WO2020135669A1 - 一种基因合成的方法 - Google Patents

一种基因合成的方法 Download PDF

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WO2020135669A1
WO2020135669A1 PCT/CN2019/128998 CN2019128998W WO2020135669A1 WO 2020135669 A1 WO2020135669 A1 WO 2020135669A1 CN 2019128998 W CN2019128998 W CN 2019128998W WO 2020135669 A1 WO2020135669 A1 WO 2020135669A1
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sequence
target gene
bases
overlapping oligonucleotide
sequences
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PCT/CN2019/128998
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English (en)
French (fr)
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李一凡
邱蔚
戴晓慧
吴政宪
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江苏金斯瑞生物科技有限公司
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Publication of WO2020135669A1 publication Critical patent/WO2020135669A1/zh

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

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  • the invention relates to a method of gene synthesis, in particular to a method of synthesizing multiple different genes simultaneously.
  • the primer design process of this method contains two enzyme digestion steps, which makes the whole process particularly cumbersome; meanwhile, the design of a single primer needs to add two enzyme digestion sites twice, resulting in a low utilization of primer length; in addition
  • This method cannot be used to synthesize sequences containing these two enzyme sites, which makes the application of the method very limited, and the downstream processing process is too complicated, which limits the wide application of this method.
  • the present invention provides a multi-gene synthesis method, which includes:
  • each target gene segment of the multiple target gene segments to be synthesized into several overlapping oligonucleotide sequences, and add a modified sequence at the end of each overlapping oligonucleotide sequence to enable it to
  • the chain form is specifically linked to the magnetic beads, and the modified sequence includes a restriction site sequence and a tag sequence, and the tag sequence is different according to different target gene fragments;
  • the overlapping oligonucleotide sequence with the modified sequence is specifically connected to the magnetic beads; the magnetic beads are connected with the reverse complementary sequence of the tag sequence, and the specific connection is through the tag sequence and its reverse The complementary sequence forms a double-stranded structure;
  • a single-stranded overlapping oligonucleotide sequence is obtained by enzyme digestion, and then polymerase chain assembly (PCA) is performed to obtain multiple target gene fragments;
  • the enzyme digestion and polymerase chain assembly are performed in a water-in-oil system.
  • At least one water-in-oil system contains only one magnetic bead in each water-in-oil system.
  • the buffer and reagents required for the digestion and PCA reaction are added before digestion, and added to the oily medium, and shaken to form a water-in-oil system.
  • each target gene fragment has universal primers at both ends.
  • each target gene fragment has specific primers at both ends.
  • the cleavage is by an endonuclease, preferably the BspQI enzyme.
  • the tag sequence contains 10-100 bases, preferably 15-70 bases, more preferably 20-40 bases, and most preferably 20-30 bases.
  • each overlapping oligonucleotide sequence contains 40-150 bases; preferably 50-130 bases; more preferably 60-110 bases; still more preferably 65-90 bases; most preferably 65-80 bases.
  • the overlapping oligonucleotide sequence comprises overlapping bases, the number of overlapping bases is 10-100; preferably 10-70; more preferably 10-50; more preferably 10-30; more preferably 10 -20; most preferably 15-16.
  • the magnetic beads are linked to the reverse complementary sequence of the tag sequence by streptavidin-biotin.
  • the multi-gene synthesis method further includes the step of purifying multiple target gene fragments after polymerase chain assembly.
  • the multi-gene synthesis method further includes the step of amplifying the obtained multiple target gene fragments.
  • overlapping oligonucleotides with modified sequences are synthesized by chip.
  • Figure 1 is a schematic diagram of overlapping oligonucleotide sequences
  • Figure 2 is an electropherogram of the target synthetic sequence
  • Figure 3 is the sequencing result of the target synthetic sequence Gene1
  • Figure 4 is the sequencing result of the target synthetic sequence Gene2
  • Figure 5 is the sequencing result of the target synthetic sequence Gene3
  • 6 is an electrophoresis diagram of a target sequence synthesized using chip primer pool technology
  • Figure 7 is the sequencing result of Gene1’ synthesized using chip oligo
  • Figure 8 is the sequencing results of Gene2’ synthesized using chip oligo
  • Figure 9 is the sequencing results of Gene3’ synthesized using chipoligo
  • Figure 10 is the sequencing results of Gene4’ synthesized using chip oligo
  • Fig. 11 is the sequencing result of Gene5' synthesized using chipoligo.
  • the invention provides a method of multi-gene synthesis, which solves the technical problem of too cumbersome in primer design in multi-gene synthesis in the prior art.
  • the present invention provides a multi-gene synthesis method.
  • the double-stranded target gene fragment is first divided into several overlapping oligonucleotide sequences, and a modified sequence is added to each overlapping oligonucleotide sequence, that is, Add the restriction site sequence and the tag sequence at its ends; synthesize the overlapping oligonucleotide sequence with the modified sequence and the reverse complementary sequence of the tag sequence respectively, and connect the magnetic beads to the reverse complementary sequence of the tag sequence;
  • the tag sequence and its reverse complementary sequence form a double-stranded structure, so that the modified overlapping oligonucleotide sequence is captured by the magnetic beads, wherein the overlapping oligonucleotide sequence exists in the form of a single strand; the single strand overlap is obtained by digestion Oligonucleotide sequence; polymerase chain assembly (PCA) to obtain multiple target gene fragments at the same time; the enzyme digestion and polymerase chain assembly (PCA) are carried out in a water-in
  • the overlapping oligonucleotide sequence is based on the principle of polymerase chain assembly (PCA), which is formed by dividing a double-stranded target gene fragment, and the oligonucleotide sequence obtained by dividing one strand is obtained by dividing it with its reverse complementary strand
  • PCA polymerase chain assembly
  • the oligonucleotide sequences are partially reverse complementary, and the reverse complementary portions are located at both ends of the oligonucleotide sequence, see FIG. 1.
  • the overlapping oligonucleotide sequence in the present invention is an oligonucleotide sequence obtained by dividing a double-stranded target gene fragment, and a complete target gene fragment can be synthesized by a PCA reaction.
  • the overlapping oligonucleotide sequence has 40-150 bases or any number of bases within the range; preferably 50-130 bases; more preferably 60-110 bases; and Preferably 65-90 bases; most preferably 65-80 bases.
  • the number of bases of overlapping oligonucleotide sequences may be specifically selected from but not limited to 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 1200 , 125, 130, 135, 140, 145, 150 bases.
  • overlapping oligonucleotide sequences have 65-80 bases.
  • the overlapping oligonucleotide sequence comprises overlapping bases, the number of overlapping bases is 10-100; preferably 10-70; more preferably 10-50; more preferably 10-30; most preferably 10 -20.
  • overlapping bases refers to bases that can complement each other at the ends of two adjacent overlapping oligonucleotide sequences.
  • overlapping oligonucleotide sequences comprise 15-16 overlapping bases.
  • the purpose of adding a modified sequence to each overlapping oligonucleotide sequence is to enable it to be specifically linked to the magnetic beads in a single-stranded form.
  • the modified sequence may be connected at the 5'end or 3'end of the overlapping oligonucleotide sequence.
  • Modified sequences include restriction site sequences and tag sequences.
  • the cleavage site sequence and tag sequence can be added at the 5'end or 3'end of each overlapping oligonucleotide sequence.
  • overlapping oligonucleotide sequences from different target gene fragments are connected with the same tag sequence, and overlapping oligonucleotide sequences from the same target gene fragments are connected with different Tag sequence, which distinguishes overlapping oligonucleotide sequences of different target gene fragments according to the tag sequence.
  • each target gene fragment is divided into several overlapping oligonucleotide sequences.
  • tag sequences for different target gene fragments, synthesize overlapping oligonucleotide sequences with tag sequences added and mix them, and then capture the overlap by forming a double-stranded structure with the reverse complementary sequence of the tag sequence on the magnetic beads and the tag sequence
  • Oligonucleotide sequences overlapping oligonucleotide sequences corresponding to different target gene fragments are sorted and distinguished by tag sequences when captured by magnetic beads.
  • a plurality of magnetic beads may be connected enough (e.g., greater than 103, greater than 104 or more than 10 5) the reverse complement of the same sequence, so that the bead can be captured from at least one All overlapping oligonucleotide sequences of the same target gene fragment.
  • Another function of the tag sequence is to connect overlapping oligonucleotide sequences and magnetic beads.
  • the reverse complementary sequence of the tag sequence is connected to the magnetic bead, and the tag sequence can form a double-stranded structure with its reverse complementary sequence, thereby connecting the overlapping oligonucleotide sequence to the magnetic bead and connecting the overlapping oligonucleotide to the magnetic bead
  • the acid sequence is in a single chain state.
  • the tag sequence has 10-100 bases, more preferably 15-70 bases, still more preferably 20-40 bases, and most preferably 20-30 bases.
  • the tag sequence has a base number selected from but not limited to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30.
  • overlapping oligonucleotide sequences with modified sequences, and/or reverse complementary sequences of tag sequences can be synthesized by any suitable means.
  • Methods for synthesizing oligonucleotide sequences are well known in the art. For example, they can be synthesized on a chip. For example, inkjet printing or photoactivation can be used.
  • oligonucleotide sequences with modified sequences After the overlapping oligonucleotide sequences with modified sequences are synthesized, they can be mixed into an oligonucleotide mixture. Overlapping oligonucleotide sequences from different target gene fragments can be mixed together and sorted out by magnetic beads with different reverse complementary sequences in subsequent steps. For example, it is possible to synthesize overlapping oligonucleotide sequences with modified sequences from different target gene fragments through the chip, and collect the synthesized sequences from the chip without collecting the overlapping oligonucleotide sequences of different target gene fragments during collection Can be mixed together and sorted by magnetic beads with different reverse complementary sequences in the subsequent steps.
  • the magnetic beads can be connected to the reverse complementary sequence of the tag sequence by any possible means. Methods for linking magnetic beads to sequences are well known in the art, and can be linked by affinity adsorption, for example.
  • the magnetic beads are connected to the reverse complementary sequence of the tag sequence through streptavidin-biotin. In some embodiments, there may be a sufficient number of streptavidin-biotin (e.g., greater than 103, greater than 104 or more than 105) on the magnetic beads, the magnetic beads may be connected such that a sufficient The reverse complementary sequence. Magnetic beads with multiple streptavidin can be prepared by themselves or obtained commercially.
  • Methods for forming a double-stranded structure between the tag sequence and its reverse complementary sequence are well known in the art, for example, the two can be formed into a double-stranded structure by annealing.
  • different magnetic beads can be separated to separately synthesize each target gene fragment.
  • the magnetic beads are separated, and the overlapping oligonucleotide sequences are cut from the magnetic beads in the separated system to obtain single-stranded overlapping oligonucleotide sequences, and then polymerase chain assembly is performed to obtain target gene fragments.
  • Each separate system can contain all overlapping oligonucleotide sequences from the same target gene segment, so a complete target gene segment can be synthesized in a separate system.
  • Different systems can contain overlapping oligonucleotide sequences from different target gene fragments, and multiple different systems can react simultaneously to obtain a variety of different target gene fragments.
  • the divided system may be a water-in-oil system.
  • the magnetic beads are separated by a water-in-oil system.
  • Each water-in-oil system contains magnetic beads connected with overlapping oligonucleotide sequences, buffers and reagents required for enzyme digestion reaction, and buffers and reagents required for polymerase chain assembly.
  • the double-stranded structure of the tag sequence and its reverse complementary sequence is separated from the single-stranded overlapping oligonucleotide sequence by enzyme cleavage to obtain a single-stranded unmodified overlapping oligonucleotide sequence, and then Perform polymerase chain assembly to obtain target gene fragments.
  • Each water-in-oil system can contain all overlapping oligonucleotide sequences from the same target gene segment, so a complete target gene segment can be synthesized in a separate system.
  • Different water-in-oil systems may contain overlapping oligonucleotide sequences from different target gene fragments. Multiple such water-in-oil systems react simultaneously to obtain multiple target gene fragments at the same time.
  • each water-in-oil system in at least one water-in-oil system contains only a number of overlapping oligonucleotide sequences of a target gene fragment, and the overlapping oligonucleotide fragments are in the water-in-oil system
  • the assembled PCA solution is assembled, and its assembly will not be affected by overlapping oligonucleotide fragments of other target gene fragments.
  • each of the at least one water-in-oil system contains only one magnetic bead.
  • the buffers and reagents required for the enzyme digestion reaction, and the buffers and reagents required for polymerase chain assembly are mixed with the magnetic beads before forming the water-in-oil system.
  • the water-in-oil system can be formed by any suitable method, for example, by mixing and shaking the buffer with a water-incompatible solvent (eg, oily medium) and shaking to form the water-in-oil system.
  • a buffer solution may be added before the digestion reaction, and the buffer containing the magnetic beads is mixed with a water-incompatible solvent (eg, an oily medium) and shaken to form a water-in-oil system, followed by the digestion reaction And polymerase chain assembly.
  • a buffer solution to the magnetic beads before the digestion reaction, and add the buffer solution containing the magnetic beads to a water-incompatible solvent such as an oily medium, or the water-incompatible A solvent such as an oily medium is added to the buffer containing magnetic beads, and shaken to form a water-in-oil system.
  • each of the at least one water-in-oil system contains only a few overlapping oligonucleotide sequences of a target gene fragment, or, further, each of the at least one water-in-oil system A water-in-oil system contains only one magnetic bead.
  • the volume ratio of the water-incompatible solvent (eg, oily medium) to the buffer during mixing may be 3:1-10:1, preferably 5:1.
  • the shaking speed may be 2000 rpm to 4000 rpm, preferably 2800 rpm.
  • the added buffer may contain the buffer and reagents required for the digestion reaction. In some embodiments, the added buffer may contain buffers and reagents required for polymerase chain assembly.
  • the oily medium may contain oil and surfactant.
  • the oil in the oily medium may be mineral oil.
  • the surfactant in the oily medium may be selected from any one or more of Span, Tween and Triton X-100.
  • the surfactant in the oily medium may be a mixture of Span, Tween, and Triton X-100.
  • the composition of the oily medium may be: Span 4.5%, Tween 80 0.4%, Triton X-100 0.05%, the remainder is mineral oil, the percentage of which is the volume/volume ratio.
  • the overlapping oligonucleotide sequences from the magnetic beads can be cleaved by using the cleavage sites linked to the overlapping oligonucleotide sequences.
  • the enzyme used for enzymatic cleavage may be any enzyme capable of cleaving a single strand, and the enzyme is selected from but not limited to endonuclease, and the endozyme is selected from but not limited to BspQI enzyme.
  • the cleavage site sequence and the tag sequence can be joined at the 5'end or the 3'end of the overlapping oligonucleotide sequence, wherein the cleavage site is located between the tag sequence and the overlapping oligonucleotide sequence.
  • PCA polymerase chain assembly
  • PCR polymerase chain reaction
  • universal primers can be connected at both ends of each target gene fragment, so that it can be amplified by using universal primers in subsequent amplification without additional primer design.
  • the universal primer F used may be CACGACTACAGTGAATAGGCAAGCG
  • the universal primer R used may be CGTCTGGGTAACATAACTATCTGGGAGG. If a common primer is used to amplify a mixture of multiple target gene fragments, the obtained product can be separated by different separation methods known in the art, such as electrophoresis.
  • specific primers can be attached to both ends of each target gene fragment.
  • the specific primers on both ends of different target gene fragments are different. After the assembly of overlapping oligonucleotide fragments is completed, different target gene fragments can be directly amplified by specific primers.
  • a purification step may also be included.
  • purification may include breaking the emulsion of the water-in-oil system first, followed by purification and recovery of the nucleic acid.
  • the method of demulsification is well known in the art, for example, oil can be removed by centrifugation and water saturated ether extraction, and the remaining aqueous phase can be purified and recovered.
  • Methods for purifying and recovering nucleic acids are well known in the art. For example, column recovery can be performed by a commercially available kit, such as a PCR cleanup recovery kit.
  • the method of the present invention may further comprise the step of further amplifying the synthesized target gene fragments.
  • each target gene fragment can be amplified by universal primers.
  • a mixture of multiple target gene fragments can be amplified by universal primers.
  • the mixture of the plurality of target gene fragments can be amplified in the same amplification reaction.
  • each target gene fragment can be amplified by specific primers.
  • specific primers can be used to amplify a mixture of multiple target gene fragments to obtain specific target gene fragments.
  • the "at least one" in the present invention may include at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 100, at least 1000, or at least 10000.
  • the gene synthesis process of the present invention can be divided into the following steps:
  • the magnetic beads of streptavidin and the reverse complementary sequence of the tag sequence linked to biotin are affinity adsorbed to form the magnetic beads of the reverse complementary sequence with the tag sequence;
  • the magnetic beads with the reverse complementary sequence of the tag sequence are mixed with the oligonucleotide mixture obtained in step 2 and slowly annealed to make the tag sequence on the overlapping oligonucleotide fragments and the tag sequence on the magnetic beads
  • the reverse complementary sequence forms a double-stranded structure, so that one magnetic bead has all the overlapping oligonucleotide sequences required for target gene synthesis;
  • PCA polymerase chain assembly
  • the emulsion is first demulsified and then purified and recovered to obtain the PCA product;
  • step 3 the order of some steps can be arbitrarily changed, for example, the step of obtaining an overlapping oligonucleotide mixture (including the above steps 1, 2)
  • the step of obtaining the magnetic beads with the reverse complement of the tagged sequence (step 3 above) can be performed in any order.
  • multi-gene synthesis is performed by the following steps:
  • the reverse complementary sequence of tag sequence 1 is defined as D1-R
  • the reverse complementary sequence of tag sequence 2 is defined as D2-R
  • biotin modification is added at the 3'end of the sequence
  • the magnetic beads with streptavidin and the reverse complementary sequence of the biotin-tag sequence are affinity adsorbed to form the magnetic beads with the reverse complementary sequence of the tag sequence;
  • biotin-modified D1-R and D2-R are incubated with streptavidin-based magnetic beads respectively, so that the biotin-modified D1-R and D2-R are adsorbed on the surface of the magnetic beads ;
  • the magnetic beads with the reverse complementary sequence of the tag sequence are mixed together with the oligonucleotide mixture, and slowly annealed to make the tag sequence on the oligonucleotide and its reverse complementary sequence form a partial double-stranded structure, so 1 magnetic bead There is all the oligonucleotides needed for the synthesis of a target gene segment;
  • the tag sequence is added to the 5'end of each overlapping oligonucleotide fragment, and many reverse complementary sequences with tag sequences are bound to the magnetic beads, the tag sequence and its reverse complementary sequence form exactly reverse complement, Therefore, all overlapping oligonucleotide fragments can be bound to the magnetic beads by annealing; the overlapping oligonucleotide fragments required for the synthesis of a target gene fragment can only be combined with specific magnetic beads;
  • the structure formed by annealing contains one BspQI site. All the overlapping oligonucleotide fragments can be cut from the magnetic beads by enzyme digestion, and the desired target gene fragments can be assembled by PCA; at least one oil droplet in the oil droplets formed by the vibration can only accommodate one magnetic bead. All overlapping oligonucleotide fragments are assembled in the PCA solution contained in the oil droplet, and their assembly will not be affected by overlapping oligonucleotide fragments of other target gene fragments;
  • the oil is removed by centrifugation and water saturated ether extraction, and the column is purified to obtain the PCA product;
  • the PCA product obtained in the previous step is used as a template, and PCR is performed using universal primer F and universal primer R to obtain the target product.
  • primer design software can be used to divide the target gene fragment into several overlapping oligonucleotide sequences.
  • primer design software is easily available to those skilled in the art, for example, it can be provided by https://primerize.stanford .edu/acquired.
  • the synthesis of the oligonucleotide sequence or the oligonucleotide fragment can be performed by any suitable method known in the art, such as conventional chemical synthesis, or synthesis by chip. Synthesizing oligonucleotides through chips is a high-throughput synthesis method. Usually, there are multiple synthesis pools on the chip. According to the designed DNA sequence, the corresponding sequences are synthesized in each pool. During synthesis, for example, it can be synthesized from the 3′ to the 5′ end of the oligonucleotide to be synthesized. A large number of oligonucleotides synthesized by the chip can be cut or eluted from the chip for subsequent applications, for example. In the present invention, for example, oligonucleotides can be synthesized using the chip primer pool of Nanjing Kingsray Biological Technology Co., Ltd.
  • the method of the present invention is simpler in primer design, and only one step of enzyme digestion is needed to obtain the primer, which is more time-saving and efficient, saves reagents, and improves the efficiency of gene synthesis.
  • the underlined part is the added sequence.
  • the specific oligo sequence becomes:
  • the three kinds of magnetic beads Gene1-R-Beads, Gene2-R-Beads, Gene3-R-Beads and other proportions were mixed together and resuspended with 20 ⁇ l of water, mixed with the oligo mix of 3 genes.
  • the following reaction was carried out in a shaker at 2200 rpm, and the annealing procedure was as follows: incubate at 50°C for 3 hours, reduce the temperature to 0.1°C/s to 40°C and maintain for 3 hours, at 0.1°C/s to 30°C and maintain for 3 hours, according to 0.1°C /s Reduced temperature to 20°C and maintained for 2 hours, at 0.1°C/s reduced to 10°C and maintained for 2 hours. After the annealing, the product was washed 3-4 times with Elution buffer to remove unbound oligo, and finally suspended with 20 ⁇ l H 2 O.
  • Example 4 100 ⁇ l of the solution system of Example 4 was added dropwise to 500 ⁇ l of oil-surfactant (Span 4.5%, Tween80 0.4%, Triton X-100 0.05%, the remainder was Mineral oil, both volume/volume percentage), and added during the process Vibrated violently in an oscillator (Vortex-5, Hailin Qilinbeier Instrument Manufacturing Company), and vortexed at a maximum speed of 2800 rpm to form a water-in-oil structure. Dispense the final water-in-oil emulsion system into PCR tubes,
  • the reaction was carried out according to the following procedure: digestion at 50°C for 90 min; pre-denaturation at 95°C for 2 min; denaturation at 95°C for 10 s, annealing at 60°C for 20 s, extension at 72°C for 40 s, 60 cycles, and finally extension reaction at 72°C for 5 min.
  • the emulsions in the several PCR tubes at the end of the reaction were recombined into a 1.5ml centrifuge tube, first centrifuged at 12000rpm for 10min, and then the upper oil layer was removed; 1.5ml of water saturated ether was added, shaken and mixed, and centrifuged at 1000rpm for 10s ,Remove the upper ether layer; Add 1.5ml water saturated ether again, shake and mix, centrifuge at 1000rpm for 10s, remove the upper ether layer, this step remove the ether layer as much as possible; use the remaining water phase by PCR Clean the recovery kit for column recovery, and finally elute with 30 ⁇ l of sterile water, leaving the product for future use.
  • the oligo sequence of the gene of interest was designed using substantially the same method as in Example 1 and Example 2. The difference is that the chip primer pool technology of Nanjing Kingsray Biotechnology Co., Ltd. is used to synthesize oligo instead of ordinary chemical synthesis, that is, high-throughput chip synthesis of oligo; in addition, specific primer sequences are added at both ends of the target gene instead of Universal primer sequence.
  • the target genes to be synthesized are Gene1', Gene2', Gene3', Gene4', and Gene5', a total of 5 genes.
  • the gene sequence to be synthesized is as follows:
  • the specific primer at the 5'end of the sequence Gene1' is ATTAGTCCTCCCGCTCAGTTTT (SEQ ID NO: 33), and the specific primer at the 3'end is TACGTATTTCTGGTCATCGGCG (SEQ ID NO: 34);
  • the specific primer at the 5'end of the sequence Gene2' is CAGCACTAACATGAGGCGAATC (SEQ ID NO: 35), and the specific primer at the 3'end is ATCGTTTACCCAGACCGCTCTT (SEQ ID NO: 36);
  • the specific primer at the 5'end of the sequence Gene3' is GACAAAGCCCGTGATTCAGGTC (SEQ ID NO: 37), and the specific primer at the 3'end is CAATGGGTGCCGGATAACTTGG (SEQ ID NO: 38);
  • the specific primer at the 5'end of the sequence Gene4' is TGATAGAACCTGGGCTCCTCAA (SEQ ID NO: 39), and the specific primer at the 3'end is GCTTGTTTGCGTGCCTTAACTA (SEQ ID NO: 40);
  • the specific primer at the 5'end of the sequence Gene5' is ATGATCGCACGAGACTGTACCC (SEQ ID NO: 41), and the specific primer at the 3'end is TAACTTGGTCCTGGACGTTGCT (SEQ ID NO: 42);
  • oligos were synthesized using the chip primer pool technology of Nanjing Kingsray Biotechnology Co., Ltd., that is, these oligos were synthesized by high-throughput chips. Subsequent biotin oligo and streptavidin magnetic beads binding, annealing, digestion and PCA, digestion, purification steps are the same as described in Example 3-6, and finally, through specific primer sequences at both ends of each gene of interest Each target gene is directly amplified by PCR. The product was subjected to electrophoretic detection and Sanger sequencing.

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Abstract

一种基因合成的方法，所述方法包含将多种靶基因分割成若干个重叠寡核苷酸序列，在重叠寡核苷酸序列的端部添加酶切位点序列和标签序列，所述标签序列因不同的靶基因而不同；使连接有标签序列的重叠寡核苷酸序列以单链形式与连接有标签序列的反向互补序列的磁珠通过双链结构特异性连接，然后在油包水体系中酶切得到单链的重叠寡核苷酸，再进行聚合酶链式组装，同时获得多种靶基因片段。

Description

一种基因合成的方法 技术领域
本发明涉及一种基因合成的方法，具体涉及一种同时合成多个不同基因的方法。
背景技术
基因合成是现代生物技术持续快速发展的重要使能技术，能否以更低的成本更高的通量获得价格更低的基因非常关键。近年来基于芯片的DNA合成技术得以快速发展，使得科研人员能够以非常低廉的价格获得大量DNA序列。同时，与之对应的也有一些DNA合成技术被开发出来，能够利用这些价格低廉的DNA文库进行基因合成(Large-scale de novo DNA synthesis:technologies and applications,Nat Methods.2014 May；11(5):499-507)。但是，到目前为止，大部分的技术都需要做depooling，之后将目的基因分别合成出来，这会带来比较高的下游处理成本。
2018年，Sriram Kosuri团队开发了一种多元基因合成的方法(Multiplexed gene synthesis in emulsions for exploring protein functional landscapes.Science.2018 Jan19；359(6373):343-347)，该方法通过精巧的oligo文库设计，使得不同基因的合成引物能够被磁珠吸附进入不同的微球，从而能够将不同的基因在同一个PCR反应体系中进行合成。然而该方法的引物设计过程含有两次酶切的步骤，致使整个流程特别繁琐；同时，单条引物的设计需要分别对两个酶切位点加入两次，致使引物长度的利用率很低；另外，该方法不能用来合成序列中含有这两个酶位点的序列，致使方法的应用非常有限，而且下游处理的流程过于复杂，限制了这个方法的广泛应用。
发明内容
本发明提供了一种多基因合成方法，所述方法包括：
(1)将待合成的多种靶基因片段中的每种靶基因片段分割成若干个重叠寡核苷酸序列，在每个重叠寡核苷酸序列端部加上修饰序列使其能够以单链形式与磁珠特异性连接，所述修饰序列包括酶切位点序列和标签序列，所述标签序列因不同的靶基因片段而不同；
(2)合成带有修饰序列的重叠寡核苷酸序列；
(3)使带有修饰序列的重叠寡核苷酸序列与磁珠特异性连接；所述磁珠上连接有标签序列的反向互补序列，所述的特异性连接是通过标签序列与其反向互补序列形成双链结构；
(4)通过酶切得到单链的重叠寡核苷酸序列，再进行聚合酶链式组装(PCA)，获得多种靶基因片段；
其中，所述酶切和聚合酶链式组装在油包水体系中进行。
在一些实施方案中，至少一个油包水体系的每个油包水体系中仅中包含一个磁珠。
在一些实施方案中，在酶切之前加入酶切和PCA反应所需的缓冲液和试剂，并将其加入到油性介质中，震荡形成油包水体系。
在一些实施方案中，每个靶基因片段两端具有通用引物。
在一些实施方案中，每个靶基因片段两端具有特异性引物。
在一些实施方案中，所述酶切是通过内切酶进行切割，所述内切酶优选BspQI酶。
在一些实施方案中，所述的标签序列含有10-100个碱基，优选15-70个碱基，更优选20-40个碱基，最优选20-30个碱基。
在一些实施方案中，每一个重叠寡核苷酸序列含40-150个碱基；优选50-130个碱基；更优选60-110个碱基；再优选65-90个碱基；最优选65-80个碱基。
在一些实施方案中，所述重叠寡核苷酸序列包含重叠碱基，所述重叠碱基数量为10-100；优选10-70；更优选10-50；更优选10-30；更优选10-20；最优选15-16。
在一些实施方案中，所述磁珠通过链霉亲和素-生物素与所述标签序列的反向互补序列连接。
在一些实施方案中，所述多基因合成方法进一步包含在聚合酶链式组装后纯化多种靶基因片段的步骤。
在一些实施方案中，所述多基因合成方法进一步包含扩增获得的多种靶基因片段的步骤。
在一些实施方案中，通过芯片合成带有修饰序列的重叠寡核苷酸。
附图说明
通过以下详细的描述并结合附图将更充分地理解本发明。
图1是重叠寡核苷酸序列示意图；
图2是目标合成序列的电泳图；
图3是目标合成序列Gene1的测序结果；
图4是目标合成序列Gene2的测序结果；
图5是目标合成序列Gene3的测序结果；
图6是使用芯片引物池技术合成的目标序列的电泳图；
图7是使用chip oligo合成的Gene1’的测序结果；
图8是使用chip oligo合成的Gene2’的测序结果；
图9是使用chip oligo合成的Gene3’的测序结果；
图10是使用chip oligo合成的Gene4’的测序结果；
图11是使用chip oligo合成的Gene5’的测序结果。
具体实施方式
本发明提供了一种多基因合成的方法，解决了现有技术中在多基因合成在引物设计方面过于繁琐的技术难题。
本发明提供一种多基因合成方法，如图1所示，首先将双链靶基因片段分割成若干个重叠寡核苷酸序列，给每个重叠寡核苷酸序列加上修饰序列，即，在其端部添加酶切位点序列和标签序列；分别合成添加有修饰序列的重叠寡核苷酸序列和标签序列的反向互补序列，将磁珠与标签序列的反向互补序列连接；使标签序列与其反向互补序列形成一段双链结构，从而使修饰的重叠寡核苷酸序列被磁珠捕获，其中重叠寡核苷酸序列以单链的形式存在；通过酶切得到单链的重叠寡核苷酸序列；再进行聚合酶链式组装(polymerase chain assembly,PCA)同时获得多种靶基因片段；所述的酶切和聚合酶链式组装(PCA)在油包水体系中进行。
本发明中，重叠寡核苷酸序列是基于聚合酶链式组装(PCA)的原理由双链靶基因片段分割而成，一条链分割得到的寡核苷酸序列与其反向互补链分割得到的寡核苷酸序列部分反向互补，反向互补的部分位于所述寡核苷酸序列的两端，参见图1。具体而言，本发明中的重叠寡核苷酸序列是由双链靶基因片段分割而成的、且可以通过PCA反应合成完整的靶基因片段的寡核苷酸序列。
在一些实施方案中，所述重叠寡核苷酸序列具有40-150个碱基或其范围内的任意的碱基数；优选50-130个碱基；更优选60-110个碱基；再优选65-90个碱基；最优选65-80个碱基。重叠寡核苷酸序列的碱基数具体可选自但不限于40，45，50，55，60，65，70，75，80，85，90，95，100，105，110，115，1200，125，130，135，140，145，150个碱基。
在一些实施方案中，重叠寡核苷酸序列具有65-80个碱基。
在一些实施方案中，所述重叠寡核苷酸序列包含重叠碱基，所述重叠碱基数量为10-100；优选10-70；更优选10-50；再优选10-30；最优选10-20。本发明中，“重叠碱基”是指两个相邻重叠寡核苷酸序列端部相接的能够互补的碱基。
在一些实施方案中，重叠寡核苷酸序列包含重叠碱基数为15-16。
本发明中，给每个重叠寡核苷酸序列加上修饰序列的目的是为了使其能够以单链形式与磁珠特异性连接。修饰序列可以连接在重叠寡核苷酸序列的5'端或3'端。修饰序列包括酶切位点序列和标签序列。基于此目的，可以在每个重叠寡核苷酸序列的5’端或3'端添加酶切位点序列和标签序列。对不同靶基因片段使用不同的标签序列，因此，来自于不同靶基因片段的重叠寡核苷酸序列连接有相同的标签序列，来自于相同靶基因片段的重叠寡核苷酸序列连接有不同的标签序列，根据标签序列区分不同靶基因片段的重叠寡核苷酸序列。
当同时合成多种靶基因片段时，将每个靶基因片段分割成若干个重叠寡核苷酸序列。针对不同的靶基因片段使用不同的标签序列，合成添加有标签序列的重叠寡核苷酸序列并混合，随后通过磁珠上的标签序列的反向互补序列与标签序列形成双链结构来捕获重 叠寡核苷酸序列，不同靶基因片段相应的重叠寡核苷酸序列在被磁珠捕获时通过标签序列被分选和区别开。
一个磁珠上只连接一种反向互补序列，因此与同一个磁珠相连的重叠寡核苷酸序列都具有相同的标签序列，即与同一个磁珠相连的重叠寡核苷酸序列均来自同一种靶基因片段，由此通过磁珠特异性捕获的方式将来自不同靶基因片段的重叠寡核苷酸序列区分开，以便于后续的进一步合成。一个磁珠上可以连接多个相同的反向互补序列，由此可以在同一个磁珠上捕获来自于同一种靶基因片段的多个重叠寡核苷酸序列。在一些实施方案中，一个磁珠上可以连接足够多个(例如多于10 3个、多于10 4个或多于10 5个)相同的反向互补序列，使得至少一个磁珠可以捕获来自同一个靶基因片段的全部重叠寡核苷酸序列。
标签序列的另一个功能是用于连接重叠寡核苷酸序列和磁珠。磁珠上连接有标签序列的反向互补序列，标签序列能够与其反向互补序列形成双链结构，由此使得重叠寡核苷酸序列与磁珠相连，连接到磁珠上的重叠寡核苷酸序列为单链状态。
在一些实施方案中，标签序列具有10-100个碱基，更优选15-70个碱基，再优选20-40个碱基，最优选20-30个碱基。
在一些实施方案中，标签序列具有的碱基数选自但不限于20，21，22，23，24，25，26，27，28，29，30。
在一些实施方案中，带有修饰序列的重叠寡核苷酸序列、和/或标签序列的反向互补序列可以通过任何适当的方式合成。合成寡核苷酸序列的方法是本领域公知的，例如可以通过芯片合成，例如可以采用喷墨打印法或者光活化的方法。
带有修饰序列的重叠寡核苷酸序列被合成后，可以混合成寡核苷酸混合物。来自不同靶基因片段的重叠寡核苷酸序列可以被混合在一起，在后续步骤中通过带有不同反向互补序列的磁珠分选出来。例如，可以通过芯片合成来自不同靶基因片段的带有修饰序列的重叠寡核苷酸序列，从芯片上收集合成的序列，在收集时无需将不同靶基因片段的重叠寡核苷酸序列区分开，可以全部混合在一起，在后续步骤中通过带有不同反向互补序列的磁珠进行分选。
磁珠可以通过任何可能的方式与标签序列的反向互补序列相连接。使磁珠与序列相连接的方法是本领域公知的，例如可以通过亲和吸附连接。在一些实施方案中，所述的磁珠通过链霉亲和素-生物素与标签序列的反向互补序列连接。在一些实施方案中，一个磁珠上可以有足够多的链霉亲和素(例如多于10 3个、多于10 4个或多于10 5个)，以使得一个磁珠可以连接足够多的反向互补序列。具有多个链霉亲合素的磁珠可以自行制备或通过商购获得。
使标签序列与其反向互补序列形成双链结构的方法是本领域公知的，例如可以通过退火使二者形成双链结构。
修饰的重叠寡核苷酸序列被磁珠捕获后，可以将不同的磁珠分隔开，分别进行每一个靶基因片段的合成。磁珠被分隔开，在分隔的体系中从磁珠上将重叠寡核苷酸序列切 割下来，得到单链的重叠寡核苷酸序列，再进行聚合酶链式组装，获得靶基因片段。每个分隔的体系中可以含有来自同一种靶基因片段的全部重叠寡核苷酸序列，因此在一个分隔的体系中可以合成完整的一种靶基因片段。不同的体系中可以包含来自不同靶基因片段的重叠寡核苷酸序列，多个不同的体系同时进行反应，可以获得多种不同的靶基因片段。
在一些实施方案中，所述分隔的体系可以是油包水体系。磁珠通过油包水体系被分隔开。每个油包水体系中包含连接有重叠寡核苷酸序列的磁珠、酶切反应所需的缓冲液和试剂、以及聚合酶链式组装所需的缓冲液和试剂。在油包水体系中通过酶切作用将标签序列与其反向互补序列的双链结构与单链重叠寡核苷酸序列分割开，得到单链的未经修饰的重叠寡核苷酸序列，再进行聚合酶链式组装，获得靶基因片段。
每个油包水体系中可以含有来自同一种靶基因片段的全部重叠寡核苷酸序列，因此在一个分隔的体系中可以合成完整的一种靶基因片段。不同的油包水体系中可以含有来自不同靶基因片段的重叠寡核苷酸序列，多个这样的油包水体系同时进行反应，可以同时获得多种靶基因片段。
在一些实施方案中，至少一个油包水体系中的每一个油包水体系只含有一种靶基因片段的若干重叠寡核苷酸序列，这些重叠寡核苷酸片段在该油包水体系中包含的PCA溶液中进行组装，其组装不会受到其它靶基因片段的重叠寡核苷酸片段的影响。在一些实施方案中，至少一个油包水体系中的每一个油包水体系只含有一个磁珠。
酶切反应所需的缓冲液和试剂、以及聚合酶链式组装所需的缓冲液和试剂在形成油包水体系之前与磁珠混合。可以通过任何合适的方法形成油包水体系，例如通过将缓冲液与水不相容的溶剂(例如油性介质)混合并震荡形成油包水体系。在一些实施方案中，可以在酶切反应之前加入缓冲液，并将含有磁珠的缓冲液与水不相容的溶剂(例如油性介质)混合并震荡形成油包水体系，之后进行酶切反应和聚合酶链式组装。在一些实施方案中，可以通过在酶切反应之前向磁珠加入缓冲液，并将含有磁珠的缓冲液加入到与水不相容的溶剂例如油性介质中，或者将与水不相容的溶剂例如油性介质加入到含有磁珠的缓冲液中，震荡形成油包水体系。
对缓冲液与水不相容的溶剂(例如油性介质)的混合物进行震荡以使得形成的油滴(油包水体系表现为油滴的形式，一个油包水体系即为一个油滴)尽量小，由此使得至少至少一个油包水体系中的每一个油包水体系只含有一种靶基因片段的若干重叠寡核苷酸序列，或者，进一步地，使得至少一个油包水体系中的每一个油包水体系只含有一个磁珠。
在一些实施方案中，混合时水不相容的溶剂(例如油性介质)与缓冲液的体积比可以是3:1-10:1，优选5:1。在一些实施方案中，震荡的转速可以是2000rpm-4000rpm，优选2800rpm。
在一些实施方案中，所加入的缓冲液可以包含酶切反应所需的缓冲液和试剂。在一些实施方案中，所加入的缓冲液可以包含聚合酶链式组装所需的缓冲液和试剂。
在一些实施方案中，所述油性介质可以包含油和表面活性剂。在一些实施方案中，所述油性介质中的油可以是矿物油。在一些实施例中，所述油性介质中的表面活性剂可以选自Span、Tween和Triton X-100的任意一种或几种。在一些实施例中，所述油性介质中的表面活性剂可以是Span、Tween和Triton X-100的混合物。在一些实施方案中，所述油性介质的组成可以是：Span 4.5％，Tween 80 0.4％，Triton X-100 0.05％，剩余为矿物油，其中的百分含量为体积/体积比。
从磁珠上将重叠寡核苷酸序列切割下来可以利用重叠寡核苷酸序列上连接的酶切位点，通过酶切进行。在一些实施方案中，酶切采用的酶可以是任意能够将单链切割的酶，所述的酶选自但不限于内切酶，所述内切酶选自但不限于BspQI酶。
在一些实施方案中，酶切位点序列和标签序列可以连接在重叠寡核苷酸序列的5'端或3’端，其中酶切位点位于标签序列和重叠寡核苷酸序列之间。
本发明中，聚合酶链式组装(polymerase chain assembly,PCA)是本领域公知的技术，它是一种基于聚合酶链式反应(polymerase chain reaction,PCR)原理的方法，是指使彼此之间部分重叠并覆盖整个目的基因的单链寡核苷酸(其示意图参见图1)互为引物和模板，在存在聚合酶的条件下通过多轮变性、退火、延伸循环，最终获得目的基因。
在一些实施方案中，在每种靶基因片段两端可以连接通用引物，使其能够在后续的扩增中采用通用引物进行扩增，而不需要额外设计引物。在一些实施方案中，所使用的通用引物F可以是CACGACTACAGTGAATAGGCAAGCG，所使用的通用引物R可以是CGTCTGGGTAACATAACTATCTGGGAGG。如果使用通用引物扩增多种靶基因片段的混合物，获得的产物可以通过本领域公知的分离方法，例如电泳，将不同靶基因片段分离。
在一些实施方案中，在每种靶基因片段两端可以连接特异性引物。不同的靶基因片段两端的特异性引物不同，当重叠寡核苷酸片段组装完成后，可以通过特异性引物直接扩增出不同的靶基因片段。
在一些实施方案中，在聚合酶链式组装之后，还可以包含纯化的步骤。在一些实施方案中，纯化可以包括先将油包水体系的乳浊液破乳，再进行核酸的纯化回收。破乳的方法是本领域公知的，例如可以通过离心和水饱和乙醚萃取去除油，剩下水相进行纯化回收。纯化回收核酸的方法是本领域公知的，例如可以通过可商购的试剂盒，例如PCR clean up回收试剂盒进行柱回收。
在一些实施方案中，本发明的方法还可以包含进一步扩增合成的靶基因片段的步骤。在一些实施方案中，可以通过通用引物扩增每种靶基因片段。在一些实施方案中，可以通过通用引物扩增多种靶基因片段的混合物。在一些实施方案中，所述多种靶基因片段的混合物可以在同一个扩增反应中被扩增。在一些实施方案中，可以通过特异性引物扩增每种靶基因片段。在一些实施方案中，可以使用特异性引物扩增多种靶基因片段的混合物，获得特定靶基因片段。
本发明中所述的“至少一个”可以包括至少1个、至少2个、至少3个、至少4个、至少5个、至少10个、至少100个、至少1000个、或至少10000个。
在一些实施方案中，本发明的基因合成流程可以分为以下几步：
1.在靶基因片段两端添加上通用引物序列或特异性引物序列，利用引物设计软件分成多个65-80bp的小重叠片段；
2.给每个小重叠片段的5’端添加上对特定基因的标签序列和BspQI酶切位点，最终形成92-102bp小片段，合成这些小重叠片段并将这些小片段混合成重叠寡核苷酸混合物(Mix)；
3.将链霉亲和素的磁珠和与生物素连接的标签序列的反向互补序列进行亲和吸附，形成带标签序列的反向互补序列的磁珠；
4.带标签序列的反向互补序列的磁珠与步骤2中得到的寡核苷酸混合物混合，进行缓慢退火，使重叠寡核苷酸片段上带有的标签序列与磁珠上的标签序列的反向互补序列形成双链结构，使得1个磁珠上有靶基因合成所需要的所有重叠寡核苷酸序列；
5.将磁珠加入到聚合酶链式组装(PCA)体系中，同时加入BspQI，将含有BspQI的PCA体系缓慢加入5倍体积的与水不相容的油性介质(oil-surfactant)中并剧烈震荡(转速为2800rpm)，形成油包水结构，；将乳浊液分装至PCR管中先进行酶切后进行重叠聚合酶链反应(PCA)；
6.PCA反应后的乳浊液先进行乳浊液破乳，再进行纯化回收，得到PCA产物；
7.利用通用引物或特异性引物进行PCR获得靶基因目的片段。
本领域技术人员应当理解，上述流程中，一些步骤在操作上并没有先后的要求，因此一些步骤的先后顺序可以任意调换，例如获得重叠寡核苷酸混合物的步骤(包括上述步骤1、2)和获得带标签序列的反向互补序列的磁珠的步骤(上述步骤3)可以任意顺序进行。
在一个优选的实施方案中，通过以下步骤进行多基因合成：
1.将需要合成的靶基因片段两端添加上通用引物序列，利用引物设计软件将待合成的片段分成多个65-80bp的小片段；
为了可以在一个体系中进行多个靶基因片段的合成，需要在待合成的靶基因片段两端分别添加上通用引物序列作为最终的序列；5’端添加通用引物序列：CACGACTACAGTGAATAGGCAAGCG；3’端添加通用引物序列：CCTCCCAGATAGTTATGTTACCCAGACG；将带有通用引物的待合成基因片段放入相应的设计软件中，设置每条重叠寡核苷酸序列的长度为65-80bp，重叠寡核苷酸序列之间的重叠碱基(overlap)长度为15bp，得到拆解后的多条重叠寡核苷酸序列。注：通用引物F：CACGACTACAGTGAATAGGCAAGCG；通用引物R：CGTCTGGGTAACATAACTATCTGGGAGG；
2.在得到的重叠寡核苷酸序列两端添加特定的序列并进行合成
在同一个靶基因片段的所有重叠寡核苷酸序列的5’端添加上BspQI位点，然后再在BspQI位点的5’端添加上一段标签序列(同一个基因的所有重叠寡核苷酸序列用同一个标签序列)；具体地，基因1的所有重叠寡核苷酸序列5’端添加上BspQI位点然后在BspQI位点5’端添加上标签序列1；基因2的所有引物5’端添加上BspQI位点然后在BspQI位点5’端添加上标签序列2；以此类推；
标签序列1的反向互补序列定义为D1-R，标签序列2的反向互补序列义为D2-R，在其序列的3’端添加生物素修饰；
将上述设计好的寡核苷酸进行合成，将合成的带有酶切位点和标签序列的重叠寡核苷酸混合成寡核苷酸混合物(Mix)；
3.将带有链霉亲和素的磁珠和生物素-标签序列的反向互补序列进行亲和吸附，形成带标签序列的反向互补序列的磁珠；
具体而言，将生物素修饰的D1-R和D2-R等分别与带有链霉亲和素的磁珠进行孵育，使生物素修饰的D1-R和D2-R等吸附于磁珠表面；
4.带标签序列的反向互补序列的磁珠与寡核苷酸混合物一起混合，进行缓慢退火使寡核苷酸上的标签序列与其反向互补序列形成部分双链结构，因此1个磁珠上有一个靶基因片段合成所需要的所有寡核苷酸；
由于每条重叠寡核苷酸片段的5’端都添加了标签序列，而磁珠上结合上很多带有标签序列的反向互补序列，标签序列与其反向互补序列形成正好是反向互补，因此可以通过退火将所有重叠寡核苷酸片段结合在磁珠上；一个靶基因片段合成所需要的重叠寡核苷酸片段只能与特定的磁珠结合在一起；
5.将结合有重叠寡核苷酸片段的磁珠加入到PCA体系中，同时加入BspQI，将含有BspQI的PCR体系缓慢加入到5倍体积的oil-surfactant中并剧烈震荡(转速为2800rpm)，形成油包水结构；
退火形成的结构中，包含1个BspQI酶切位点。通过酶切可以将所有的重叠寡核苷酸片段从磁珠上切割下来，通过PCA可以组装成所需的靶基因片段；震荡形成的油滴中至少存在一个油滴仅能容纳一个磁珠，所有的重叠寡核苷酸片段在该油滴包含的PCA溶液中进行组装，其组装不会受到其它靶基因片段的重叠寡核苷酸片段的影响；
6.乳浊液破乳，纯化回收，得到PCA产物；
具体地，通过离心和水饱和乙醚萃取去除油，柱纯化，得到PCA产物；
7.利用通用引物进行PCR获得目的片段；
具体地，以上一步得到的PCA产物为模板，使用通用引物F和通用引物R进行PCR获得目的产物。
本发明中，可以利用常规的引物设计软件将靶基因片段分割成若干个重叠寡核苷酸序列，这样的引物设计软件是本领域技术人员容易获得的，例如可以由 https://primerize.stanford.edu/获得。
本发明中，寡核苷酸序列或寡核苷酸片段的合成可以通过本领域已知的任何适当的方式进行，例如传统的化学合成，或者通过芯片合成。通过芯片合成寡核苷酸是一种高通量的合成方式，通常在芯片上具有多个合成池，按照设计的DNA序列，在每个池中合成相应的序列。合成时例如可由待合成寡核苷酸的3'向5'端合成。通过芯片合成的大量寡核苷酸例如可以从芯片上剪切或洗脱下来用于后续应用。本发明中，例如可以使用南京金斯瑞生物科技有限公司的芯片引物池合成寡核苷酸。
本发明的方法在引物设计上更简便，引物的获得仅需要一步酶切步骤，更省时高效，并且节省试剂，提高了基因合成的效率。
下面通过实施例，并结合附图，对本发明的技术方案作进一步详细的说明，但本发明不限于下面的实施例。
实施例1对3个需要合成的基因进行分段
首先对需要进行合成的3个DNA序列进行编号，分别命名为Gene1，Gene2，Gene3。为了方便后续使用通用引物进行扩增，分别在每个基因两端添加通用引物序列：
5’端添加通用引物序列：CACGACTACAGTGAATAGGCAAGCG(SEQ ID NO:1)；
3’端添加通用引物序列：CCTCCCAGATAGTTATGTTACCCAGACG(SEQ ID NO:2)；
下划线的部分是添加的序列。
Gene1
Figure PCTCN2019128998-appb-000001
Gene2
Figure PCTCN2019128998-appb-000002
Gene3
Figure PCTCN2019128998-appb-000003
Figure PCTCN2019128998-appb-000004
在对应的引物设计软件中，将添加了通用引物区域的基因序列输入，设定overlap长度为15-16bp，单条引物长度为65-80bp，即可得到基因合成所需要的重叠寡核苷酸(oligo)序列，由于oligo较多，以下仅展示Gene1通过软件设计的8条oligo序列：
Gene1_1
Figure PCTCN2019128998-appb-000005
Gene1_2
Figure PCTCN2019128998-appb-000006
Gene1_3
Figure PCTCN2019128998-appb-000007
Gene1_4
Figure PCTCN2019128998-appb-000008
Gene1_5
Figure PCTCN2019128998-appb-000009
Gene1_6
Figure PCTCN2019128998-appb-000010
Gene1_7
Figure PCTCN2019128998-appb-000011
Gene1_8
Figure PCTCN2019128998-appb-000012
实施例2在得到的oligo序列两端添加特定的序列并进行合成
为了能从合成的oligo mix中，把其中某一个基因的所有oligo独立地分离出来，需要在同一个基因的所有oligo的5’端添加上一段特异性序列，并在oligo与特异性序列之间添加上BspQI识别位点：gctcttca。
具体的oligo序列变为：
Gene1_1
Figure PCTCN2019128998-appb-000013
Figure PCTCN2019128998-appb-000014
Gene1_2
Figure PCTCN2019128998-appb-000015
Gene1_3
Figure PCTCN2019128998-appb-000016
Gene1_4
Figure PCTCN2019128998-appb-000017
Gene1_5
Figure PCTCN2019128998-appb-000018
Gene1_6
Figure PCTCN2019128998-appb-000019
Gene1_7
Figure PCTCN2019128998-appb-000020
Gene1_8
Figure PCTCN2019128998-appb-000021
以上为展示Gene1中的8条oligo的最终序列，下划线部分为5’端添加的序列。
根据上述的oligo序列，设计反向互补的连接生物素的oligo，下表1为3个基因5’端添加的序列及其对应的生物素修饰R序列。
表1 5’端添加的序列及其对应的生物素修饰R序列
  序列
Gene1 ATAGATGCCGTCCTgctcttca(SEQ ID NO:22)
Gene2 GTGGGTAAATGGTAgctcttca(SEQ ID NO:23)
Gene3 CGACGGGGAGTATAgctcttca(SEQ ID NO:24)
   
Gene1-R tgaagagcAGGACGGCATCTAT(SEQ ID NO:25)-Biotin
Gene2-R tgaagagcTACCATTTACCCAC(SEQ ID NO:26)-Biotin
Gene3-R tgaagagcTATACTCCCCGTCG(SEQ ID NO:27)-Biotin
通过化学合成的方法合成上述设计的所有序列。
实施例3生物素oligo与链霉亲和素磁珠结合
取5μl链霉亲和素磁珠加入清洗缓冲液(10mM Tris-HCl,pH 7.5、1mM EDTA、2.0 M NaCl)清洗3次，使用45μl 1*HF buffer(NEB的Phusion聚合酶的buffer)重悬，加入100pmol Gene1-R引物，使终体积为50μl。在室温25℃震荡(2200rpm)过夜，第二天使用清洗缓冲液清洗去除未与磁珠结合的生物素oligo。通过此种方式得到3种带有生物素oligo的磁珠(Gene-R-Beads)，分别命名为Gene1-R-Beads、Gene2-R-Beads、Gene3-R-Beads。
实施例4退火
将3种磁珠Gene1-R-Beads、Gene2-R-Beads、Gene3-R-Beads等比例混合在一起用20μl水重悬，与3个基因的oligo mix混合在一起。配制以下50μl体系：5*HF Buffer 10μl，3种连接有生物素oligo的磁珠混合物2μl，3个基因的oligo mix 0.9pmol(单条引物浓度，加入的引物需要过量)，H 2O至50μl。在振荡器2200rpm中进行如下反应，退火程序如下：50℃孵育3小时，按照0.1℃/s降温至40℃并维持3小时，按照0.1℃/s降温至30℃并维持3小时，按照0.1℃/s降温至20℃并维持2小时，按照0.1℃/s降温至10℃并维持2小时。退火结束后，使用洗脱缓冲液(Elution buffer)对产物进行清洗3-4次，将未结合上的oligo去除，最终使用20μl H 2O悬浮。
实施例5酶切和PCA
配制如下酶切和PCA体系：5*HF buffer 20μl、dNTPs(10mM each)2μl、BSA(20mg/ml)2μl、Phusion(2U/μl聚合酶NEB Inc.)2.5μl、通用引物F(25μM)2μl、通用引物R(25μM)2μl、BspQI 4μl、无菌水补充至100μl体系。
将100μl实施例4的溶液体系逐滴加入到500μl的oil-surfactant(Span 4.5％，Tween80 0.4％，Triton X-100 0.05％，剩余为Mineral oil，均为体积/体积百分比)，并加入过程中在振荡器(Vortex-5，海门市其林贝尔仪器制造公司)中剧烈震荡，以最大转速2800转/分进行涡旋，形成油包水的结构。将最终的油包水的乳浊液体系分装至PCR管中，
按照以下程序进行反应：50℃酶切90min；95℃预变性2min；95℃变性10s，60℃退火20s，72℃延伸40s，60个循环，最后72℃延伸反应5min。
实施例6纯化
将反应结束的几个PCR管中的乳浊液重新合并至1.5ml离心管中，首先12000rpm离心10min，结束后将上层的油层去除；加入1.5ml水饱和乙醚，振荡混匀，1000rpm离心10s后，将上层的乙醚层去除；再次加入1.5ml水饱和乙醚，振荡混匀，1000rpm离心10s后，将上层的乙醚层去除，此步骤尽可能将乙醚层去除干净；将剩下的水相使用PCR clean up回收试剂盒进行柱回收，最后使用30μl无菌水洗脱，产物留待备用。
实施例7利用通用引物进行PCR获得目的片段
将上述的柱纯化得到的产物作为模板，按照以下体系继续PCR：5*HF buffer 10μl、dNTPs(10mM each)1μl、Phusion(2U/μl,NEB Inc.)0.5μl、通用引物F(25μM)1μl、通用 引物R(25μM)1μl、无菌水补充至50μl体系。反应程序为：98℃预变性30s；98℃变性10s,70℃退火15s,72℃延伸45s，25个循环；最后72℃延伸反应5min。反应结束后，将产物进行电泳检测及Sanger测序，结果见图2-5。从图3-5测序结果可以看出，测序结果与目标靶基因序列完全一致，本实施例成功地完成了三条基因的同时合成。
实施例8利用芯片引物池技术(chip oligo pool)合成基因
使用与实施例1和实施例2基本相同的方法设计目的基因的oligo序列。不同之处在于，使用南京金斯瑞生物科技有限公司的芯片引物池技术合成oligo代替普通的化学合成，即芯片高通量合成oligo；另外，在目的基因两端添加特异性引物序列，而不是通用引物序列。
本实施例需要合成的目的基因为Gene1’、Gene2’、Gene3’、Gene4’、Gene5’，共5条基因。要合成的基因序列如下：
Gene1’：
Figure PCTCN2019128998-appb-000022
Gene2’：
Figure PCTCN2019128998-appb-000023
Gene3’：
Figure PCTCN2019128998-appb-000024
Figure PCTCN2019128998-appb-000025
Gene4’：
Figure PCTCN2019128998-appb-000026
Gene5’：
Figure PCTCN2019128998-appb-000027
Figure PCTCN2019128998-appb-000028
在每个目的基因两端添加特异性引物序列，以便于可以通过PCR直接扩增出目的基因。其中：
序列Gene1’的5’端特异性引物为ATTAGTCCTCCCGCTCAGTTTT(SEQ ID NO:33)，3’端特异性引物为TACGTATTTCTGGTCATCGGCG(SEQ ID NO:34)；
序列Gene2’的5’端特异性引物为CAGCACTAACATGAGGCGAATC(SEQ ID NO:35)，3’端特异性引物为ATCGTTTACCCAGACCGCTCTT(SEQ ID NO:36)；
序列Gene3’的5’端特异性引物为GACAAAGCCCGTGATTCAGGTC(SEQ ID NO:37)，3’端特异性引物为CAATGGGTGCCGGATAACTTGG(SEQ ID NO:38)；
序列Gene4’的5’端特异性引物为TGATAGAACCTGGGCTCCTCAA(SEQ ID NO:39)，3’端特异性引物为GCTTGTTTGCGTGCCTTAACTA(SEQ ID NO:40)；
序列Gene5’的5’端特异性引物为ATGATCGCACGAGACTGTACCC(SEQ ID NO:41)，3’端特异性引物为TAACTTGGTCCTGGACGTTGCT(SEQ ID NO:42)；
在对应的引物设计软件中，将添加了特异性引物区域的基因序列输入，设定overlap长度为15-16bp，单条寡核苷酸(oligo)长度为65-80bp，得到基因合成所需要的重叠寡核苷酸序列。在同一个基因的所有oligo的5’端添加上一段特异性序列，并在oligo与特异性序列之间添加上BspQI识别位点：gctcttca。设计反向互补的连接生物素的oligo，其反向互补序列与oligo 5'端的特异性序列和BspQI识别位点互补。下表2为5个基因的oligo的5’端添加的序列及其对应的生物素修饰R序列。
表2 oligo 5’端添加的序列及其对应的生物素修饰R序列
Gene1’ ACATGAAACGATGGgctcttca(SEQ ID NO:43)
Gene2’ GATTCCCTTGACTTgctcttca(SEQ ID NO:44)
Gene3’ CCCTAAAACAGACCgctcttca(SEQ ID NO:45)
Gene4’ CGCGATAAACGAAGgctcttca(SEQ ID NO:46)
Gene5’ AACGACAAGCAGACgctcttca(SEQ ID NO:47)
Gene1’-R tgaagagcCCATCGTTTCATGT(SEQ ID NO:48)-Biotin
Gene2’-R tgaagagcAAGTCAAGGGAATC(SEQ ID NO:49)-Biotin
Gene3’-R tgaagagcGGTCTGTTTTAGGG(SEQ ID NO:50)-Biotin
Gene4’-R tgaagagcCTTCGTTTATCGCG(SEQ ID NO:51)-Biotin
Gene5’-R tgaagagcGTCTGCTTGTCGTT(SEQ ID NO:52)-Biotin
使用南京金斯瑞生物科技有限公司的芯片引物池技术合成这些oligo，即通过芯片高通量合成这些oligo。后续的生物素oligo与链霉亲和素磁珠结合、退火、酶切和 PCA、酶切、纯化步骤与实施例3-6所述相同，最后，通过每个目的基因两端的特异性引物序列通过PCR直接扩增出每一个目的基因。将产物进行电泳检测及Sanger测序。
电泳结果见图6，左边第一条泳道为marker，分子量从上至下分别为3000bp，2000bp，1500bp，1000bp，750bp，500bp，250bp，100bp；泳道2，3，4，11，13分别为Gene1’、Gene2’、Gene3’、Gene4’、Gene5’。图7-11为5条基因的测序结果，图7中gene2代表gene1’，图8中gene3代表gene2’，图9中gene4代表gene3’，图10中gene13代表gene4’，图11中gene15代表gene5’。
本发明的实施方式并不限于上述实施例所述，在不偏离本发明的精神和范围的情况下，本领域普通技术人员可以在形式和细节上对本发明做出各种改变和改进，而这些均被认为落入了本发明的保护范围。

Claims (13)

  1. 一种多基因合成方法，所述方法包括：
    (1)将待合成的多种靶基因片段中的每种靶基因片段分割成若干个重叠寡核苷酸序列，在每个重叠寡核苷酸序列端部加上修饰序列使其能够以单链形式与磁珠特异性连接，所述修饰序列包括酶切位点序列和标签序列，所述标签序列因不同的靶基因片段而不同；
    (2)合成带有修饰序列的重叠寡核苷酸序列；
    (3)使带有修饰序列的重叠寡核苷酸序列与磁珠特异性连接；所述磁珠上连接有标签序列的反向互补序列，所述的特异性连接是通过标签序列与其反向互补序列形成双链结构；
    (4)通过酶切得到单链的重叠寡核苷酸序列，再进行聚合酶链式组装，获得多种靶基因片段；
    其中，所述酶切和聚合酶链式组装在油包水体系中进行。
  2. 权利要求1所述的方法，其中至少一个油包水体系的每个油包水体系中仅包含一个磁珠。
  3. 权利要求1或2的方法，其中在酶切之前加入酶切和PCA反应所需的缓冲液和试剂，并将其与油性介质混合，震荡形成油包水体系；
    优选地，与油性介质混合的液体的体积与油性介质的体积比为1:5；震荡的转速为2800rpm。
  4. 权利要求1-3任一项所述的方法，其中每个靶基因片段两端具有通用引物。
  5. 权利要求1-3任一项所述的方法，其中每个靶基因片段两端具有特异性引物。
  6. 权利要求1-5任一项所述的方法，其中所述酶切是通过内切酶进行切割，所述内切酶优选BspQI酶。
  7. 权利要求1-6任一项所述的方法，其中所述的标签序列含有10-100个碱基，优选15-70个碱基，更优选20-40个碱基，最优选20-30个碱基。
  8. 权利要求1-7任一项所述的方法，其中所述重叠寡核苷酸序列含40-150个碱基；优选50-130个碱基；更优选60-110个碱基；更优选65-90个碱基；最优选65-80个碱基。
  9. 权利要求1-8任一项所述的方法，其中所述重叠寡核苷酸序列包含重叠碱基，所述重叠碱基数量为10-100；优选10-70；更优选10-50；更优选10-30；更优选10-20；最优选15-16。
  10. 权利要求1-9任一项所述的方法，其中所述磁珠通过链霉亲和素-生物素与所述标签序列的反向互补序列连接。
  11. 权利要求1-10任一项所述的方法，进一步包含在聚合酶链式组装后纯化多种靶基因片段的步骤。
  12. 权利要求1-11任一项所述的方法，进一步包含扩增获得多种靶基因片段的步骤。
  13. 权利要求1-12任一项所述的方法，其中使用芯片合成带有修饰序列的重叠寡核苷酸序列。
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