High-throughput automatic gene construction method of oligonucleotide pool based on chip synthesis

Abstract

The invention relates to a high-throughput automatic gene construction method of an oligonucleotide pool based on chip synthesis. On the basis of the high-throughput gene construction automation device and system, a high-throughput automatic gene construction scheme is further designed to achieve overall collaboration, an operation process capable of improving efficiency, reducing cost, improving the automation degree and completing process standardization in a laboratory / production workshop is developed, the working efficiency and applicability are remarkably improved, and the application prospect is wide. The high-throughput automatic construction that the initial DNA raw material is at the fmol level and the final gene product is at the 300bp to Kb level can be met.

Description

Technical Field

[0001] The invention belongs to the technical field of gene synthesis and relates to a high-throughput automated gene construction method based on an oligonucleotide pool synthesized by a chip. Background Art

[0002] With the emergence of automated pipetting workstations and integrated experimental equipment, a large number of repetitive tasks in the field of synthetic biology can now be efficiently completed through integrated liquid handling workstations. This not only reduces the need for manual labor, but also improves overall efficiency.

[0003] Currently, there are three types of oligonucleotide synthesis technologies: the first is column-synthesized oligonucleotides, the second is chip-synthesized oligonucleotides, and the third is enzymatically synthesized oligonucleotides. Among the three, column-synthesis technology is more mature, with high yield and low error rate, but high cost and low throughput. Chip synthesis technology has high throughput and low cost, but the error rate is slightly higher than the former and the yield is low. Both of these have synthesis length restrictions, generally less than 150nt, and some technologies can be as high as 230nt. The reaction conditions of enzymatic synthesis are relatively environmentally friendly, and the synthesis length can be as high as 280bp or longer, with high purity, but the synthesis cycle is long, the cost is high, the error rate is also high, and it has not been truly commercialized.

[0004] Since the length of de novo DNA synthesis is limited by oligonucleotide synthesis technology, it is necessary to assemble longer genes or genomes from DNA fragments. DNA synthesis and assembly technology can be mainly divided into two categories according to the method used, polymerase cycle assembly (PCA) and ligase chain reaction (LCR). There are also some methods for producing larger assemblies, including overlap amplification polymerase chain reaction (OE-PCR), circular polymerase extension cloning (CPEC), Golden Gate, Gibson, sequence ligation independent cloning (SLIC), etc. The assembly of longer DNA fragments is usually carried out in vivo, usually using organisms such as Escherichia coli, Bacillus subtilis and yeast. The development of cheap, high-throughput and reliable gene synthesis methods will greatly promote the progress of biology and biotechnology, but the current reliance on column-synthesized oligonucleotides as a DNA source limits the further reduction of gene synthesis costs, while the use of oligonucleotides synthesized from DNA microchips can reduce costs by at least one order of magnitude.

[0005] Therefore, it is necessary to develop a highly parallel gene synthesis method using oligonucleotide pools synthesized on DNA microchips as raw materials and convert it into a high-throughput automated long-chain gene construction system to further improve efficiency, reduce costs, and achieve standardization of production processes. Summary of the invention

[0006] In view of the deficiencies in the prior art and actual needs, the present invention provides a high-throughput automated gene construction method based on a chip-synthesized oligonucleotide pool, and develops a high-throughput automated gene construction solution with high efficiency, low cost, high degree of automation, standardization and wide adaptability based on a gene automated construction device and system.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a high-throughput automated gene construction method based on a chip-synthesized oligonucleotide pool, the high-throughput automated gene construction method comprising the following steps:

[0009] (S1) splitting the target gene sequence, and designing corresponding barcode primers and amplification primers according to the split sequence;

[0010] (S2) synthesizing a primer pool containing the barcode primer and amplification primer, and performing primer enrichment and separation and purification;

[0011] (S3) digesting the barcode primer obtained in step (S2), performing gene splicing using the digestion product, and amplifying using amplification primers after splicing to obtain the target gene;

[0012] In the present invention, based on the high-throughput gene construction automation device and system, a high-throughput automated gene construction scheme is further designed to achieve overall synergy, which can meet the automated high-throughput construction of 300bp to Kb-level genes.

[0013] Preferably, the reaction processes involved in steps (S1) to (S3) are performed on a gene automation construction device and system, and the gene automation construction device and system include:

[0014] A frame (1) is provided on the frame (1): a mobile unit (2) and an enzyme-labeled concentration detection unit (3) capable of docking with the mobile unit (2), an electrophoresis unit (4), a filtration purification unit (5), a storage unit (6) capable of storing a preset solvent, a centrifugal unit (7), a constant temperature oscillation unit (8), a film sealing unit (9) and a PCR instrument (10); the frame (1) is also provided with a gel cutting unit (20) and a monitoring component; the gel cutting unit (20) is capable of docking with the storage unit (6); the monitoring component is capable of being electrically connected to the above structures respectively, and configures primers, configures gene splicing reaction liquid, runs the splicing reaction, configures gene product amplification reaction liquid, runs the amplification reaction, performs agarose gel electrophoresis, cuts target gene bands, purifies electrophoretic PCR products, performs enzyme-labeled quantification and concentration uniformity, configures enzyme-linked reaction liquid and runs the enzyme-linked reaction according to preset information; the mobile unit (2) is equipped with a liquid pipette head (26) for taking and placing solvents.

[0015] Preferably, step (S1) specifically includes: (S1-1) importing the target gene sequence data into the sequence analysis software and performing a sequence splitting operation; (S1-2) designing amplification primers and barcode primers corresponding to the split sequences.

[0016] In the present invention, a barcode sequence is designed in the splicing primer after splitting, which is convenient for subsequent identification and separation of different sequences, allowing the use of a high-throughput primer synthesis method (such as chip synthesis method) to synthesize a primer pool and then perform selective amplification, enrichment, separation and purification, which is conducive to reducing the cost of primer synthesis. The specific sequence and number of barcode sequences can be designed according to the number of target genes to be constructed.

[0017] Preferably, step (S2) specifically includes:

[0018] (S2-1) starting the automated equipment for high-throughput splicing and assembly;

[0019] (S2-2) selecting and executing an automated splicing program according to the splitting scheme of step (S1);

[0020] (S2-3) Importing the relevant parameter table into the gene automated construction device and system, and placing the required reagents and consumables in designated locations;

[0021] (S2-4) Starting the gene template primer pool enrichment program, performing primer enrichment, and purifying the enriched product.

[0022] Preferably, step (S3) specifically includes:

[0023] (S3-1) After the product of step (S2-4) is quantified by microplate reader, the uniformity of the sample concentration is calculated;

[0024] (S3-2) performing an enzyme digestion procedure and a negative pressure filtration purification procedure to obtain a primer without a barcode end;

[0025] (S3-3) transferring the product of step (S3-2), calculating the uniformity of the sample concentration, entering the automated splicing program, and continuing to run until the reaction process is completed;

[0026] (S3-4) The product of step (S3-3) is transferred and amplified using amplification primers to obtain the target gene.

[0027] Preferably, the high-throughput automated gene construction method further comprises the step of constructing a plasmid, including assembling the target gene into the target plasmid, and specifically linearizing the expression plasmid and connecting it to the target gene.

[0028] Preferably, the primer pool enrichment procedure comprises:

[0029] Pre-denaturation: 95-98°C, 3-5min;

[0030] Denaturation: 95-98°C, 8-12s, annealing: 53-55°C, 13-16s, extension: 70-73°C, 13-16s, 18-25 cycles;

[0031] Thorough extension: 70-73℃, 3-5min.

[0032] The specific primer pool enrichment system can be:

[0033] Components concentration volume Primer pool ~50-200 fmol / μL 2μL DNA polymerase reaction solution 2× 25μL Barcode primer (forward) 5-10pmol / μL 2μL Barcode primer (reverse) 5-10pmol / μL 2μL Sterilized water / Make up to 50 μL

[0034] Preferably, the enzyme digestion procedure comprises:

[0035] Incubate at 37-65°C (e.g., 37°C, 50°C, 55°C, 60°C, or 65°C, etc.) for 60-90 minutes. The optimal reaction temperature depends on the optimal reaction temperature of the selected enzyme.

[0036] Specifically, the enzyme digestion system can be:

[0037] Components concentration volume Type IIS restriction enzyme buffer 10× 5μL Type IIS restriction endonuclease 10U / μL 1μL BSA 0.8mg / mL 1μL Sterilized water / Make up to 50 μL

[0038] IIS type restriction endonucleases can be used according to the oligos that need to be split, and the design software automatically proofreads and matches them. Currently available IIS type restriction endonucleases include A1WI, BbsI, BbsI-HF, BccI, BcoDI, BfuAI, BmrI, BsaI-HF, BsmAI, BsmBI-v2, BspMI, BspQI, BsrDI, BtsCI, BtsI-v2, BtsI-MutI, EarI, Esp3I, BsaI, FauI, HpyAV, MlyI, Mn1I, PleI, SapI, SfaNI, BciVI, and PaqCI.

[0039] Preferably, the automated splicing procedure comprises:

[0040] Pre-denaturation: 95-98°C, 3-5min;

[0041] Denaturation: 95-98°C, 8-12s, annealing: 60-65°C, 13-16s, annealing: 45-50°C, 13-16s, extension: 70-73°C, 13-16s, 18-25 cycles;

[0042] Thorough extension: 70-73℃, 3-5min.

[0043] Specifically, the automated splicing system can be:

[0044] Components concentration volume Enzyme digestion products 20-40 ng / μL 12.5μL DNA polymerase 2× 12.5μL Sterilized water / Make up to 25 μL

[0045] Preferably, the amplification procedure comprises:

[0046] Pre-denaturation: 95-98°C, 3-5min;

[0047] Denaturation: 95-98°C, 8-12s, annealing: 60-65°C, 13-16s, annealing: 55-60°C, 13-16s, extension: 70-73°C, 30-60s, 25-30 cycles;

[0048] Thorough extension: 70-73℃, 3-5min.

[0049] Specifically, the amplification system can be:

[0050] Components concentration volume Splicing products / 2.5μL DNA polymerase reaction solution 2× 12.5μL Forward Primer 10pmol / μL 1.25μL Reverse primer 10pmol / μL 1.25μL Sterilized water / Make up to 25 μL

[0051] In the present invention, a primer pool enrichment program, an automated splicing program, an amplification program and a system are designed to improve the applicability of the overall process, and more target genes can be automatically constructed, which is conducive to achieving high-throughput construction.

[0052] In the present invention, the constructed gene sequence can be a sequence shown in SEQ ID NO.1 to SEQ ID NO.4, the primer pool sequence can be a sequence shown in SEQ ID NO.5 to SEQ ID NO.96, the barcode primer sequence can be a sequence shown in SEQ ID NO.97 to SEQ ID NO.108, and the gene-specific amplification primer can be a sequence shown in SEQ ID NO.109 to SEQ ID NO.116.

[0053] Preferably, the high-throughput automated gene construction method based on the oligonucleotide pool synthesized on a chip is performed on an automated gene construction device and system, and specifically comprises:

[0054] (S1-1) Importing the target gene sequence data into the sequence analysis software and performing sequence splitting operations;

[0055] (S1-2) designing amplification primers and barcode primers corresponding to the split sequences;

[0056] (S2-1) starting the automated equipment for high-throughput splicing and assembly;

[0057] (S2-2) selecting and executing an automated splicing program according to the splitting scheme of step (S1);

[0058] (S2-3) Importing the relevant parameter table into the gene automated construction device and system, and placing the required reagents and consumables in designated locations;

[0059] (S2-4) starting the gene template primer pool enrichment program, performing primer enrichment, and purifying the enriched product; the mobile unit (2) is docked with the storage unit (6) to configure the gene product amplification reaction solution; the mobile unit (2) is docked with the sealing unit (9) and the PCR instrument (10) to run the amplification reaction; the mobile unit (2) is docked with the electrophoresis unit (4) and the storage unit (6) to perform agarose gel electrophoresis, and the gel cutting unit (20) is docked with the storage unit (6) to cut the target gene band; the mobile unit (2) is docked with the storage unit (6), the constant temperature oscillation unit (8), the suction filtration purification unit (5) and the centrifugal unit (7) to purify the electrophoresis PCR product;

[0060] (S3-1) After transferring the product of step (S2-4) to the ELISA instrument for quantification, the sample concentration is calculated for uniformity; the mobile unit (2) is docked with the ELISA concentration detection unit (3) to perform ELISA quantification and make the concentration uniform;

[0061] (S3-2) performing an enzyme digestion procedure and a negative pressure filtration purification procedure to obtain a primer without a barcode end; the mobile unit (2) is docked with the storage unit (6) to configure an enzyme digestion reaction solution; the mobile unit (2) is docked with a sealing unit (9) and a PCR instrument (10) to run an enzyme digestion reaction; the mobile unit (2) is docked with the storage unit (6), the filtration purification unit (5) and the centrifugal unit (7) to purify the primer without a barcode end;

[0062] (S3-3) transferring the product of step (S3-2), calculating the uniformity of the sample concentration, entering the automated splicing program, and continuously running until the reaction process is completed; the mobile unit (2) is docked with the enzyme label concentration detection unit (3) to perform enzyme label quantification and make the concentration uniform; the mobile unit (2) is docked with the storage unit (6) to configure the gene splicing reaction solution; the mobile unit (2) is docked with the sealing unit (9) and the PCR instrument (10) respectively to run the splicing reaction;

[0063] (S3-4) transferring the product of step (S3-3), performing an amplification procedure using amplification primers, and obtaining a target gene; the mobile unit (2) is docked with the storage unit (6) to configure a gene product amplification reaction solution; the mobile unit (2) is docked with the sealing unit (9) and the PCR instrument (10) to run the amplification reaction;

[0064] (S4) Entering the target gene electrophoresis separation and purification program, calculating the uniformity of the sample concentration, and entering the target gene assembly to the target plasmid program; the mobile unit (2) is respectively docked with the electrophoresis unit (4) and the storage unit (6) to perform agarose gel electrophoresis, and the gel cutting unit (20) is docked with the storage unit (6) to cut the target gene band; the mobile unit (2) is respectively docked with the storage unit (6), the constant temperature oscillation unit (8), the suction filtration purification unit (5) and the centrifugal unit (7) to purify the electrophoresis PCR product; the mobile unit (2) is docked with the enzyme label concentration detection unit (3) to perform enzyme label quantification and make the concentration uniform; the mobile unit (2) is docked with the storage unit (6) to configure the enzyme linkage reaction solution; the mobile unit (2) is respectively docked with the sealing unit (9) and the PCR instrument (10) to run the enzyme linkage reaction.

[0065] In the present invention, the calculation of sample concentration homogenization refers to the sample being subjected to enzyme-labeled quantitative analysis. Based on the obtained concentration value, the software automatically calculates the amount of pure water that needs to be added, and automatically instructs the pipetting unit to move the required amount of water into the sample to dilute it to the concentration required for the next reaction procedure. The above mentioned sample concentration homogenization uses the same formula: W = ((C*E) / (X / V))-E, and the meaning of each symbol in the formula is as follows:

[0066] E is the volume after purification and recovery (μL);

[0067] C is the concentration after purification and recovery (ng / μL);

[0068] X is the required amount of DNA (ng) fixed;

[0069] V is the required DNA volume (μL) fixed;

[0070] Y is the maximum total concentration (ng / μL) to be adjusted;

[0071] W is the water (μL) to be added in the end.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] Based on the high-throughput gene construction automation device and system, the present invention further designs a high-throughput automated gene construction scheme based on the oligonucleotide pool synthesized by chip to achieve overall synergy, and develops an operating procedure that can improve efficiency, reduce costs, enhance the degree of automation, and complete process standardization in the laboratory / production workshop, which significantly improves work efficiency and applicability and can meet the automated high-throughput construction of 300bp to Kb-level genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1Schematic diagram of the high-throughput automated gene construction protocol.

[0075] Figure 2 This is the electrophoresis result of the gene constructed in Example 2.

[0076] Figure 3 Schematic diagram of automated apparatus and system for high-throughput gene sequencing.

[0077] Figure 4 Layout of structures on the substrate for high-throughput gene production automation devices and systems;

[0078] Figure 5 Schematic diagram of the third moving part and support frame of the automated device and system for high-throughput gene construction;

[0079] Figure 6 Schematic diagram of the consumables unit for constructing an automated device and system for high-throughput genetics;

[0080] In the figure, 1-frame; 11-base plate; 12-support frame; 13-support plate; 14-third moving part; 141-belt transmission part; 1411-driving wheel; 1412-driven wheel; 1413-belt; 142-guide part; 1421-guide rail; 1422-slider; 143-driving motor; 15-fan filter unit;

[0081] 2-mobile unit; 21-first mobile part; 22-second mobile part; 23-96 channel liquid transfer part; 24-8 channel liquid transfer part; 25-carrying clamp; 26-liquid transfer head; 27-liquid transfer head box;

[0082] 3-enzyme label concentration detection unit; 31-384 enzyme label plate;

[0083] 4-electrophoresis unit; 41-peristaltic pump; 42-electrophoresis apparatus;

[0084] 5- suction filtration purification unit; 51- suction filtration plate;

[0085] 6-storage unit; 61-liquid storage tank; 62-PCR plate; 63-deep well plate; 64-waste liquid tank;

[0086] 7-centrifugal unit; 71-centrifuge; 72-first centrifugal plate; 73-second centrifugal plate;

[0087] 8- Constant temperature oscillation unit;

[0088] 9-film sealing unit;

[0089] 10-PCR instrument;

[0090] 20-rubber cutting unit; 201-rubber cutting head; 202-visual inspection component; 203-rubber cutting head box;

[0091] 30-consumable unit; 310-consumable stack; 3101-consumable rack; 3102-installation cabin; 3103-consumable motor; 320-consumable extraction mechanism; 3201-driving member; 32011-first driving member; 32012-second driving member; 3202-clamping claw; 3203-lifting mechanism;

[0092] 40- Panoramic camera;

[0093] 50-Abandoned barrel.

[0094] Figure 7 This is the electrophoresis result of the gene constructed in Example 3. DETAILED DESCRIPTION

[0095] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0096] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0097] Based on the high-throughput gene construction automation device and system, the present invention further designs a high-throughput automated gene construction scheme based on the oligonucleotide pool synthesized on the chip to achieve overall synergy, significantly improve work efficiency and applicability, and reduce costs. Specifically, the process of the high-throughput automated gene construction scheme is as follows Figure 1 As shown, it mainly includes the following steps:

[0098] (1) Pretreatment

[0099] 1) Import high-throughput gene sequence data into sequence analysis tools (such as AnalysisTool (software registration numbers are 2023SR1470468 and 2023SR1386947)), etc.), and perform sequence splitting operations;

[0100] 2) Use sequence splitting software to optimize the codons of the introduced gene sequences and accurately evaluate the complex sequences to determine whether the high-throughput automated gene construction scheme is suitable for the splicing of the introduced sequences;

[0101] 3) Automatically output the designed split sequence and the corresponding barcode primers and amplification primers;

[0102] (2) Gene template primer pool enrichment

[0103] 1) Start high-throughput splicing and assembly automation equipment to prepare for efficient sequence splicing operations;

[0104] 2) Select and execute the correct automated splicing program based on the optimal splicing solution determined in the pre-processing stage;

[0105] 3) Follow the system prompts to import the relevant parameter table into the system and place the required reagents and consumables in the designated locations;

[0106] 4) Start the gene template primer pool enrichment program and continue running until the primary and secondary amplification reaction processes are completed.

[0107] (3) Electrophoresis purification

[0108] 1) The system automatically enters electrophoresis separation, gel image collection, automatically identifies and determines the correct product bands, and performs precise gel cutting and recovery operations;

[0109] 2) Subsequently, the system automatically enters the negative pressure filtration purification step to obtain the purified spliced ​​template product without the barcode end;

[0110] (4) Enzyme digestion and splicing

[0111] 1) After transferring to the microplate reader for quantification, the system will automatically calculate and execute the sample concentration homogenization procedure to ensure that the sample concentration meets the downstream application;

[0112] 2) The system automatically enters the enzyme digestion program and the negative pressure filtration purification program to obtain the splicing template product without the barcode end;

[0113] 3) Transfer again, the system will automatically calculate and execute the sample concentration homogenization procedure again to ensure that the sample concentration meets the downstream application;

[0114] 4) The system automatically enters the plate automatic splicing program and continues to run until the reaction process is completed.

[0115] (5) Plasmid construction

[0116] 1) The system automatically enters the electrophoresis separation and purification procedure of the splicing product;

[0117] 2) Subsequently, the system automatically enters the program of assembling the splicing products into the target plasmid, completing the construction of the plasmid containing the target gene.

[0118] Example 1

[0119] This example performs gene splitting sequence design and determines the biochemical conditions for high-throughput gene construction.

[0120] 1. Design of multiple gene template primer pools

[0121] The sequence splitting software AnalysisTool was used to verify the endonuclease recognition sites of the four gene sequences 5q, 5h, 6q and 6h, and then the sequences were split. Then all the split primers were automatically assigned the first-level barcode and the second-level barcode for identification of different sequences.

[0122] 2. According to the above design, a primer pool is synthesized, including 2 pairs of first-level barcode primers, 4 pairs of second-level barcode primers, and 4 pairs of gene-specific primers, wherein the first-level barcode primers are used to amplify the first-level sub-primer pool from the total primer pool, the second-level barcode primers are used to amplify the second-level sub-primer pool from the first-level sub-primer pool, and the gene-specific primers are used to amplify a single specific gene from the second-level sub-primer pool after the splicing reaction.

[0123] 3. Verify the conditions and steps of gene synthesis reaction, which are divided into first-level template amplification, second-level template amplification, and target gene splicing and amplification after enzyme cutting.

[0124] 4. Purify and recover the splicing products, and perform first-generation sequencing after continuous transfer. Determine the gene splicing effect based on the electrophoresis gel image and sequencing results.

[0125] 5. Assemble the splicing products into the target plasmid.

[0126] The first-level template amplification reaction system and procedure in the 5q, 5h, 6q and 6h gene synthesis steps are shown in Tables 1 and 2.

[0127] Table 1

[0128]

[0129] Table 2

[0130]

[0131] The secondary template amplification reaction system and procedure in the 5q, 5h, 6q and 6h gene synthesis steps are shown in Tables 3 and 4.

[0132] Table 3

[0133]

[0134] Table 4

[0135]

[0136]

[0137] The enzyme digestion reaction system and procedure in the 5q, 5h, 6q and 6h gene synthesis steps are shown in Figures 5 and 6.

[0138] Table 5

[0139] Components concentration volume <![CDATA[rCutSmart TM Buffer]]> 10× 5μL Bts1-v2 endonuclease (NEB) 10U / μL 1μL BSA 0.8mg / mL 1μL Sterilized water / Make up to 50 μL

[0140] Table 6

[0141] Reaction temperature Reaction time 37℃ 1.5h

[0142] The target gene splicing reaction system and procedure in the 5q, 5h, 6q and 6h gene synthesis steps are shown in Tables 7 and 8.

[0143] Table 7

[0144]

[0145] Table 8

[0146]

[0147] The target gene amplification reaction system and procedure in the 5q, 5h, 6q and 6h gene synthesis steps are shown in Tables 9 and 10.

[0148] Table 9

[0149]

[0150]

[0151] Table 10

[0152]

[0153] The reaction system and procedure for assembling the 5q, 5h, 6q and 6h genes into plasmids are shown in Table 11, and the reaction was carried out at 22° C. for 1 hour.

[0154] Table 11

[0155] Components concentration volume Purified splicing product 6ng / μL 5μL Linearized plasmid 4ng / μL 2.5μL T4 DNA Ligase Buffer 10× 1μL 50% PEG 4000 50%(w / v) 1μL T4 DNA ligase (Thermo Scientific) 5U / μL 0.5μL

[0156] The complete sequences of the double-stranded DNA of 5q, 5h, 6q and 6h are shown in Table 12.

[0157] Table 12

[0158]

[0159]

[0160] Sequences of the 5q, 5h, 6q, and 6h primer pools (oligo 1 to oligo 92, corresponding to SEQ ID NO.5 to SEQ ID NO.96 in sequence):

[0161] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGcacaggaaaccggttatgagcacaaaaaagaaaccattaacacaagagcagcttgaggacgcacCACTGCatTGGACGCAACTCGCTttgcgatagcctcgca, oligo1;

[0162] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGaagccaagttcatttttctttttttcataaattgctttaaggcgacgtgcgtcctcaagctgctcCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca, oligo2;

[0163] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGagaaaaatgaacttggcttatcccaggaatctgtcgcagacaagatggggatggggcagtcaggCACTGCatTGGACGCAACTCGCTtgcgatagcctcgca, oligo3;

[0164] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGgttataagcatttaatgcattgatgccattaaataaagcaccaacgcctgactgccccatccccaCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca, oligo4;

[0165] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGtgcattaaatgcttataacgccgcattgcttgcaaaaattctcaaagttagcgttgaagaatttaCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca, oligo5;

[0166] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGctaaccgcttcatacatctcgtagatttctctggcgattgaagggctaaattcttcaacgctaacCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo6;

[0167] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGagatgtatgaagcggttagtatgcagccgtcacttagaagtgagtatgagtaccctgttttttctCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo7;

[0168] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGtggtaaaggttctaagctcaggtgagaacatccctgcctgaacatgagaaaaaacagggtactcaCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo8;

[0169] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGgagcttagaacctttaccaaaagtgatgcggagagatgggtaagcacaaccaaaaaagccagtgaCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo9;

[0170] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGtggtgcggtcatggaattaccttcaacctcaagccagaatgcagaatcactggcttttttggttgCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo10;

[0171] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGtaattccatgaccgcaccaacaggctccaagccaagctttcctgacggaatgttaattctcgttgCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo11;

[0172] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGagtctggctatgcagaaatcacctggctcaacagcctgttcagggtcaacgagaattaacattccCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo12;

[0173] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGatttctgcatagccagacttgggggtgatgagtttaccttcaagaaactgatcagggatagcggtCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo13;

[0174] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGatgggatcattgggtactgtgggtttagtggttgtaaaaacacctgaccgctatccctgatcagtCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo14;

[0175] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGcagtacccaatgatcccatgcaatgagagttgttccgttgtggggaaagttatcgctagtcagtgCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo15;

[0176] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGagagcggggttatttatgctgcagtcagccaaacgtctcttcaggccactgactagcgataacttCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo16;

[0177] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGgcataaataaccccgctcttacacattccagccctgaaaaagggcatcaaattaaaccacacctaCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo17;

[0178] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGttctcctttactcatggtacccatttaaattcctttgcatacaccataggtgtggtttaatttgaCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo18;

[0179] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGtaccatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggcgCACTGCaTGGACGCAACTCGCTtgcgatagcctcgca,oligo19;

[0180] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGcaccttcaccctctccactgacagagaatttttgcccattaacatcgccatctaattcaacaagCACTGCatTGGACGCAACTCGCTtgcgatagcctcgca,oligo20;

[0181] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactCACTGCatgcatTGGACGCAACTCGCTtgcgatagcctcgca,oligo21;

[0182] GCCTTGATTGGCGCTtcgggcgtaactcgaGCAGTGaagtagtgacaagtgttggccatggaacaggtagcttcccagtagtgcaaataaatttaaggCACTGCatgcTGGACGCAACTCGCTtgcgatagcctcgca,oligo22;

[0183] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGgccaacacttgtcactacttggagagaacttgctttagacacagttcaggtgaagaaagacCACTGCatgcaTAGCGATAACGCCGCtgcgatagcctcgca,oligo23;

[0184] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGttttagcccctactgagcttatagttaacagaagtttccaggtctttcttcacctgaactCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo24;

[0185] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGctataagctcagtaggggctaaaagcatgttaatcggcataaaaaggcaatgagatcttaCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo25;

[0186] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGatgaagggcagaagaggggcggggagctgtgtgccctaagatctcattgcctttttatgcCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo26;

[0187] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGccccgcccctcttctgcccttcatccttctttcaatcagcagggaccgtgcactctcttgCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo27;

[0188] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGgttttctgtggtgctggatgcacctctgttttctgtggtggctccaagagagtgcacggtcccCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo28;

[0189] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGatccagcaccacagaaaacagagccaccacagaaaacagagggtgactgtcatcccctccagtCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo29;

[0190] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGtgctctctcctcctgctctgctgcagctgggagtgtgcagagactggaggggatgacagtcCACTGCatgcaTAGCGATAACGCCGCtgcgatagcctcgca,oligo30;

[0191] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGagagcaggaggagagagcacagcctgcaatgctaatttgccaggagctcacctgcctgcgCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo31;

[0192] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGacagcccggcctctggcctcactggcgtctgtgcccagtgacgcaggcaggtgagctcctCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo32;

[0193] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGagtgaggccagaggccgggctgtgctggggcctgagctgggtggtggggagagagtctctcccCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo33;

[0194] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGgtccccttaaacatgctcctcctgcacggaagagacaggggcaggggagagactctctccccaCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo34;

[0195] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGggagcatgtttaaggggacgggttcaaagctggtcacatccccaccgaaaaagcccatggacaCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo35;

[0196] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGacttgcccctcctgtggcactggacaagctagtgggcttttcgttgtccatgggctttttcggCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo36;

[0197] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGtgccacaggaggggcaagtggaggaggagaggtggcggtgctccccactccacttccagtcgtCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo37;

[0198] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGggtgacagatagggaacgaggatgaagggaagggagagccagtgacgactggaagtggagtggCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo38;

[0199] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGtcgttccctatctgtcaccatttcctgtcgtcgtttcctctgaatgtctcaccctgccctcccCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo39;

[0200] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGaggatacgacaggggtgaggctacagacaggggacttgcaagcagggagggcagggtgagacaCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo40;

[0201] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGctcacccctgtcgtatcctgactacaataacagcttctgggtgtccctggcatccactctctcCACTGCatgTAGCGATAACGCCGCtgcgatagcctcgca,oligo41;

[0202] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGgaaggttcctcaggcatccgtcacggaagggacaagaagggagagagagtggatgccagggCACTGCatgcaTAGCGATAACGCCGCtgcgatagcctcgca,oligo42;

[0203] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGggatgcctgaggaaccttccccaaactcttctgtcccatccctgccctgctcaaaatccaCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo43;

[0204] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGcaggacttcaagttgattcaggcgtgttagggagctgtgattggattttgagcagggcagCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo44;

[0205] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGgaatcaacttgaagtcctgtcttgagtaatccgtgggccctaactcactcatcccaactcCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo45;

[0206] GCCTTGATTGGCGCTtgcgcggaacgacatGCAGTGggaggctccctggcagggtgtggggcaaggaagtgagtgaagagttgggatgagtgagttCACTGCatgcatTAGCGATAACGCCGCtgcgatagcctcgca,oligo46;

[0207] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGagtgaattgacgcgtattgggattggtctcttggtgctatcaccgcaagggataaatatctaacaCACTGCaTACCGGCGACGAACAggcccatgttgacca,oligo47;

[0208] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGcatgcaaccattatcaccgccagaggtaaaatagtcaacacgcacggtgttagatatttatcccttCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo48;

[0209] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGcggtgataatggttgcatgtactaaggaggttgtatggaacaacgcataaccctgaaagattatgcCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo49;

[0210] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGggtaggtgagagatcttttagctgtcttggtttgcccaaagcgcattgcataatctttcagggttaCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo50;

[0211] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGaaaagatctctcacctaccaaacaatgcccccctgcaaaaaataaattcatataaaaaacatacagCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo51;

[0212] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGatttatgtcaacaccgccagagataatttatcaccgcagatggttatctgtatgttttttatatgaCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo52;

[0213] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGtggcggtgttgacataaataccactggcggtgatactgagcacatcagcaggacgcactgaccaccCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo53;

[0214] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGaatgctgcccttcttcagggcttaatttttaagagcgtcaccttcatggtggtcagtgcgtcctgcCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo54;

[0215] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGcctgaagaagggcagcattcaaagcagaaggctttggggtgtgtgatacgaaacgaagcattggtCACTGCaTACCGGCGACGAACAggcccatgttgacca,oligo55;

[0216] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGtggtgatggctgctgcccatgaattccctccttaatttttaaccaatgcttcgtttcgtaCACTGCatgcatTACCGGCGACGAACAggcccatgttgacca,oligo56;

[0217] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGtgggcagcagccatcaccatcatcaccacagccaggatccaatggtgagcaagggcgaggCACTGCatgcatTACCGGCGACGAACAggcccatgttgacca,oligo57;

[0218] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGcttgaagcgcatgaactccttgatgatggccatgttatcctcctcgcccttgctcaccatCACTGCatgcatTACCGGCGACGAACAggcccatgttgacca,oligo58;

[0219] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatCACTGCatgcatTACCGGCGACGAACAggcccatgttgacca,oligo59;

[0220] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGtggcggtctgggtgccctcgtaggggcggccctcgccctcgccctcgatctcgaactcgtggccgtCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo60;

[0221] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGgagggcacccagaccgccaagctgaaggtgaccaagggcggccccctgcccttcgcctgggacatcCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo61;

[0222] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGgggtgcttcacgtaggccttggagccgtacatgaactgaggggacaggatgtcccaggcgaagggcCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo62;

[0223] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo63;

[0224] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGcacggtcaccacgccgccgtcctcgaagttcatcacgcgctcccacttgaagccctcggggaaggaCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo64;

[0225] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGcggcggcgtggtgaccgtgacccaggactcctcccttcaggacggcgagttcatctacaaggtgaaCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo65;

[0226] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGgcattacggggccgtcggaggggaagttggtgccgcgcagcttcaccttgtagatgaactCACTGCatgcatTACCGGCGACGAACAggcccatgttgacca,oligo66;

[0227] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGtccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccCACTGCTACCGGCGACGAACAggcccatgttgacca,oligo67;

[0228] AGGTAGCGTTCCCGTacgtctaggcgcgatGCAGTGtcagcttcagcctctgcttgatctcgcccttcagggcgccgtcctcggggtacatccgctcggagCACTGCaTACCGGCGACGAACAggcccatgttgacca,oligo68;

[0229] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacCACTGCatgcaTAGCGGACCCGACTAggcccatgttgacca,oligo69;

[0230] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGacgttgtaggcgccgggcagctgcacgggcttcttggccttgtaggtggtcttgacctcaCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo70;

[0231] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggacCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo71;

[0232] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGgccctcggcgcgttcgtactgttccacgatggtgtagtcctcgttgtgggaggtgatgtcCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo72;

[0233] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGagtacgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaagtaaaCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo73;

[0234] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGattactttctgttcgacttaagcattatgcggccgcaagcttttacttgtacagctcgtcCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo74;

[0235] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGaagtcgaacagaaagtaatcgtatgagctcgacaccatcgaatggcgcaaacctttcgcgCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo75;

[0236] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggtttgcgccattCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo76;

[0237] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGgaagagagtcaattcagggtggtgaatgtataagaaggagatatacatatgagcacaaaaCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo77;

[0238] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGaaggcgacgtgcgtcctcaagctgctcttgtgttaatggtttcttttttgtgctcatatgtatatCACTGCaTAGCGGACCCGACTAggcccatgttgacca,oligo78;

[0239] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGtgaggacgcacgtcgccttaaagcaatttatgaaaaaaagaaaaatgaacttggcttatccCACTGCatgcaTAGCGGACCCGACTAggcccatgttgacca,oligo79;

[0240] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGcctgactgccccatccccatcttgtctgcgacagattcctgggataagccaagttcatttCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo80;

[0241] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGtggggatggggcagtcaggcgttggtgccttatttaatggcatcaatgcattaaatgcttatCACTGCatgcTAGCGGACCCGACTAggcccatgttgacca,oligo81;

[0242] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGttcaacgctaactttgaggattcttgtaagcgatgcggcgttataagcatttaatgcattgCACTGCatgcaTAGCGGACCCGACTAggcccatgttgacca,oligo82;

[0243] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGcctcaaagttagcgttgaagaatttagcccttcaatcgccagagaaatctacgagatgtaCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo83;

[0244] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGcatactcacttctaagtgacggctgcatactaaccgcttcatacatctcgtagatttctcCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo84;

[0245] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGtcacttagaagtgagtatgagtaccctgttttttctcatgttcaggcagggatgctctcaCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo85;

[0246] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGcacctttccgcaccacctttggtaaaggttctaagctcaggtgagagcatccctgcctgaCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo86;

[0247] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGaaggtggtgcggaaaggtgggtaagcacaaccaaaaaagccagtgattctgcattctggcCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo87;

[0248] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGggagcctgttggtgctgtcatggaattaccttcaacctcaagccagaatgcagaatcactCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo88;

[0249] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGgacagcaccaacaggctccaagccatcttttcctgacggaatgttaattctcgttgacccCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo89;

[0250] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGgtctggctatgcagaaatcacctggctcaacagcctgctcagggtcaacgagaattaacaCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo90;

[0251] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGgatttctgcatagccagactcgggggtggtgagtttaccttcaagaaactgatcagggatCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo91;

[0252] AGGTAGCGTTCCCGTtgctaccgcgtgtgaGCAGTGgggtactgtgggtttagtggttgtaaaaacacctgaccgctatccctgatcagtttcttgCACTGCatgcatTAGCGGACCCGACTAggcccatgttgacca,oligo92;

[0253] The barcode primers and gene-specific amplification primers of 5q, 5h, 6q and 6h are shown in Table 13.

[0254] Table 13

[0255]

[0256]

[0257] Example 2

[0258] This example uses primers and procedures designed in the example to construct genes.

[0259] 1. Prepare a primer pool with a concentration of ~2 fmol / μL using column synthesis.

[0260] 2. Perform the first round of amplification on the primer pool using the first-level primer 1, and then perform the second round of amplification on the first-round amplification products using the second-level primers 1 and 2 to obtain the 5q and 5h splicing primer pools, respectively; at the same time, perform the first round of amplification on the primer pool using the first-level primer 2, and then perform the second round of amplification on the first-round amplification products using the second-level primers 3 and 4 to obtain the 6q and 6h splicing primer pools, respectively.

[0261] 3. After purifying the primer pool, perform enzyme digestion to remove the barcode end, and then purify again.

[0262] 4. Use the purified products for gene splicing and amplification reactions. After the reaction is completed, the products are recovered and sent for first-generation sequencing.

[0263] The results of the splicing run are as follows Figure 2 As shown, the size of the constructed gene was in line with expectations.

[0264] The first-generation sequencing result data are shown in Table 14. It can be seen that the present invention can successfully construct and obtain the target gene, and the phenomenon of incomplete correct cloning usually originates from base errors in the template DNA itself, or in the PCA / PCR amplification process, due to the natural error rate of the polymerase, the gene sequence is successfully spliced ​​and assembled, but there are errors in some bases, which are not errors introduced by the scheme of the present invention.

[0265] Table 14

[0266] Sample name Number of sequencing samples Exactly the right sample amount Incorrect sample size 5q 5 4 1 5h 4 2 2 6q 5 2 3 6h 6 3 3

[0267] Example 3

[0268] This example further performs automated high-throughput gene construction on an automated device and system based on the gene splicing and assembly conditions designed in Example 1.

[0269] The schematic diagram of the automation device and system structure is as follows Figure 3 As shown, it includes a frame 1 and a mobile unit 2, an enzyme label concentration detection unit 3, an electrophoresis unit 4, a suction filtration and purification unit 5, a storage unit 6 capable of storing a solvent, a centrifugal unit 7, a constant temperature oscillation unit 8, a sealing unit 9, a PCR instrument 10, a gel cutting unit 20 and a monitoring component arranged on the frame 1. Among them, the monitoring component is electrically connected to the mobile unit 2, the enzyme label concentration detection unit 3, the electrophoresis unit 4, the suction filtration and purification unit 5, the storage unit 6 capable of storing a preset solvent, the centrifugal unit 7, the constant temperature oscillation unit 8, the sealing unit 9, the PCR instrument 10 and the gel cutting unit 20, respectively, so that the mobile unit 2 can be docked with the enzyme label concentration detection unit 3, the electrophoresis unit 4, the suction filtration and purification unit 5, the storage unit 6 capable of storing a solvent, the centrifugal unit 7, the constant temperature oscillation unit 8, the sealing unit 9 and the PCR instrument 10, and the gel cutting unit 20 can be docked with the storage unit 6. Among them, the mobile unit 2 is equipped with a liquid pipette head 26 to take and put the solvent. The monitoring component configures primers, configures gene splicing reaction solution, runs the splicing reaction, configures gene product amplification reaction solution, runs the amplification reaction, performs agarose gel electrophoresis, cuts target gene bands, purifies electrophoretic PCR products, performs enzyme labeling quantification and concentration uniformity, configures enzyme-linked reaction solution, and runs the enzyme-linked reaction according to preset information.

[0270] The structural layout on the substrate is shown in the figure Figure 4 As shown, the schematic diagram of the third moving component and the support frame is as shown Figure 5 As shown, the schematic diagram of the consumable unit is as follows Figure 6 shown.

[0271] The mobile unit 2 includes a first mobile part 21, a second mobile part 22, a 96-channel pipetting part 23, an 8-channel pipetting part 24 and a carrying clamp 25, all of which are electrically connected to the monitoring part. The 96-channel pipetting part 23 is connected to the end of the first mobile part 21 to achieve horizontal movement, the 8-channel pipetting part 24 and the carrying clamp 25 are connected to the end of the second mobile part 22 to achieve horizontal movement, and the 96-channel pipetting part 23, the 8-channel pipetting part 24 and the carrying clamp 25 can each move in the vertical direction, so that the 96-channel pipetting part 23, the 8-channel pipetting part 24 and the carrying clamp 25 can be independently controlled to work. The 96-channel pipetting part 23 and the 8-channel pipetting part 24 are both equipped with a pipette head 26. The mobile unit 2 also includes a pipette head box 27, which is used to store the pipette head 26, and the pipette head 26 can be installed on the mobile unit 2.

[0272] The rubber cutting unit 20 includes a rubber cutting head 201, a blue light lamp and a visual detection component 202. A rubber cutting head box 203 is provided on the frame 1. A plurality of rubber cutting heads 201 are provided in the rubber cutting head box 203. The first movable component 21 can be docked with the rubber cutting head box 203 so that the rubber cutting head 201 can be replaceably arranged at the end of the first movable component 21. The rubber cutting head 201 and the visual detection component 202 are both arranged at the end of the first movable component 21 to realize horizontal movement, and the rubber cutting head 201 can realize vertical movement; the blue light lamp is provided on the frame 1 and can illuminate the storage unit 6. The visual detection component 202 is used to take pictures of the storage unit 6. The rubber cutting head 201 is docked with the storage unit 6 for rubber cutting.

[0273] The suction filtration purification unit 5 includes a suction filtration plate 51 and a recovery plate, the centrifugal unit 7 includes a centrifuge 71, a first centrifugal plate 72 and a second centrifugal plate 73, and the mobile unit 2 can move the suction filtration plate 51 to the recovery plate and the centrifuge 71. The electrophoresis unit 4 includes an electrophoresis instrument 42 and an agarose gel plate for accommodating agarose gel. The sealing unit 9 includes a sealing instrument.

[0274] The frame 1 is provided with a base plate 11, the moving unit 2 is provided above the base plate 11, the base plate 11 is provided with a support frame 12, a support plate 13 and a third moving component 14, the support frame 12 is slidably connected to the base plate 11 and connected to the output end of the third moving component 14, the support plate 13 is provided above the support frame 12, and the third moving component 14 is electrically connected to the monitoring component. A part of the liquid pipetting head box 27, the rubber cutting head box 203, the enzyme label concentration detection unit 3, the electrophoresis unit 4, the suction filtration purification unit 5, the storage unit 6, the centrifugal unit 7, the constant temperature oscillation unit 8, the film sealing unit 9 and the PCR instrument 10 are provided on the base plate 11, and the other part is provided on the support plate 13.

[0275] The third moving part 14 includes a driving motor 143, two belt transmission parts 141 and two guide parts 142. The belt transmission part 141 includes a driving wheel 1411, a driven wheel 1412 and a belt 1413. The belt 1413 is wound around the driving wheel 1411 and the driven wheel 1412. The driving motor 143 is connected to the driving wheel 1411 of the belt transmission part 141. The guide part 142 includes a guide rail 1421 and a slider 1422 slidably connected to the guide rail 1421. The guide rail 1421 is connected to the base plate 11. The slider 1422 is connected to the support frame 12. The support frame 12 and the support plate 13 form a gate shape. The two ends of the support frame 12 are correspondingly connected to the belts 1413 of the two belt transmission parts 141 and the sliders 1422 of the two guide parts 142. The third moving part 14 is arranged below the base plate 11. The support frame 12 passes through the base plate 11, which further makes the structure more compact in the vertical direction.

[0276] The base plate 11 is divided into 44 working positions, of which T1-T10 positions are all located on the support plate 13, and the remaining positions are all located on the base plate 11. The positions and corresponding functions are as follows: T1, T3-8, T10 are room temperature PCR plates, T2 is a 384 ELISA plate, T9 and T35 are centrifugal plates, T11 is a 96-channel agarose gel electrophoresis tank, T12-T15 are gel cutting head boxes, T16 is a waste liquid tank, and T17-19 are room temperature shaking tanks. Liquid storage tank, T20-T28 is the 4℃ reaction liquid configuration working area, which can be used to place liquid storage tank, deep well plate, liquid adding tank and PCR plate, T29-T34, T45-46 are pipette head boxes, T36 is a waste bucket, T37 is a film sealing instrument, T38 is a 96-channel constant temperature oscillator, T39-40 is a 96-channel PCR instrument, T41 is the consumable unit docking point, T42-43 are filtration plates, and T44 is a special well plate centrifuge for automation equipment.

[0277] The consumable unit 30 includes a consumable stack 310 and a consumable extraction mechanism 320, both of which are electrically connected to the monitoring component. The consumable stack 310 includes a consumable rack 3101 and a consumable motor 3103. The consumable rack 3101 is provided with a plurality of installation cabins 3102, which are used to place the pipette head box 27. The consumable motor 3103 drives the consumable rack 3101 to rotate so that the corresponding installation cabin 3102 rotates to a preset position to dock with the consumable extraction mechanism 320. The driving member 3201 may include a first driving member 32011 and a second driving member 32012, which respectively realize the movement of the clamping jaw 3202 in the horizontal and vertical directions.

[0278] (1) Pretreatment

[0279] 1) Import high-throughput gene sequence data into the sequence analysis tool - AnalysisTool to perform sequence splitting operations;

[0280] 2) Use the sequence splitting software AnalysisTool to perform codon optimization on the imported gene sequence and accurately evaluate the complex sequence to determine whether the automated construction scheme is suitable for the splicing of the imported sequence;

[0281] Automatically output the designed split sequence and the corresponding barcode primers and amplification primers.

[0282] (2) Gene template primer pool enrichment

[0283] 1) Start high-throughput splicing and assembly automation equipment to prepare for efficient sequence splicing operations;

[0284] 2) Select and execute the correct automated splicing program based on the optimal splicing solution determined in the pre-processing stage;

[0285] 3) Follow the system prompts to import the relevant parameter table into the system and place the required reagents and consumables in the designated locations;

[0286] 4) Start the gene template primer pool enrichment program and continue running until the primary and secondary amplification reaction processes are completed.

[0287] (3) Electrophoresis purification

[0288] 1) The system automatically enters electrophoresis separation, gel image collection, automatically identifies and determines the correct product bands, and performs precise gel cutting and recovery operations;

[0289] 2) Subsequently, the system automatically enters the negative pressure filtration purification step to obtain the purified spliced ​​template product containing the barcode end;

[0290] (4) Enzyme digestion and splicing

[0291] 1) After transferring to the microplate reader for quantification, the system will automatically calculate and execute the sample concentration homogenization procedure to ensure that the sample concentration meets the downstream application;

[0292] 2) The system automatically enters the enzyme digestion program and the negative pressure filtration purification program to obtain the splicing template product without the barcode end;

[0293] 3) Transfer again, the system will automatically calculate and execute the sample concentration homogenization procedure again to ensure that the sample concentration meets the downstream application;

[0294] 4) The system automatically enters the plate automatic splicing program and continues to run until the reaction process is completed.

[0295] (5) Plasmid construction

[0296] 1) The system automatically enters the electrophoresis separation and purification procedure of the splicing product;

[0297] 2) Subsequently, the system automatically enters the program of assembling the splicing products into the target plasmid, completing the construction of the plasmid containing the target gene.

[0298] Specifically, the steps include:

[0299] 1. A primer pool with a concentration of ~30 fmol / uL was prepared based on chip synthesis method, which contained 96 gene (including 5q, 5h, 6q, 6h, etc., 500-800bp) split template primers;

[0300] 2. Using the primer pool as a template, the construction of high-throughput genes is started, including the above-mentioned primer pool enrichment, electrophoresis purification, enzyme cutting, splicing and plasmid construction. After the high-throughput splicing and assembly program is started, the gene synthesis process will be automatically carried out, and the preset parameters are input into the monitoring component to perform the following steps: the mobile unit is docked with the storage unit to configure the primers; the mobile unit is docked with the storage unit to configure the gene splicing reaction solution; the mobile unit is docked with the sealing unit and the PCR instrument respectively to run the splicing reaction; the mobile unit is docked with the storage unit to configure the gene product amplification reaction solution; the mobile unit is docked with the sealing unit respectively The membrane unit is docked with the PCR instrument to run the amplification reaction; the mobile unit is docked with the electrophoresis unit and the storage unit respectively to perform agarose gel electrophoresis; the gel cutting unit is docked with the storage unit to cut the target gene band; the mobile unit is docked with the storage unit, the constant temperature oscillation unit, the filtration purification unit and the centrifugal unit respectively to purify the electrophoresis PCR product; the mobile unit is docked with the enzyme label concentration detection unit to perform enzyme label quantification and make the concentration uniform; the mobile unit is docked with the storage unit to configure the enzyme-linked reaction solution; the mobile unit is docked with the sealing unit and the PCR instrument respectively to run the enzyme-linked reaction. This process covers the parallel splicing and assembly of 96 different genes;

[0301] The results of the splicing run are as follows Figure 7 As shown, the band marked with a red frame is the desired target band, which represents the successfully spliced ​​gene fragment. It can be seen that most genes can be successfully constructed. The system automatically identifies the band (red frame) and performs gel excision recovery, quantification and subsequent plasmid assembly. After analysis, most of the genes that did not splice out the target product are because the gene itself has some more complex structures or the designed gene-specific primers are not suitable. The data of this analysis can be used as the setting parameters for the subsequent gene splicing condition restrictions.

[0302] The exemplary sequencing results of 5q, 5h, and 6q are shown in Table 15, and correct splicing products can be obtained.

[0303] Table 15

[0304] Sample name Number of sequencing samples Exactly the right sample amount Incorrect sample size 5q 4 3 1 5h 4 1 3 6q 4 3 1

[0305] In summary, the present invention is based on a high-throughput gene construction automation device and system, and further designs a high-throughput automated gene construction scheme based on a chip-synthesized oligonucleotide pool to achieve overall synergy, and develops an operating procedure that can improve efficiency, reduce costs, increase the degree of automation, and complete process standardization in the laboratory / production workshop, significantly improving work efficiency and applicability, and can meet the automated high-throughput construction of 300bp to Kb-level genes.

[0306] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A high-throughput automated gene construction method based on a chip-synthesized oligonucleotide pool, characterized in that: The high-throughput automated gene construction method comprises the following steps: (S1) splitting the target gene sequence, and designing corresponding barcode primers and amplification primers according to the split sequence; (S2) synthesizing a primer pool containing the barcode primer and amplification primer, and performing primer enrichment and separation and purification; (S3) digesting the barcode primer obtained in step (S2), performing gene splicing using the digestion product, and performing amplification using amplification primers after splicing to obtain the target gene.

2. The high-throughput automated gene construction method based on chip-synthesized oligonucleotide pool according to claim 1, characterized in that: The reaction processes involved in steps (S1) to (S3) are performed on a gene automation construction device and system, and the gene automation construction device and system include: A frame (1), wherein the frame (1) is provided with: a mobile unit (2) and an enzyme-labeled concentration detection unit (3) capable of docking with the mobile unit (2), an electrophoresis unit (4), a filtration purification unit (5), a storage unit (6) capable of storing a preset solvent, a centrifugal unit (7), a constant temperature oscillation unit (8), a film sealing unit (9) and a PCR instrument (10); the frame (1) is also provided with a gel cutting unit (20) and a monitoring component, wherein the gel cutting unit (20) is capable of docking with the storage unit (6), and the monitoring component is capable of being electrically connected to the above structures, and configures primers, configures gene splicing reaction liquid, runs the splicing reaction, configures gene product amplification reaction liquid, runs the amplification reaction, performs agarose gel electrophoresis, cuts target gene bands, purifies electrophoretic PCR products, performs enzyme-labeled quantification and concentration uniformity, configures enzyme-linked reaction liquid and runs the enzyme-linked reaction; the mobile unit (2) is provided with a liquid pipette head (26) for taking and placing solvents; Preferably, step (S1) specifically includes: (S1-1) importing the target gene sequence data into the sequence analysis software and performing a sequence splitting operation; (S1-2) designing amplification primers and barcode primers corresponding to the split sequences.

3. The high-throughput automated gene construction method based on the oligonucleotide pool synthesized on a chip according to claim 1 or 2, characterized in that: Step (S2) specifically includes: (S2-1) starting the automated equipment for high-throughput splicing and assembly; (S2-2) selecting and executing an automated splicing program according to the splitting scheme of step (S1); (S2-3) Importing the relevant parameter table into the gene automated construction device and system, and placing the required reagents and consumables in designated locations; (S2-4) Starting the gene template primer pool enrichment program, performing primer enrichment, and purifying the enriched product.

4. The high-throughput automated gene construction method based on a chip-synthesized oligonucleotide pool according to any one of claims 1 to 3, characterized in that: Step (S3) specifically includes: (S3-1) After the product of step (S2-4) is quantified by microplate reader, the normalized sample concentration is calculated; (S3-2) performing an enzyme digestion procedure and a negative pressure filtration purification procedure to obtain a primer without a barcode end; (S3-3) transferring the product of step (S3-2), calculating the uniformity of the sample concentration, entering the automated splicing program, and continuing to run until the reaction process is completed; (S3-4) The product of step (S3-3) is transferred and amplified using amplification primers to obtain the target gene.

5. The high-throughput automated gene construction method based on the oligonucleotide pool synthesized on a chip according to any one of claims 1 to 4, characterized in that: The high-throughput automated gene construction method also includes a step of constructing a plasmid, including assembling the target gene into the target plasmid.

6. The high-throughput automated gene construction method based on chip-synthesized oligonucleotide pool according to claim 3, characterized in that: The primer pool enrichment procedure includes: Pre-denaturation: 95-98°C, 3-5min; Denaturation: 95-98°C, 8-12s, annealing: 53-55°C, 13-16s, extension: 70-73°C, 13-16s, 18-25 cycles; Thorough extension: 70-73℃, 3-5min.

7. The high-throughput automated gene construction method based on chip-synthesized oligonucleotide pool according to claim 4, characterized in that: The enzyme digestion procedure includes: Incubate at 37-65°C for 60-90 min.

8. The high-throughput automated gene construction method based on chip-synthesized oligonucleotide pool according to claim 4, characterized in that: The automated splicing procedure includes: Pre-denaturation: 95-98°C, 3-5min; Denaturation: 95-98°C, 8-12s, annealing: 60-65°C, 13-16s, annealing: 45-50°C, 13-16s, extension: 70-73°C, 13-16s, 18-25 cycles; Thorough extension: 70-73℃, 3-5min.

9. The high-throughput automated gene construction method based on chip-synthesized oligonucleotide pool according to claim 4, characterized in that: The amplification procedure includes: Pre-denaturation: 95-98°C, 3-5min; Denaturation: 95-98°C, 8-12s, annealing: 60-65°C, 13-16s, annealing: 55-60°C, 13-16s, extension: 70-73°C, 30-60s, 25-30 cycles; Thorough extension: 70-73℃, 3-5min.

10. The high-throughput automated gene construction method based on a chip-synthesized oligonucleotide pool according to any one of claims 1 to 9, characterized in that: The high-throughput automated gene construction method is performed on an automated gene construction device and system, and specifically includes: (S1-1) Importing the target gene sequence data into the sequence analysis software and performing sequence splitting operations; (S1-2) designing amplification primers and barcode primers corresponding to the split sequences; (S2-1) starting the automated equipment for high-throughput splicing and assembly; (S2-2) selecting and executing an automated splicing program according to the splicing scheme of step (S1); (S2-3) Importing the relevant parameter table into the gene automated construction device and system, and placing the required reagents and consumables in designated locations; (S2-4) starting the gene template primer pool enrichment program, performing primer enrichment, and purifying the enriched product; the mobile unit (2) is docked with the storage unit (6) to configure the gene product amplification reaction solution; the mobile unit (2) is docked with the sealing unit (9) and the PCR instrument (10) to run the amplification reaction; the mobile unit (2) is docked with the electrophoresis unit (4) and the storage unit (6) to perform agarose gel electrophoresis, and the gel cutting unit (20) is docked with the storage unit (6) to cut the target gene band; the mobile unit (2) is docked with the storage unit (6), the constant temperature oscillation unit (8), the suction filtration purification unit (5) and the centrifugal unit (7) to purify the electrophoresis PCR product; (S3-1) After transferring the product of step (S2-4) to the ELISA instrument for quantification, the sample concentration is calculated for uniformity; the mobile unit (2) is docked with the ELISA concentration detection unit (3) to perform ELISA quantification and make the concentration uniform; (S3-2) executing an enzyme digestion procedure and a negative pressure filtration purification procedure to obtain primers without barcode ends; the mobile unit (2) is docked with the storage unit (6) to configure an enzyme digestion reaction solution; the mobile unit (2) is docked with the sealing unit (9) and the PCR instrument (10) to run an enzyme digestion reaction; the mobile unit (2) is docked with the storage unit (6), the filtration purification unit (5) and the centrifugal unit (7) to purify the primers without barcode ends; (S3-3) transferring the product of step (S3-2), calculating the uniformity of the sample concentration, entering the automatic splicing procedure, and continuously running until the reaction process is completed; the mobile unit (2) is docked with the enzyme label concentration detection unit (3) to perform enzyme label quantification and make the concentration uniform; the mobile unit (2) is docked with the storage unit (6) to configure a gene splicing reaction solution; the mobile unit (2) is docked with the sealing unit (9) and the PCR instrument (10) to run a splicing reaction; (S3-4) transferring the product of step (S3-3), and performing an amplification procedure using amplification primers to obtain the target gene; The mobile unit (2) is docked with the storage unit (6) to configure a gene product amplification reaction solution; the mobile unit (2) is docked with the sealing unit (9) and the PCR instrument (10) to run the amplification reaction; (S4) entering the target gene electrophoresis separation and purification program, calculating the uniformity of the sample concentration, and entering the target gene assembly into the target plasmid program; The mobile unit (2) is docked with the electrophoresis unit (4) and the storage unit (6) respectively to perform agarose gel electrophoresis, and the gel cutting unit (20) is docked with the storage unit (6) to cut the target gene band; the mobile unit (2) is docked with the storage unit (6), the constant temperature oscillation unit (8), the suction filtration purification unit (5) and the centrifugal unit (7) respectively to purify the electrophoresis PCR product; the mobile unit (2) is docked with the enzyme label concentration detection unit (3) to perform enzyme label quantification and make the concentration uniform; the mobile unit (2) is docked with the storage unit (6) to configure the enzyme linkage reaction solution; the mobile unit (2) is docked with the sealing unit (9) and the PCR instrument (10) respectively to run the enzyme linkage reaction.

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