 3' end of all primers to ensure that the primers have a 3' free hydroxyl group after aminolysis. The primers grouped according to the target gene are synthesized in specific areas on the chip, and a certain blank area is left between the areas to avoid cross contamination during extraction (as shown in Figure 1). In order to further reduce the possibility of cross contamination, a series of hydrophobic groups can be synthesized in the blank area to increase the surface energy. The synthesized chip needs to use gas aminolysis to cut the primers from the chip surface and retain them in the corresponding synthesis position. Then use a sampler to drop an appropriate amount of distilled water to the primer clusters in each area to dissolve the primers (as shown in Figure 2), and transfer the distilled water dissolved with the primers to a 96-well plate for corresponding gene synthesis. In order to improve the efficiency of primer transfer, repeated blowing and suction can be used during extraction to enhance the dissolution effect. The transfer needle needs to be cleaned between transfers of different primer clusters to avoid cross contamination.

Methods for synthesizing primers on a chip are known in the art, such as inkjet printing or photoactivation.

In the present invention, a primer cluster refers to a collection of all primers encoding the same target gene fragment, and different primer clusters encode different target gene fragments. The primers here can also be called short nucleic acid fragments, which refer to single-stranded short nucleic acid fragments that partially overlap each other and cover the entire target gene fragment. These short nucleic acid fragments serve as primers and templates for each other, and can be subjected to multiple rounds of denaturation, annealing, and extension cycles in the presence of polymerase to ultimately obtain the target gene. In the present invention, all primers in a primer cluster can, for example, synthesize the target gene fragment by polymerase chain assembly (PCA).

When performing primer synthesis, each primer can be synthesized multiple times in the region to which it belongs, for example, 3 to 8 times. For a primer, "synthesized multiple times" means that the primer is synthesized at multiple positions in the region.

In the present invention, the target gene fragment can be any target synthesized gene fragment. For example, the aforementioned target gene fragment can be a complete short gene with a length of several hundred bp. The aforementioned target gene fragment can also be a part of a longer gene. For example, for a long gene, it can be split into multiple short gene fragments during synthesis. These short gene fragments can be the target gene fragments of the present invention. Each short gene fragment is further split into multiple primers, and each short gene fragment is synthesized using the method of the present invention, and then these short gene fragments are assembled into a complete long gene.

In the present invention, different primer clusters are synthesized in different regions of the chip. Different regions of the chip can be divided in any appropriate manner, for example, the rows and columns of each region can be determined. The shape and size of each region can be determined according to specific needs. A blank area can be left between each region to avoid cross contamination during extraction. The size of the blank area can be determined by a person with ordinary knowledge in the relevant technical field according to the situation. A series of hydrophobic groups can also be synthesized on the blank area to increase the surface energy, thereby further avoiding cross contamination during extraction.

In the present invention, in order to perform subsequent gene synthesis, it is necessary to make the primer cluster have a 3' free hydroxyl group after being cut off from the chip. Various possible methods can be used to make the cut primer contain a 3' free hydroxyl group. Some of these methods are known in the art, such as by aminolysis. Ideally, at least one linker that can be cut by aminolysis and at least 3 dTs as protective bases can be added to the 3' end of the primer during synthesis. When aminolysis is performed, the linker and dT can be cut off to form a free hydroxyl group at the 3' end of the primer. In some embodiments, 2, 3, 4 or 5 linkers that can be cut by aminolysis and 3, 4, 5 or 6 dTs as protective bases are added to the 3' end of the primer. In some embodiments, two additional linkers that can be cleaved by aminolysis and five dTs are added to the 3' end of the primer as protective bases. The aforementioned linker can be any structure that can generate a 3' free hydroxyl group in the primer after aminolysis, and is ideally a structure based on a phosphoramidite structure that can provide a 3' free hydroxyl group, such as succinyl hexamethylenetetramidite. In the present invention, the purpose of the aforementioned "linker" is to form a free hydroxyl group at the 3' end of the primer by aminolysis. The purpose of adding at least three dTs to the 3' end of the primer outside the linker is to serve as a protective base.

In the present invention, the method of aminolysis is known in the art, and the aminating agent that is particularly suitable for the present invention may be, for example, a non-aqueous aminating agent, such as ammonia, monomethylamine, etc. The aminolysis may be carried out, for example, in a high-pressure reaction vessel filled with the aminating agent. In some embodiments, the aminolysis is carried out at 25°C to 120°C and 20 to 120 psi for 15 minutes to 4 hours; ideally, the aforementioned aminolysis is carried out at 60°C to 90°C and 20 to 60 psi for 1 to 4 hours; more ideally, the aforementioned aminolysis is carried out at 80°C and 40 psi for 3 hours.

Psi is a unit of pressure known in this field. According to the known technology in this field, 1psi=6.895kPa.

In the present invention, any liquid transfer workstation that can dissolve and transfer the primers synthesized on the chip can be used, for example, a sampler can be used. In some embodiments, the aforementioned liquid transfer workstation is a Biodot AD 1500 sample station. In some embodiments, the aforementioned aminolyzed chip can be placed on the sampling platform, and after position correction, the solvent used to dissolve the primer can be transferred.

In the present invention, the solvent used to dissolve the primers can be any solvent that can be used for PCR reaction, such as TE solution or distilled water, preferably distilled water. In some embodiments, the process of dissolving the primers includes using a liquid transfer workstation (such as a sampler) to drop 10-1000nL of solvent (such as distilled water) into each primer cluster, standing for 0.5-3 minutes, and then using a needle to blow and aspirate to dissolve it. Ideally, 50nL of distilled water is added to each primer cluster, standing for 1 minute, and then using a needle to blow and aspirate to dissolve it.

The primers on the chip can be fully dissolved by methods known in the art, such as using a needle to blow and aspirate 1 to 3 times (e.g., 2 times) to fully dissolve the primers. In some embodiments, for example, a Biodot sampler can be used to add distilled water to each primer cluster and then let it stand for a period of time (e.g., 1 minute), and then blow and aspirate 1 to 3 times (e.g., 2 times) with a needle.

In the present invention, the independent reaction container suitable for gene synthesis can be a hole of a porous plate or any other suitable independent reaction container. The porous plate can be selected from a 96-well plate, a 24-well plate, and a 384-well plate. In some embodiments, the step of transferring the dissolved primer cluster to an independent reaction container includes adjusting the needle height of the liquid transfer workstation to the surface of the chip and aspirating the dissolved liquid in the chip and transferring it to the hole of the porous plate. In some embodiments, part of the primer cluster can be transferred first, and then the sampling needle is cleaned and the remaining primer clusters are repeatedly transferred. For example, part of the primer cluster can be transferred first, and then the sampling needle is cleaned using a cleaning program and the transfer operation is repeated until all primer clusters are completely transferred.

In the present invention, the gene synthesis reaction may include adding a gene synthesis reaction solution and/or F/R primers of the target gene fragment to each well to perform a PCR reaction. The gene synthesis reaction solution may be selected from a PCA reaction solution and a PCR reaction solution. For example, the PCA reaction solution may be added first to cluster the primers for assembly reaction, and then the PCR reaction solution and F/R primers of the target gene fragment may be added to perform a PCR reaction. Before performing the gene synthesis reaction, the solvent in the reaction container may be drained, and then the gene synthesis reaction solution and/or F/R primers of the target gene fragment may be added, or the gene synthesis reaction solution and/or F/R primers of the target gene fragment may be directly added to the reaction container. In the present invention, polymerase chain assembly (PCA) is a method based on the principle of polymerase chain reaction (PCR), which refers to making single-stranded oligonucleotides that partially overlap each other and cover the entire target gene serve as primers and templates for each other, and finally obtain the target gene through multiple rounds of denaturation, annealing, and extension cycles in the presence of polymerase. In some cases, the product obtained by polymerase chain assembly can be amplified using primers that can bind to both ends of the product sequence, for example, by PCR reaction.

In the present invention, the F/R primers of the target gene fragment refer to the forward and reverse primers used to amplify the entire target gene fragment.

In the present invention, "drying" refers to the operation of removing the solvent in the reaction container while retaining the solute, i.e., the primer cluster. The solvent can be dried by various methods known in the art, for example, the solvent in the reaction container can be dried by reduced pressure drying.

The following is a further detailed description of the technical means of the present invention through embodiments and combined with drawings, but the present invention is not limited to the following embodiments.

Example 1 Synthesis of 19 target genes

This embodiment will use this platform to synthesize 19 target genes, the lengths of which are shown in Table 1 below:

Two fragments, Gene_Frag 4 and Gene_Frag 13, are used as examples for detailed explanation.

Synthesis of target gene of Gene_Frag 4

The target gene sequence of Gene_Frag 4 (SEQ ID NO.1) is as follows:

It was split into corresponding primers, and the primer sequences after adding a cleavable linker and a dT protective base at the 3' end were shown in Table 2 (W represents the cleavable linker, which is succinyl hexylamine phosphoramidite, and Gene_Frag 4_F and Gene_Frag 4_R are conventional primers synthesized using a 25nmol synthesis column):

Using CustomArray's B3P synthesizer, primers from Gene_Frag 4_1 to Gene_Frag 4_16 were synthesized on the chip according to the preset positions. The arrangement of the chip is shown in the enlarged view in Figure 3, and the arrangement of primers synthesized on the microarray scanner (8*11) is shown in Table 3.

After the synthesis is completed, the chip is placed in a high-pressure gas phase ammonolysis apparatus, using monomethylamine as the ammonolysis gas, and ammonolysis is carried out at 80°C and 40psi for 3 hours. After the chip is cooled, it is taken out and transferred to the standard glass slide holder of the Biodot AD 1500 sampler.

Use the Biodot sampler to add 50nL of distilled water to each primer cluster. After standing for one minute, adjust the height of the Biodot sampler needle to the chip surface and absorb 60nL of volume and transfer it to the 96-well plate, then add 1μL of distilled water. Use the cleaning program to clean the sampling needle and repeat the above steps until all primer clusters are completely transferred.

Prepare PCA reaction system: 5*HF buffer 10μL, dNTPs 1μL, NEB-Phusion 0.5μL, BSA (20mg/mL) 5μL, H2O 33.5μL. And PCR reaction system: 5*HF buffer 2μL, dNTPs 0.2μL, NEB-Phusion 0.1μL, BSA (20mg/mL) 1μL, Gene_Frag 4_F (10μM) and Gene_Frag 4_R (10μM) 0.3μL each, H2O 3.6μL.

After the solvent in the sample in the 96-well plate was depressurized and dried by a vacuum centrifuge concentrator (Concentrator plus, eppendorf), 2.5μL PCA reaction mixture was added to each well containing the primer cluster and transferred to the 384-well for PCA reaction. The reaction conditions were: 98℃ 3min, 35 cycles (98℃ 10s, 58℃ 10s, 72℃ 20s), 72℃ 5min. Then, 2.5μL PCA product was transferred to 7.5μL PCR reaction system for PCR reaction. The reaction conditions were: 98℃ 3min, 40 cycles (98℃ 10s, 64℃ 10s, 72℃ 35s), 72℃ 5min.

Transform the above reaction solution (i.e. PCR product) into Top10 competent cells and follow the chemical competent cell transformation steps. After recovery, take 100μL of bacterial solution and spread it on Kan resistance color plate. Place it in a 37℃ incubator and culture it overnight.

The white spots on the color-developing plate were tested with bacterial detection primers, and 10 positive clones were selected for sequencing and the sequencing results were analyzed. The sequencing results of Gene_Frag 4 are shown in Figure 4.

Gene_Frag 13 target gene synthesis

The target gene sequence of Gene_Frag 13 (SEQ ID NO.20) is as follows:

It was split into corresponding primers, and the primer sequences after adding a cleavable linker and a dT protective base at the 3' end are shown in Table 4 (W represents the cleavable linker, which is succinyl hexylamine phosphoramidite, and Gene_Frag 13_F and Gene_Frag 13_R are conventional primers synthesized using a 25nmol synthesis column):

Using CustomArray's B3P synthesizer, the primer sequences from Gene_Frag 13_1 to Gene_Frag 13_12 were synthesized on the chip according to the preset positions. Chip arrangement See the enlarged image in Figure 3, and the arrangement of primers synthesized on the microarray scanner (8*11) is shown in Table 5.

After the synthesis is completed, the chip is placed in a high-pressure gas phase ammonolysis apparatus, using monomethylamine as the ammonolysis gas, and ammonolysis is carried out at 80°C and 40psi for 3 hours. After the chip is cooled, it is taken out and transferred to the standard glass slide holder of the Biodot AD 1500 sampler.

Use the Biodot sampler to add 50nL of distilled water to each primer cluster. After standing for one minute, adjust the height of the Biodot sampler needle to the chip surface and absorb 60nL of volume and transfer it to the 96-well plate, then add 1μL of distilled water. Use the cleaning program to clean the sampling needle and repeat the above steps until all primer clusters are completely transferred.

Prepare PCA reaction system: 5*HF buffer 10μL, dNTPs 1μL, NEB-Phusion 0.5μL, BSA (20mg/mL) 5μL, H ₂ O 33.5μL. And PCR reaction system: 5*HF buffer 2μL, dNTPs 0.2μL, NEB-Phusion 0.1μL, BSA (20mg/mL) 1μL, Gene_Frag 13_F (10μM) and Gene_Frag 13_R (10μM) 0.3μL each, H ₂ O 3.6μL.

After the solvent in the sample in the 96-well plate was depressurized and dried by a vacuum centrifuge concentrator (Concentrator plus, eppendorf), 2.5μL PCA reaction mixture was added to each well containing the primer cluster and transferred to the 384-well for PCA reaction. The reaction conditions were: 98℃ 3min, 35 cycles (98℃ 10s, 58℃ 10s, 72℃ 20s), 72℃ 5min. Then, 2.5μL PCA product was transferred to 7.5μL PCR reaction system for PCR reaction. The reaction conditions were: 98℃ 3min, 40 cycles (98℃ 10s, 64℃ 10s, 72℃ 35s), 72℃ 5min.

Transform the above reaction solution (i.e. PCR product) into Top10 competent cells and follow the chemical competent cell transformation steps. After recovery, take 100μL of bacterial solution and spread it on Kan resistance color plate. Place it in a 37℃ incubator and culture it overnight.

The white spots on the color-developing plate were tested with bacterial detection primers, and 10 positive clones were selected for sequencing and the sequencing results were analyzed. The sequencing results of Gene_Frag 13 are shown in Figure 5.

All 19 target genes were synthesized and subjected to electrophoresis. The electrophoresis diagram is shown in Figure 6.

Example 2 Comparison of the Chip Primer Surface Extraction Gene Synthesis Method with the Traditional Method

The traditional method uses the CustomArray synthesizer for high-throughput gene synthesis, referring to the gene synthesis method in Eroshenko N, Kosuri S, Marblestone AH, Gene Assembly from Chip-Synthesized Oligonucleotides. Curr Protoc Chem Biol. 4: 1-17, March 2012, and compares the time of the specific steps of the traditional method and the method of Example 1 in the synthesis step, aminolysis and purification step, extraction step, and expansion step, as shown in Table 6:

The implementation of the present invention is not limited to the above-mentioned embodiments. Without departing from the spirit and scope of the present invention, a person with ordinary knowledge in the relevant technical field can make various changes and improvements to the present invention in form and details, and these are considered to fall within the scope of protection of the present invention.

         
SEQUENCE LISTING
<![CDATA[<110> Nanjing Jinsirui Science & Technology Biology Corp., a Chinese company]]>
<![CDATA[<120> A high-throughput gene synthesis method based on surface extraction of primers on a chip]]>
<![CDATA[<130> 1]]>
<![CDATA[<150> CN 201811313909.7]]>
<![CDATA[<151> 2018-11-06]]>
<![CDATA[<160> 34 ]]>
<![CDATA[<170> PatentIn version 3.5]]>
<![CDATA[<210> 1]]>
<![CDATA[<211> 732]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4]]>
<![CDATA[<400> 1]]>
atgaggagcc ggaagctcac aggtgcagtg cggtcttcag cgcgcttgaa agcacgaagt 60
tgttcggcag ccaggttggc ctctgcccag gaagttgctg gttccacgtc tgccaagaca 120
gcatgtctga cttcctcatc ccacaaagcc acagacacgc gaacgtccaa gaagttcaaa 180
tgtgacaaag gacatcttgt gaagtcagaa ttacagaagc ttgtccctaa gaatgacagc 240
gcttctttgc caaaagtgac acctgagacc ccttgtgaaa atgagtttgc tgaaggcagt 300
gccttgcttc caggcagcga ggctggcgtt tctgtgcagc agggggctgc aagtcttcct 360
ctcggtggct gcagagttgt gagtgactct cgcttagcaa agactagaga tggcctgtcc 420
gtgccaaaac acagtgccgg gtccggagca gaagaatcca acagcagctc cactgtgcag 480
aagcagaatg agccagggct acagacagag gatgtgcaga agccaccact tcagatggac 540
aacagcgtct ttctagatga cgacagcaat cagccaatgc ccgtgagccg gttctttgga 600
aacgttgagc tcatgcagga cctcccacct gcgtcttcat cttgtccttc aatgagcaga 660
cgagagttca gaaaaatgca tttcagagcc aaagatgatg atgatgacga cgacgatgat 720
gcagaaatgt ag 732
<![CDATA[<210> 2]]>
<![CDATA[<211> 72]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_1]]>
<![CDATA[<400> 2]]>
atgaggagcc ggaagctcac aggtgcagtg cggtcttcag cgcgcttgaa agcacgaagt 60
tgttcwwttt tt 72
<![CDATA[<210> 3]]>
<![CDATA[<211> 71]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_2]]>
<![CDATA[<400> 3]]>
acgtggaacc agcaacttcc tgggcagagg ccaacctggc tgccgaacaa cttcgtgctt 60
tcaawwtttt t 71
<![CDATA[<210> 4]]>
<![CDATA[<211> 67]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_3]]>
<![CDATA[<400> 4]]>
gttgctggtt ccacgtctgc caagacagca tgtctgactt cctcatccca caaagccaca 60
wwttttt 67
<![CDATA[<210> 5]]>
<![CDATA[<211> 71]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_4]]>
<![CDATA[<400> 5]]>
acaagatgtc ctttgtcaca tttgaacttc ttggacgttc gcgtgtctgt ggctttgtgg 60
gatgwwtttt t 71
<![CDATA[<210> 6]]>
<![CDATA[<211> 68]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_5]]>
<![CDATA[<400> 6]]>
tgtgacaaag gacatcttgt gaagtcagaa ttacagaagc ttgtccctaa gaatgacagc 60
gwwttttt 68
<![CDATA[<210> 7]]>
<![CDATA[<211> 62]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_6]]>
<![CDATA[<400> 7]]>
cacaaggggt ctcaggtgtc acttttggca aagaagcgct gtcattctta gggacwwttt 60
tt 62
<![CDATA[<210> 8]]>
<![CDATA[<211> 70]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_7]]>
<![CDATA[<400> 8]]>
acctgagacc ccttgtgaaa atgagtttgc tgaaggcagt gccttgcttc caggcagcga 60
ggcwwttttt 70
<![CDATA[<210> 9]]>
<![CDATA[<211> 72]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_9]]>
<![CDATA[<400> 9]]>
gcagccaccg agaggaagac ttgcagcccc ctgctgcaca gaaacgccag cctcgctgcc 60
tggaawwttt tt 72
<![CDATA[<210> 10]]>
<![CDATA[<211> 72]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_9]]>
<![CDATA[<400> 10]]>
tcctctcggt ggctgcagag ttgtgagtga ctctcgctta gcaaagacta gagatggcct 60
gtccgwwttt tt 72
<![CDATA[<210> 11]]>
<![CDATA[<211> 62]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_10]]>
<![CDATA[<400> 11]]>
tggattcttc tgctccggac ccggcactgt gttttggcac ggacaggcca tctctwwttt 60
tt 62
<![CDATA[<210> 12]]>
<![CDATA[<211> 69]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_11]]>
<![CDATA[<400> 12]]>
ccggagcaga agaatccaac agcagctcca ctgtgcagaa gcagaatgag ccagggctac 60
agwwttttt 69
<![CDATA[<210> 13]]>
<![CDATA[<211> 67]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_12]]>
<![CDATA[<400> 13]]>
acgctgttgt ccatctgaag tggtggcttc tgcacatcct ctgtctgtag ccctggctca 60
wwttttt 67
<![CDATA[<210> 14]]>
<![CDATA[<211> 71]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_13]]>
<![CDATA[<400> 14]]>
cagatggaca acagcgtctt tctagatgac gacagcaatc agccaatgcc cgtgagccgg 60
ttctwwtttt t 71
<![CDATA[<210> 15]]>
<![CDATA[<211> 69]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_14]]>
<![CDATA[<400> 15]]>
gatgaagacg caggtgggag gtcctgcatg agctcaacgt ttccaaagaa ccggctcacg 60
ggwwttttt 69
<![CDATA[<210> 16]]>
<![CDATA[<211> 72]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_15]]>
<![CDATA[<400> 16]]>
ccacctgcgt cttcatcttg tccttcaatg agcagacgag agttcagaaa aatgcatttc 60
agagcwwttt tt 72
<![CDATA[<210> 17]]>
<![CDATA[<211> 71]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_16]]>
<![CDATA[<400> 17]]>
ctacatttct gcatcatcgt cgtcgtcatc atcatcatct ttggctctga aatgcatttt 60
tctgwwtttt t 71
<![CDATA[<210> 18]]>
<![CDATA[<211> 65]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_F]]>
<![CDATA[<400> 18]]>
atgaggagcc ggaagctcac aggtgcagtg cggtcttcag cgcgcttgaa agcacgaagt 60
tgttc 65
<![CDATA[<210> 19]]>
<![CDATA[<211> 64]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 4_R]]>
<![CDATA[<400> 19]]>
ctacatttct gcatcatcgt cgtcgtcatc atcatcatct ttggctctga aatgcatttt 60
tctg 64
<![CDATA[<210> 20]]>
<![CDATA[<211> 500]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13]]>
<![CDATA[<400> 20]]>
ccgagctcgg atccgccacc atatgggcat ggagggtctt ctccagaact ccactaactt 60
cgtcctcaca ggcctcatca cccatcctgc cttccccggg cttctctttg caatagtctt 120
ctccatcttt gtggtggcta taacagccaa cttggtcatg attctgctca tccacatgga 180
ctcccgcctc cacacaccca tgtacttctt gctcagccag ctctccatca tggataccat 240
ctacatctgt atcactgtcc ccaagatgct ccaggacctc ctgtccaagg acaagaccat 300
ttccttcctg ggctgtgcag ttcagatctt cctctacctg accctgattg gaggggaatt 360
cttcctgctg ggtctcatgg cctatgaccg ctatgtggct gtgtgcaacc ctctacggta 420
ccctctcctc atgaaccgca gggtttgctt attcatggtg gtcggctcct gggttggtgg 480
ttccttggat gggttcatgc 500
<![CDATA[<210> 21]]>
<![CDATA[<211> 72]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_1]]>
<![CDATA[<400> 21]]>
ccgagctcgg atccgccacc atatgggcat ggagggtctt ctccagaact ccactaactt 60
cgtccwwttt tt 72
<![CDATA[<210> 22]]>
<![CDATA[<211> 66]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_2]]>
<![CDATA[<400> 22]]>
gaagcccggg gaaggcagga tgggtgatga ggcctgtgag gacgaagtta gtggagttcw 60
wttttt 66
<![CDATA[<210> 23]]>
<![CDATA[<211> 66]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_3]]>
<![CDATA[<400> 23]]>
gccttccccg ggcttctctt tgcaatagtc ttctccatct ttgtggtggc tataacagcw 60
wttttt 66
<![CDATA[<210> 24]]>
<![CDATA[<211> 64]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_4]]>
<![CDATA[<400> 24]]>
cgggagtcca tgtggatgag cagaatcatg accaagttgg ctgttatagc caccacawwt 60
tttt 64
<![CDATA[<210> 25]]>
<![CDATA[<211> 62]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_5]]>
<![CDATA[<400> 25]]>
tccacatgga ctcccgcctc cacacaccca tgtacttctt gctcagccag ctctcwwttt 60
tt 62
<![CDATA[<210> 26]]>
<![CDATA[<211> 67]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_6]]>
<![CDATA[<400> 26]]>
gcatcttggg gacagtgata cagatgtaga tggtatccat gatggagagc tggctgagca 60
wwttttt 67
<![CDATA[<210> 27]]>
<![CDATA[<211> 62]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_7]]>
<![CDATA[<400> 27]]>
cactgtcccc aagatgctcc aggacctcct gtccaaggac aagaccattt ccttcwwttt 60
tt 62
<![CDATA[<210> 28]]>
<![CDATA[<211> 66]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_8]]>
<![CDATA[<400> 28]]>
cagggtcagg tagaggaaga tctgaactgc acagcccagg aaggaaatgg tcttgtcctw 60
wttttt 66
<![CDATA[<210> 29]]>
<![CDATA[<211> 62]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_9]]>
<![CDATA[<400> 29]]>
ttcctctacc tgaccctgat tggaggggaa ttcttcctgc tgggtctcat ggcctwwttt 60
tt 62
<![CDATA[<210> 30]]>
<![CDATA[<211> 63]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_10]]>
<![CDATA[<400> 30]]>
gggtaccgta gagggttgca cacagccaca tagcggtcat aggccatgag acccagwwtt 60
ttt 63
<![CDATA[<210> 31]]>
<![CDATA[<211> 65]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_11]]>
<![CDATA[<400> 31]]>
aaccctctac ggtaccctct cctcatgaac cgcagggttt gcttattcat ggtggtcgww 60
ttttt 65
<![CDATA[<210> 32]]>
<![CDATA[<211> 62]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_12]]>
<![CDATA[<400> 32]]>
gcatgaaccc atccaaggaa ccaccaaccc aggagccgac caccatgaat aagcawwttt 60
tt 62
<![CDATA[<210> 33]]>
<![CDATA[<211> 65]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_F]]>
<![CDATA[<400> 33]]>
ccgagctcgg atccgccacc atatgggcat ggagggtctt ctccagaact ccactaactt 60
cgtcc 65
<![CDATA[<210> 34]]>
<![CDATA[<211> 55]]>
<![CDATA[<212> DNA]]>
<![CDATA[<213> Artificial sequence]]>
<![CDATA[<220>]]>
<![CDATA[<223> Gene_Frag 13_R]]>
<![CDATA[<400> 34]]>
gcatgaaccc atccaaggaa ccaccaaccc aggagccgac caccatgaat aagca 55

      

Claims (43)

A high-throughput gene synthesis method based on chip primer surface extraction is characterized in that it includes classifying primers into clusters and synthesizing them in different areas of the chip, cutting the primer clusters on the chip by aminolysis, then using a liquid transfer workstation to dissolve the primers of different clusters and transfer each dissolved primer cluster to an independent reaction container suitable for gene synthesis, and performing a gene synthesis reaction under conditions suitable for gene synthesis to obtain the synthesized target gene fragment. The method as described in claim 1, wherein the primers classified by cluster are primer clusters encoding multiple target gene fragments, wherein each primer cluster contains all primers encoding the same target gene fragment. A method as described in claim 1 or 2, wherein different primer clusters are synthesized in different regions of the chip, and blank areas are left between the different regions. The method as described in claim 3, wherein a plurality of hydrophobic groups are synthesized on the aforementioned blank area. The method as described in claim 3, wherein when synthesizing primers on a chip, at least one linker arm that can be cleaved by aminolysis and at least three dTs as protective bases are additionally added to the 3' end of each primer; the aforementioned linker arm is selected from any structure that can generate a 3' free hydroxyl group in the primer after aminolysis. The method as described in claim 5, wherein, when synthesizing primers on a chip, two additional linkers that can be cleaved by aminolysis and five dTs as protective bases are added to the 3' end of each primer; the aforementioned linker is selected from a structure based on a phosphoramidite structure and capable of providing a free hydroxyl group at the 3' end. The method as described in claim 5, wherein the aforementioned aminolysis method is aminolysis using a non-aqueous aminating agent. The method as described in claim 7, wherein the aforementioned aminating agent is selected from ammonia or monomethylamine. The method as described in claim 7, wherein the aforementioned aminolysis is carried out in a high-pressure reaction vessel filled with aminating agent. The method as described in claim 9, wherein the aforementioned aminolysis is carried out at 25°C to 120°C and 20 to 120 psi for 15 minutes to 4 hours. The method as described in claim 1, wherein the aforementioned liquid transfer workstation is a sampler, and the solvent used to dissolve the aforementioned primers is selected from solvents that can perform PCR reactions. The method as described in claim 11, wherein the solvent used to dissolve the primer is TE solution or distilled water. The method as described in claim 11, wherein the primer dissolution comprises using a sampler to drop 10~1000nL of solvent into each primer cluster, leaving it alone for 0.5~3 minutes and then using a needle to blow and aspirate to dissolve it. The method as described in claim 1 or 2, wherein the aforementioned independent reaction container suitable for gene synthesis is a hole of a porous plate; the aforementioned step of transferring to the independent reaction container suitable for gene synthesis includes adjusting the needle height of the liquid transfer workstation to the chip surface and sucking the dissolved liquid in the chip and transferring it to the porous plate. The method as described in claim 14, wherein after the solution in the chip is transferred to the porous plate, the sampling needle is further cleaned by a cleaning procedure, and then the primer dissolution and transfer steps are repeated until all primer clusters are completely transferred. The method as described in claim 1 or 2, wherein the aforementioned gene synthesis reaction under conditions suitable for gene synthesis comprises adding a gene synthesis reaction solution and/or F/R primers of the target gene fragment to each independent reaction container to perform a synthesis reaction; the aforementioned gene synthesis reaction solution is selected from a PCA reaction solution and a PCR reaction solution. The method as described in claim 1 or 2, wherein the aforementioned gene synthesis reaction comprises adding PCA reaction solution to each reaction container to allow the primer cluster to perform an assembly reaction, and then adding PCR reaction solution and F/R primers of the target gene fragment to perform a PCR reaction. A high-throughput gene synthesis method based on primer surface extraction on a chip is characterized by comprising the following steps: 1) synthesizing primer clusters encoding multiple target gene fragments on a chip, wherein each primer cluster contains all primers encoding the same target gene fragment, and primer clusters encoding different target gene fragments are synthesized in different regions of the chip; 2) aminolyzing the synthesized chip to cut the primers from the chip, and making the cut primers contain 3' free hydroxyl groups; 3) dripping a solvent to each primer cluster to fully dissolve the primers in the primer cluster, collecting each dissolved primer cluster and transferring them to independent reaction containers; 4) adding a gene synthesis reaction solution to the reaction container to perform gene synthesis; 5) recovering the reaction product to obtain multiple target gene fragments. The method as described in claim 18, wherein a blank area is left between the aforementioned different areas in the aforementioned step 1). The method as described in claim 19, wherein a plurality of hydrophobic groups are synthesized on the aforementioned blank area. A method as described in any one of claims 18 to 20, wherein, in the aforementioned step 1), when synthesizing primers on a chip, at least one linker arm that can be cleaved by aminolysis and at least three dTs are additionally added to the 3' end of each primer as a protective base; the aforementioned linker arm is selected from any structure that can generate a 3' free hydroxyl group in the primer after aminolysis. The method as described in claim 21, wherein, when synthesizing primers on a chip, two additional linkers that can be cleaved by aminolysis and five dTs as protective bases are added to the 3' end of each primer; the aforementioned linker is selected from a structure based on a phosphoramidite structure and capable of providing a free hydroxyl group at the 3' end. A method as described in any one of claims 18 to 20, wherein the aforementioned step 2) comprises aminolysis using a non-aqueous aminating agent. The method as described in claim 23, wherein the aforementioned aminating agent is selected from ammonia or monomethylamine. The method as described in claim 23, wherein the aforementioned aminolysis is carried out in a high-pressure reaction vessel filled with aminating agent. The method as described in claim 23, wherein the aforementioned aminolysis is carried out at 25°C to 120°C and 20 to 120 psi for 15 minutes to 4 hours. A method as described in any one of claim items 18 to 20, wherein the solvent in the aforementioned step 3) is selected from a solvent that can perform a PCR reaction; the aforementioned primer dissolution comprises using a sampler to drop 10-1000 nL of solvent into each primer cluster, standing for 0.5-3 minutes, and then using a needle to blow and aspirate to dissolve it. The method as described in claim 27, wherein the solvent is TE solution or distilled water. A method as described in any one of claims 18 to 20, wherein the independent reaction container in the aforementioned step 3) is a hole of a porous plate. A method as described in any one of claims 18 to 20, wherein the step of transferring to an independent reaction container in step 3) includes adjusting the needle height of the liquid transfer workstation to the chip surface and sucking the dissolved liquid in the chip and transferring it to the porous plate. The method as described in claim 30, wherein after the solution in the chip is transferred to the porous plate by aspiration, the sampling needle is further cleaned by a cleaning procedure, and then step 3) is repeated until all primer clusters are completely transferred. A method as described in any one of claim items 18 to 20, wherein the gene synthesis reaction solution in the aforementioned step 4) is selected from a PCA reaction solution and a PCR reaction solution; the aforementioned gene synthesis comprises adding the prepared PCA reaction solution to each reaction container to cluster the primers for assembly reaction, and then adding the PCR reaction solution and the F/R primers of the target gene fragment to perform a PCR reaction. A method for synthesizing primer clusters for high-throughput gene synthesis, characterized in that it includes: classifying primers by clusters, synthesizing different primer clusters in different areas of a chip, cutting the primer clusters on the chip by aminolysis, then using a liquid transfer workstation to dissolve the primers of different clusters, and collecting each dissolved primer cluster. The method as described in claim 33, wherein each of the aforementioned primer clusters includes all primers encoding the same target gene fragment, the aforementioned chip includes a primer cluster synthesized to encode multiple target gene fragments, and the primer clusters encoding different target gene fragments are synthesized in different regions of the chip, and blank regions are left between the aforementioned different regions. The method as described in claim 34, wherein a plurality of hydrophobic groups are synthesized on the aforementioned blank area. A method as described in any one of claim 33 to 35, wherein when synthesizing primers on a chip, at least one linker arm that can be cleaved by aminolysis and at least three dTs as protective bases are additionally added to the 3' end of each primer; the aforementioned linker arm is selected from any structure that can generate a 3' free hydroxyl group in the primer after aminolysis. The method as described in claim 36, wherein, when synthesizing primers on a chip, two additional linkers that can be cleaved by aminolysis and five dTs as protective bases are added to the 3' end of each primer; the aforementioned linker is selected from a structure based on a phosphoramidite structure and capable of providing a free hydroxyl group at the 3' end. A method as described in any one of claim items 33 to 35, wherein the solvent for dissolving the primers is distilled water; the primer dissolution comprises using a sampler to drop 10 to 1000 nL of distilled water into each primer cluster, standing for 0.5 to 3 minutes, and then using a needle to blow and aspirate to dissolve. A method as described in any one of claims 33 to 35, wherein the aforementioned synthesis method further comprises transferring each dissolved primer cluster to a separate reaction container. The method as described in claim 39, wherein the aforementioned independent reaction container is a hole of a porous plate. The method as described in claim 40, wherein the step of transferring to an independent reaction container includes adjusting the height of the sampler needle to the surface of the chip and sucking the dissolved liquid in the chip and transferring it to the porous plate. A method as described in claim 41, wherein after the solution in the chip is transferred to the porous plate by aspirating, the sampling needle is further cleaned by a cleaning procedure, and then the primer dissolution and transfer steps are repeated until all primer clusters are completely transferred. A method for synthesizing a primer cluster for high-throughput gene synthesis, characterized in that it comprises the following steps: 1) synthesizing a primer cluster encoding a plurality of target gene fragments on a chip, wherein the primer cluster encoding each target gene fragment comprises all primers encoding the target gene fragment, and the primer clusters encoding different target gene fragments are synthesized in different fixed regions of the chip respectively, and a blank region is left between the aforementioned different regions; 2) aminolyzing the synthesized chip to cut the primer from the chip, and making the cut primer contain a 3' free hydroxyl group; 3) dripping a solvent to each primer cluster to fully dissolve the primer in the aforementioned primer cluster, and collecting each dissolved primer cluster.

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