TWI232867B - Apparatus and methods for massively parallel oligonucleotide synthesis - Google Patents

Abstract

Apparatus and method are described for parallel oligonucleotide synthesis by the number of hundreds of different sequences and lengths at a time. Standard phosphoramitide chemistry is employed and the synthesis reactions take place in the container compliant with the industrial standard microplate format to allow using the readily available automated instruments for subsequent processing. Key parameters in reducing synthesis volume in small reaction wells are discussed. Solutions to the difficulties in low volume and large number synthesis reactions are provided in this invention.

Description

1232867 A7 B7 V. Description of the invention (Ί) This invention is partly supported and supported by the National Science Council and Research Institute of Taiwan, Republic of China. Brief description of the invention The present invention relates to a device and method for synthesizing a large number of oligonucleotide acids by multiple channels, and more particularly to a device and method for synthesizing hundreds or thousands of oligonucleotide acids in a solid phase at the same time. BACKGROUND OF THE INVENTION Due to the development of chemical synthesis of oligonucleotide, synthetic biopolymers have played an important role in many fields, such as molecular biology, forensic science, and medical diagnostics. In recent years, due to many large-scale projects in genetic research, such as gene polymorphism identification and recording (scoring), STS content mapping, II-ray hybridization mapping, primer walking in DNA sequencing, human genome project (Human Genome Project), and others require a variety of different oligonucleotides, so the requirements for existing oligonucleotides have been significantly increased. Due to the emergence of multi-channel analysis technologies such as DNA microarrays, new requirements for many different oligonucleotic acids have been developed. Among the various synthetic oligonucleotide costs, the existing synthetic methods appear to be too high. Reducing the consumption and cost of reagents for conjugated or unmodified nucleic acids is helpful for the application of various oligonucleotides. For example, in the application of oligonucleotide microarrays, the 5'-end of each oligonucleotide is modified with a spacer and an amine linker that immobilizes the oligonucleotide on a solid substrate. The cost of amine linkers and spacers is much higher than that of riboimine phosphate. Furthermore, the scale of existing commercial formulations that are less than 40 nanomoles is sufficient for most applications. PCR amplification usually requires a picoole amount of oligonucleotide primers. For each probe, this paper size applies Chinese National Standard (CNS) A4 specification (210X 297 $) 1232867 A7

______ B7 V. Description of the invention (2) — '^ An oligonuclear acid microarray with less than 1 picomolar is required. The impact of high-yield synthesis is of great importance in the application of genetic biotechnology, which requires hundreds of thousands of oligonucleotides. While seeking to synthesize various oligonucleotides, two types of multi-channel systems are currently used to synthesize at least dozens of oligonucleotides at one time. The first type uses a 1-dimensional movement control system with 8 screw f valves and injection nozzles that deliver a reagent to 8 rows of 96-well microdisks. An example of this is the Am0s system, which is a 9b pipe synthesizer developed by Lashkad and colleagues (pr0c ㈣% USA · I995, $ 2,7912_7915). At the same time, 8S solenoid valves are needed in the AM0s system to inject other reagents such as modified bases (not easy to operate). When other reagents such as labeling reagents or modified bases are needed, additional 8 valves and 8 nozzles are required. The inert reaction gas system in the AMOS system is maintained by inflating the Teflon sheet with a hermetic seal. The minimum injection volume of the reagent is about 10 mechanical liters. The output in 4.5 hours was a 20-mer (20-mer) 96 oligo: ¾: acid. The advantages of AMOS design are: (1) high output efficiency, using 8 nozzles to deliver reagents to 8 holes at the same time; (2) low argon consumption due to the small volume of the reaction chamber; (3) simple one-time movement design. The shortcomings of the AM〇s system are: ○) 8 valves and nozzles are required for each reagent, and it is not easy to use other reagents such as modified bases; (2) Only 96-well can be used. In order to use 384 holes, it is necessary to arrange the nozzle positions, and the number of solenoid valves and nozzles must be doubled to more than one hundred, which makes the system operation and maintenance quite difficult. The second type is a two-dimensional movement control system with a valve and a micro-disc injection nozzle that facilitates the delivery of reagents to the pores. The 96 wells are raster scanned by a movement control system below the ejection nozzle for receiving reagents. Examples of this type 1232867 A7 B7 _____ 5. Description of the invention (3) is a MerMade synthesizer developed by Rayner and colleagues (Genome Res ·, 1998, 8, 741-747). The MerMade system uses a two-dimensional movement control system and a nozzle for each reagent. Using two 96-well plates, the yield was increased to 192 oligonucleotides in 17 hours. The minimum reaction volume of the system is approximately 60 microliters. The advantages are: (1) disks of different formats can be used; (2) it is easy to add other reagents to the system by adding a valve and a nozzle for each other reagent. The disadvantages are: (1) For synthesis using a 384-moving disc (see below), the actual injection volume is too large. (2) The 2-disk design takes up a lot of space and the machine size is 3 X 3 X 6 feet. * Many multi-channel oligonucleotide synthesizers are commercially available. The ABI 3900 can synthesize 48 oligonucleotides at the same time, and can be converted several times to synthesize 288 oligonucleotides within 9 hours. GfeneMachines' Polyplex can be simultaneously 96 oligonucleotides are synthesized, and LCDR / MerMade sold by BioAutomation can synthesize 96x2 oligonucleosides simultaneously. Oligonucleotide synthesis method and device-based two patents have been announced as conventional techniques. The AMOS system is USP 5,529,765, and the MerMade system is USP 5,368,823 and 5,541,314. All oligosynthesizers are based on original or by Beaucage et al. (Tetrahedron I.ett. 1981, 22, 1859-1862) and Adams et al. (J. Am. Chem. Sac., 1983, 105, 661-663 ) Development of modified imine phosphate chemicals shall prevail. A patent based on imine phosphate chemicals (USP 4,415,732) was granted to Caruthers et al. In 1983, and a C-I-P patent (USP 4,973,679) was granted to the same inventor in 1990. Related patents were granted to Caruthers et al. (1992, USP 5,132,418). Urdea et al. (USP 4,517,338) was the main patent on the use of controlled pore glass (CPG) that performs solid-phase imine phosphate chemicals. This paper scale applies Chinese National Standard (CNS) A4 specification (210X297 public dollars) A7 B7 1232867 V. Description of invention () 4 In order to complete the genome of the model organism and the sequencing of the human gene plan, a large number of gene-specific probes and PCR primers to amplify all genes in the organism to study their functions, cellular roles, and other characteristics of genes. For example, me / awogas / er fruit rope has about 14,000 genes, and C. e / eM seems to have about 18,000 genes. To amplify all the bee fly genes in the genome, about 28,000 PCR primers are required. The previous technique required about 6 months to complete the synthesis of specific PCR primers for all genes of the above organisms. Assessing 100,000 human genes to acquire skills requires more than a year. _ One way to improve the yield of synthesis is to increase the number of pipeline synthesis more than described in the previous technology. In order to increase the number of pipeline synthesis, a disk or a reaction slot larger than the standard microdisk format can be used, or a standard microdisk format with higher density holes such as 384-wells can be used. Using disks larger than the standard microdisk format can overcome difficulties in post-synthesis processing, such as yield quantification and product purification. For large quantities of oligonucleotide, post-synthetic processing requires automated instruments. Almost all commercially available automation instruments are designed to use standard microdisks. Unused automated liquid processing or pipetting equipment for post-synthesis processing cannot perform hundreds of multi-channel oligonucleotide synthesis at the same time. For a standard microdisk, the diameter of the reaction wells in a 96-well is about 7 mm and the pitch is 9 mm. The array of 384 wells with the diameter of the reaction well reduced to 3.5 mm and the pitch of the well reduced to 4.5 mm presents many difficulties in synthesis. The 1536-hole format hole size was further reduced to 2.25 mm. Synthesis problems often occur, such as cross-contamination of adjacent reaction wells, large changes in yield between wells, splashing of reactants and synthetic supports, and so on. Therefore, by using standard 384-well or 1536-well microplates to increase the paper size, the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1232867 A7 B7 is used. 5. Description of the invention (5) plus the number of pipeline synthesis, To increase the yield of synthesis is not entirely worthless. Previous techniques have not taught methods and devices for obtaining high-yield oligonucleotides by using hundreds of reaction wells with a diameter less than 96-well disk format simultaneously. Many problems arose with the aforementioned prior art. The first is the release of synthetic supports (controlled pore glass, CPG) during the reaction. The detachment occurred randomly distributed over the entire disk at a rate of about 2% (see AMOS). Harmful effects can occur when the support is detached from the hole. Unwanted results may include (1) zero yield of pores lost by CPG beads due to detachment, (2) subsequent blockage of the vacuum f-line due to detached CPG beads, and (3) detachment of CPG beads Excessive vacuum in the holes caused exhaust in other holes to fail. These undesired effects can reduce the performance of the synthesizer, or further make the entire disc synthesis fail. This effect becomes more pronounced as the pore density increases, as discussed in the 384-well plate. The second problem is that a small change in reagent volume or waste discharge rate in each reaction well may have a devastating effect on a well that is faster than the others that can be used for the entire disc synthesis. Another problem with the prior art is that the minimum volume of reagent injection for multi-channel synthesizers is too large for 384-well or higher density synthesis. The minimum injection volume is 10 μl for the AMOS system and 20 μl for the MerMade system. This volume will be too large for proper mixing of reagents during the reaction because the pore diameter is limited to 3.5 mm smaller than a standard 384-well microdisk. The operating performance of the present invention is superior to the previous artist as follows: (1) the minimum synthesis failure rate; (2) the amount of organic waste is low; (3) the synthesis scale is low; (4) the standard microdisk can be flexibly used, such as 384- Wells and 96-wells; (5) The difference in synthetic yield during the reaction is small. Experimental results show that no single synthesis failure was found. Use This paper size applies to China National Standard (CNS) A4 (210 X 297 g g 1232867 A7)

A specific example of the filter tip as a reaction hole can effectively support CPG beads for synthesis without cPG bead loss. The absence of synthesis failures makes the system more negligible and simplifies the subsequent synthesis processing steps. Since the inside diameter of the reaction holes is small, the waste generated by the synthetic base in each hole is about 0.33 liters. The results showed that the synthesis scale of less than 10 nmoles can be performed with high quality (Fig. 7) 'and each base agent only requires 036 mg of imine phosphate. The invention has achieved novel low chemical usage and low waste generation. Reduction of reagent consumption f is very important for the synthesis of expensive imine phosphates such as capping markers, modifying bases, RNA imine phosphates and separators. The cost of these reagents may be several tens of times higher than the average DNA nucleic acid imine phosphate. Reducing reagent consumption is of great benefit in reducing overall costs. '' Object and Summary of the Invention One object of the present invention is to provide a device and a method for synthesizing hundreds of oligonuclear acids simultaneously in a multi-channel format. Another object of the present invention is to provide a device and method for feeding a small amount of reagents into hundreds of reaction wells in a microdisk format. Yet another object of the present invention is to teach a device and method for leaving oligonucleotide synthesis reagents in hundreds of wells in a microdisk format. Still another object of the present invention is to teach a device and method for minimizing the difference in output of hole-to-hole in a multi-channel synthesis of micro-disk format. Yet another object of the present invention is to teach a method for preventing the synthetic support from splashing during the synthesis process. The foregoing and other objects of the present invention can be achieved by the devices and methods described herein for multi-channel synthesis of hundreds of oligonuclear acids. This device is included in the standard Zhangjiazhuang Standard (CNS) A_4 specification (21 () χ 297 public ^ " 1232867 A7 B7 V. Description of the invention (7) Equipment for forming hundreds of reaction holes in the microdisk format, And a device for transporting microdisks with a minimum of solid reaction beads splashing under the reagent nozzle, and a device for minimizing the difference in the yield of well-to-well synthesis. The drawings briefly describe the foregoing and other objects of the present invention. It can be more easily understood through the following description and accompanying drawings, in which: Figure 1 is a schematic diagram illustrating the basic components of the device. Figure 2A is a reaction 'hole formed by a filter tip having a melt or a film to support CPG beads Specific example. Figure 2B is another specific example of a high-density reaction plate consisting of a crown plate, a medium (film) that maintains CPG beads, and a base plate. Figure 3 shows the relative size of the fluid flow rate, gas flow rate, and discharge rate. The general diagram of the effect of the completeness of the reagent discharge in the reaction well. Figure 4 is a general schematic diagram illustrating the cross section of the reaction well and its effect on the splash of CPG beads and the formation of bubbles in the reaction well. Figure 5 shows the CPG beads Splash height A diagram of the valve injection pressure function of the reagent injection volume, which also illustrates how to determine the optimal injection pressure and guest product. Figure 6 shows the valve injection pressure of the acetonitrile volume required to wash the CPG beads attached to the wall of the reaction well Function diagram. Figure 7 is a general diagram of HPLC chromatography for the synthesis of oligonucleotides on different scales. The specific example is described in detail with reference to Figure 1. The device consists of 5 main components: (1) reaction plate assembly (assembly) ) And vacuum accessories: special 384-mini version components This paper size is applicable to China National Standard (CNS) A4 specifications (210X 297 male 1232867 A7 B7) V. Description of the invention (8 as a reaction box, and each hole is an independent reaction tank. After each synthesis step, the vacuum accessories discharge the reagents with a vacuum pump. ⑺Reagents and tubes: store reagents and send the reagents from the bottle to the spiral tube and the ejection nozzle. ⑺Reagent valve accessories: a set of solenoid valves and fast response Drive circuit to control the injection volume to less than 2 microliters per injection. (4) Injection nozzle and moving system: computer-controlled 2 • dimension reagent rotation system; the micro plate under the nozzle is moved 'Are collected from the wells in each injection nozzle of reagent (5) Control software: autonomous read oligonucleotide Chang acid profile results and control software programs of the synthesizing program.

The reaction plate accessories, injection nozzles and translation stages are placed in a closed chamber filled with a gas such as argon or nitrogen to provide an inert gas for synthesis. I Referring to Figure 1, the synthesis reaction takes place in an internally constructed 384-well reaction disk assembly, and the reaction disk is a standard microdisk format. All reagent nozzles are arranged in sequence and assembled together on the nozzle holder. The seat is designed to support 20 different nozzles and is fixed to the translation stage. The nozzle holder scans the microdisk in the _ dimension by moving the translation stage. The reaction plate and vacuum accessories are erected on another translation stage of the two-dimensional mobile system. These two translation stages together constitute the X-γ scanning of the nozzles on the entire reaction plate assembly. To ensure that the injection volume of the components is about 2 microliters, the focus is on the operation of the quick response valve. Faster reaction times will also result in higher injection rates. For the actual application of the device, an emission rate higher than 20Hz is required. FIG. 2A shows a first specific example of a 384-well reaction plate accessory. The size of the 384-hole anti-paper paper is in accordance with Chinese National Standard (CNS) A4 specifications (210 X 21 1232867 A7 B7. V. Description of the invention (9) The tray accessories are used to independently support 384 glass frits equipped with filters. It consists of a carrier plate with a pointed pipette tip. The inner diameter of the pipette tip is approximately 2.2 mm and the tip distance is 4.5 mm. The holder plate is fixed with a vacuum fitting as shown in Figure 1. Between the holder and the vacuum fitting Gaskets ensure vacuum tightness. Vacuum fittings are connected to vacuum compacts that can also be used as waste containers. Reagents are injected from the top of each hole and evacuated. The carrier plate is manufactured by placing the pipette tip against each carrier plate. No tip detachment or vacuum leak was found during the synthesis, thus ensuring a reliable and consistent yield. When the number of oligonucleotides synthesized is less than 384, it can be advantageous to use a tip with a hidden tip in a substrate that does not require synthesis. Support plate, so no vacuum loss and waste of reagents occur. The second specific example of the reaction plate accessory is shown in Figure 2B. The accessory is composed of three parts, the crown plate, the medium to maintain the CPG beads, and the base plate. Make up. The The coronal plate includes a conical hole with a diameter of about 2 mm at the bottom and a depth of about 15 mm. The medium to properly maintain CPG beads must meet the flow characteristics of the gas flow rate lower than the flow velocity of the fluid applied to the vacuum port for discharge. The hydrophilic membrane The bubble point is usually higher than the vacuum used for the discharge, and is ideal for this purpose. The base plate is shaped as a constricted opening with holes to help collect the product without contaminating the holes. Figure 3 illustrates the discharge process. The importance of properly maintaining the medium of the CPG beads and the selected pumping speed. In this example, two reaction holes are described, but the principle of porosity is the same. Between the two reaction holes, the hole 1 has a high waste rate of Π liters / minute. Material discharge (fluid flow rate through the melt or film), and hole 2 has a lower discharge rate of F2 liters / minute. That is Fl & F2 This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 mm_ Love ^ 1232867 A7 B7 V. Description of the Invention (1Q) The pumping rate for discharge is P1 liter / minute, and the gas flow rate of hole 1 and hole 2 is A1 liter / minute and A2 liter / minute. After the reagent is sent in , Hole 1 and hole 2 are both詨 Received the same amount of reagent. During the discharge process, the waste flows through the holes at the flow rate of F1 and F2. Due to the difference in flow rate, the hole 1 is first emptied, and then air is passed through the hole 1 at the rate of A1. At this stage, different flow conditions will produce different results: (1) If Al < F1, the empty hole 1 will not be lower than the suction port of hole 2, and the hole 2 can be discharged without problems. (2) If Al > F1 and P > A1, because a large amount of gas flows through hole 1 at a flow rate of A1, the suction port on hole 2 is reduced. However, a high pumping rate can maintain a weak but easy-to-pump port on hole 2. And, the holes can still be discharged at the expense of longer discharge time and a large amount of high cost argon. (3) If Al > F1, and P and A1, the suction port on hole 2 is close to zero, and the discharge time of hole 2 is unbearable. The above example shows that the difference in emission rate will change the synthesis efficiency and yield. For disks with multiple reaction wells, if part of the wells are not completely discharged before the next synthesis reagent is added, the remaining waste will interfere with the synthesis chemistry, resulting in low or no yield. Referring to Figure 4, the importance of proper hole contouring is described. The upper plate in Figure 4 shows shallow reaction wells, in which CPG beads are easily washed away during reagent delivery. The mid-layer plate shows deep-narrow reaction pores, bubbles are easily formed therein, and are enclosed by surface tension. Bubbles are disturbed by reagent flow, which can lead to yield changes or synthesis failures. The lower plate of Figure 4 shows the appropriate profile for effectively maintaining CPG beads to deliver reagents that do not contain air bubbles. The appropriate inner diameter of the reaction hole is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 public attenuation ^ 1232867 A7 B7) for this paper size. 5. Description of the invention (11) High synthesis quality is very important. A smaller inner diameter helps reduce Consumption of reagents, but also easy to produce bubbles due to surface tension. The smooth surface of the tip of many commercially available filters is good enough to avoid the formation of bubbles. The decision of the appropriate pore profile is illustrated in Figure 7. In the prototype device, 'all reagents are packed in Argon-pressurized glass bottles are transported through a tube with an inner diameter of 0.03 inches or less. Reagents are sent from the bottle to a small solenoid valve (LFVX 050884A from Lee) using iron fluoride. Long tube. The volume of the hole of the conveying line from the bottle to the valve to the injection nozzle can be kept to a minimum by using the shortest possible tube length. The injection volume of each reagent is made by each solenoid valve and the time k. A solenoid valve drive circuit with fast response time is necessary to ensure a low injection volume. A 384-well disk requires a lower injection volume than a well disk. Excess reagents may not be questionable for yield, especially the coupling borrow section. Coupling stage The three components (that is, oral support, imine phosphate, and tetrazole (the activator of the coupling reaction need to be completely mixed, and can be effectively coupled. Excessive reagents will cause proper mixing and use the same amount of liquid.) The present invention People have inspected the injection characteristics and found that the total volume needs to be less than 15 microliters, which can effectively combine the ancient winter and Japanese people. This will make the imine phosphate volume less than 5 microliters. The injection nozzle is provided with 2G dyne / cm low Teflon cutting Teflon tube. The nozzle needs to use a low surface tension material to minimize the residual residual amount accumulated on the mouth of the nozzle after each shot. Spray angle big end (other-factor. Μ tip is not deburred after cutting, it will cause the reagent to accumulate at the tip of the nozzle. To avoid this, pinch the tip: Remove the burr by heating the tip of the Teflon tube. After deburring, end

1232867 A7 B7 V. Description of the invention (12 The spraying angle is large and the surface is smooth and the shape makes the reagent accumulated during the injection of the reagent is # I ^ 上 凡 1 is small, so it provides a consistent injection volume, and the distance between the reaction holes Cold dyeing with each other is the smallest. Furthermore, the injection degree of the cylinder is also very important for the success rate of the synthesis. The shot, the speed, the less the accumulation at the tip of a spray angle. On the other hand, the more the injection speed, the more CPG beads will be made. Out of the reaction hole. Therefore, it is necessary to determine the optimal injection speed to achieve a balance between the accumulation of reagents at the tip of the nozzle and the splashing of CPG beads. Take the best injection speed (selection is shown in Figure 5. The CPG beads are from the money) The tendency is directly proportional to the injection speed. The injection speed is determined by the injection pressure p and the inner diameter of the nozzle. The lower the injection speed, the lower the CPG beads, but the less the reagent consumption accumulates, the "reagent will stay at the nozzle tip until the reagent grows Until the surface tension can no longer support the specific size of the droplet. When the injection speed increases, the coefficient of variation (CV) curve of the injection volume reaches a minimum, but therefore the CPG bead splash height increases. Deeper reaction holes can be used to retain the cpG beads from the money and avoid punching out of the holes. 'Deep reaction holes' have another effect on minimizing the change in synthetic yield. This is due to the bead of the heart. The particles (although staying inside the reaction well) are tightly attached to the inner wall of the reaction well, and before the next synthesis step, these CPG beads need to be washed to the bottom of the well to ensure proper reaction efficiency. The higher the splash height It means that a larger washing volume is required to make these spheroids beads: This effect causes a large consumption of reagents, a longer reagent delivery time, and a longer discharge time. These factors need to be considered to determine the appropriate injection Figure 6 shows the increased splashing cpG beads when the injection speed is increased from the edge of the river _ reference standard half (CNS) A4 specifications (2ΐ〇χ297, "1232867 A7 -----_______________ B7 V. Description of the invention (C " ------ The volume of CH3CN required to go down. According to this, the waste discharge time and total synthesis time will be increased. In order to determine the optimal injection speed, the cv of the f-in volume is measured as a function of the injection speed. Because of the injection speed For many parameters (such as injection pressure, ID, and reagent viscosity), but for each reagent it is necessary to choose a small acceptable injection speed, and the minimum injection pressure may vary with different reagents. The overall optimal injection speed for all reagents is then selected by The acceptable injection pressure for all reagents is determined. As an example, Figure 5 demonstrates that the cv of the trichloroacetic acid (TCA) injection volume is measured as a function of the injection pressure. In Figure 5, the cv of the injection volume suddenly increased to below The cumulative pressure of the reagent. The cv curve conforms to the following exponential function: y = A * exp [. (Xx〇) / t] Formula! Where y is the injection volume of cv 4 A = 2.2, amplitude xo = 1, branch t = 0.53, the attenuation constant of the curve. Therefore, in order to achieve a CV of 0.1, the injection pressure is calculated as (2.53 * t) + x〇 ~ = 2.5 (psi). Set this value to a. The injection pressure a is the optimal injection pressure at which the TCA reaches the injection volume CV of 0.1. Similarly, the optimal injection pressure and overall injection speed of each reagent can be measured. Once the injection pressure is measured, the reaction hole depth can be measured as follows. CPG bead splatter height is measured using the required injection pressure. Example The paper size is applicable to Chinese National Standard (CNS) A4 specification (210 X 297 public form ^ 1232867 A7) Note (“) ~ ----- In the TCA system, the injection pressure is the main input, as shown in Figure 5. , And the average subtracted height is measured by repeating several injections. At 4.5 PS1 injection pressure, the average low output height obtained at 99% confidence is 48 ± 16 mm. In this example, the depth of the reaction hole needs to be at least 4.8 Mm + 16 mm = 64 mm. The 'easy-to-use' program in the prototype device was written in Visual Basic (Microsoft). Using 纟 to enter "Synthetic parameters are $, the program will calculate and issue movement instructions for the translation stage, and for the solenoid The valve issues an injection instruction. The user provides the sequence information in the simple building case. The computer program reads the information and performs the synthesis process in a fully automatic manner. When the 384 reaction wells are not fully used in operation, the program will skip the unused wells The reagents are released to save reagents. The user can easily add synthetic parameters 'such as reagent injection volume, reaction time, and modified base agents'. These parameters can be stored in the plan for subsequent use.

I Example 12 The inventors performed the 384 oligonucleotide & synthesis in different sequences and lengths using the prototype device described above. Synthetic support

Research or Chemgenes)) is suspended in chloroform / dibromomethane at a ratio of 1.1 (v / v) (ratio of 1.1 (v / v)) before being pipetted to the reaction wells. Drain the solvent. The imine wall acid salt (Chemg or class is in anhydrous acetonitrile (Aldrich, 0.001% water content) and the bottle containing the imine phosphate is connected to the synthesizer. The synthesizer room is first synthesized. Rinse with nitrogen for 30 minutes. The imine phosphate synthesis chemical contains four steps, namely deblocking, coupling, capping and oxidation. The synthesis parameters in this example are as follows:

1232867 A7 B7 V. Description of the invention (15) The tongue 'is filled with 40 microliters of TCA into each well, and then washed with 10 microliters of cH3CN. For the coupling stage, to ensure the highest achievable coupling efficiency, the coupling reaction was performed twice with 4 microliters of imine phosphate and 8 microliters of tetrazole each time. Each reaction was performed for 60 seconds before the reagent was discharged. A total of 8 microliters of imine phosphate and 16 microliters of activator were injected into each reaction well, followed by injection of $ microliters of end caps into each well. The reaction was performed for 20 seconds before the reagent was discharged. Then 5 microliters of oxidant was sent to each well. The reaction was performed for 30 seconds before the reagent was discharged. Due to the diameter of the reaction well (about 2.2mm as mentioned above), 5 microliters of reagent is enough to cover all beads and complete the reaction in each stage. After the oxidation stage, the reaction wells were washed with CNSCN, and the reaction was recombined from the deblocking stage. After the synthesis is completed, the reaction wells are thoroughly washed with acetonitrile (CH3CN) to remove the residual reagents in the reaction wells. Oligonucleotide was separated from the solid support into each well by injecting 60 microliters of base, solution (32% ammonium hydroxide or 40% methylamine, depending on the CPG beads used). The reaction is performed at room temperature for 5 to 60 minutes. The reaction plate assembly was mounted on a polypropylene collection tray, and the two trays were placed on a centrifuge to precipitate a crude product. The collection dish was then sealed and incubated for 8 hours at 55 to remove the protecting groups on the oligonucleotide molecule. The tray was cooled to room temperature again before the seal was removed. The crude product is dried in a vacuum chamber, redissolved in deionized water and set aside. Oligonucleotides can be dried in another way. The n-butanol extraction shows that it can produce oligonucleotides with sufficient quality for PCR, sequencing, etc. The tray accessories can be reused several times without degrading the quality of the synthetic product. The reaction plate accessories used were first immersed in acetone and shaken with ultrasound for 30 minutes. Each well is in order of 400 microliters of acetonitrile, 60 microliters of deionized water, acetonitrile, and the size of this paper. Applicable to China National Standard (CNS) A4 specifications (210 X 297 public compound j) 8 1232867 A7 B7 V. Description of the invention (16) Washed with 40% methylamine. The UV absorbance of the eluate of each well measured at 2 60 nm is about 0.01, which is about the same as that of the solvent. Therefore, the above process can be used to recover the reaction plate assembly. To measure the purity of synthetic oligonucleotides, high performance liquid chromatography (HPLC) was performed on a reversed C8 column. The HPLC system includes two Shimadzu LC-6A pumps, SCL-6A for controlling the pump, and a photodiode array as a detector. A personal computer was used to control the HPLC system and a chromatogram was obtained. Real-time monitoring and analysis of UV-Vis absorption spectra to help identify spikes. The mobile phase separated by HPLC consisted of reagent A 0.1M triethylacetic acid (TEAA) and reagent B: acetonitrile. The eluent composition of Reagent B was 8%, 20%, and 40% at 0, 24, and 34 minutes, respectively. The full-length oligonucleotide product dissolves in about 30 minutes, and the unqualified sequence dissolves in 14-18 minutes. To measure the synthetic yield, the oligonucleotide synthesized in the 384-well format was moved to four 96-well plates (Corning Coaster No. 3635), and the absorbance of each oligonucleotide was measured by a disk reader ( SpectraMax Plus, Molecular Device Corporation). The absorption spectrum of 200 nm to 400 nm was measured for each synthesized product. The final product amount was evaluated using the absorbances of 260 nm, 280 nm, and 320 nm. The results showed that each well in the dish contained oligonucleotides. No CPG bead loss occurred during the synthesis, and no synthesis failure was found. The actual yield of 384 oligonucleotide synthesized at 5 nmole nominal specifications is about 6 nmole. The coefficient of variation (CV) of the hole-to-hole yield change was measured to be 23%. When a 96-well reaction disk was loaded on the same device, a smaller yield of CV was found to be 15%. By comparison, the CV of the oligonucleotide synthesized in the 96-well format by previous techniques is 23% to 25%. These findings first explained that the density of the reaction pores was increased by a factor of four, that is, the paper size of the 96-well sterilization paper was adapted to the Chinese National Standard (CNS) A4 specification (210X 297 public attack ^ 1232867 V. Description of the invention (17 Likou to 3 8 4-L -V 'Yesterday, °' without sacrificing yield variation is not entirely worthless. For the method described in the present invention, it is possible to achieve lower hole-to-hole yield differences than previous techniques. Mouth The yield rate of V is based on the ratio of the full-length product yield to the total product yield. When Tian uses cpG beads of melons, the average step yield of oligonucleotide of length An nucleic acid is calculated according to the following formula: # [ NF / NT] 1 / n Formula 2, Zhong Ding and Dao Ding are the yields of all and full-length products. The amount can be determined by reverse HPLC. In order to measure the yield of the step, the claw sequence oligonucleotide ,, 7: 5 mole to 20 nanomole synthesis. The total product was randomly selected from several wells. The relative yields of all and full-length sequences were determined by HPLC. Figure 8 shows several Ηρχχ chromatography of products of different wells. The average step yield of each well is calculated according to formula 2 and shown in each chromatogram. The step yield f is greater than 99 / ί > Achieved when the synthesis scale is not larger than 〇〇Νmol. The pore-to-pore difference in the step yield can be ignored. Since the device is optimal for the synthesis of a variety of small amounts of oligonucleotides, It is not surprising that the yield of the 20 nanomole synthesis step is low. The results prove that the synthesis parameters need to be optimized for different scale synthesis. Example 2 Increase the output of the synthesizer can use only one synthesis disk translation control system Multiple reagent delivery system. The 1536-channel (384x4) synthesizer uses a set of reagent storage tanks, a translation control system, four combination instrument accessories, and four sets of 384-channel synthesizers as described in Example 1. Solenoid valve and nozzle for reagent delivery. In order to increase 12-reagent synthesis process, additional paper size is applicable to Chinese National Standard (CNS) A4 specification (210X 297 public ^ j 1232867 A7 B7 V. Description of the invention (18) For the instrument accessories, 12 additional reagent delivery solenoid valves and 3 additional reagent distribution valves are required. Compared with previous technologies such as the AMOS system, one additional synthesis module requires about 100 additional solenoid valves. The invention increases significantly increase production more flexible on the synthesis of additional modules. This paper scales applicable Chinese National Standard (CNS) A4 size (210 X 297 male g

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Patent Application No. 88 • Replacement of the scope of interest (1994! Month) A8 B8 C8 D8 i 丨 m3 A device that operates in parallel or more oligonucleotides, the device includes: a reaction plate accessory 'which contains 3 8 4 or more reaction holes and supports the reaction Hole-bearing plate 'where the diameter of the tip of the reaction hole is 3.5 mm or less and the distance between each reaction hole is 4.5 mm or less; a vacuum accessory' which contains one or more vacuum pumps, where The vacuum accessory is connected to the carrier plate to fix the carrier plate; a mobile system 'which includes a computer-controlled reagent delivery system and one or more reagent nozzles', wherein the reagent nozzle is located above the reaction disk and the mobile system and the reaction disk Connect and move the reaction disc to collect the reagents injected into the reaction holes from the reagent nozzles; a reagent valve accessory, which includes one or more solenoid valves and solenoid valve drive circuits, wherein the solenoid valve drive circuit is connected to the solenoid Tube valve and the moving system; one or more reagent storage tanks and tubes, wherein the tube is connected to the reagent storage tank, the solenoid valve and the reagent nozzle, and the reagent is delivered from the reagent storage tank to the spiral tube by the tube And said reagent nozzle; and a computer system, comprising software for controlling the controlled oligonucleotide synthesis procedures diclofenac oligonucleotide Chang acid and reads the results. 2. The device according to the scope of the patent application, wherein the reaction hole is composed of a crown plate, a medium to maintain a synthetic support, and a base plate, wherein the crown plate includes a cone with a diameter of about 2 mm at the bottom and a depth of about 15 mm. Shaped hole. 3. The device according to item 2 of the scope of patent application, wherein the bubble point of the medium for maintaining synthetic receipts is greater than the pressure difference of the vacuum fittings. 69922-940131.DOC This paper size is applicable to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1232867 A8 B8 C8 D8 Patent application scope 4. If the device of the second scope of the patent application is applied, the synthetic support is Controlled Pore Glass (CPG). 5. The device according to item 丨 of the patent application range, wherein the rate of the vacuum pump is greater than the gas flow rate through the medium. 6. The device as claimed in claim 1 in which the reagent nozzle has a low surface tension. 7. The device according to item 1 of the patent application range, wherein the reagent nozzle is a blunt-cut Teflon tube having a low surface tension of 20 dyne / cm. 8. For the device in the scope of the patent application, the reagent ejection speed of the reagent nozzle must minimize the accumulation of reagents at the mouth of the nozzle and the spillage of synthetic support. 9. The device according to item 1 of the scope of patent application, wherein the ejection volume of the reagent is about 2 microliters. '10 · —A method for synthesizing hundreds or more oligonucleotides using a device such as the one described in the patent application, wherein the method includes the following steps: The computer program of the device controls the synthesis of oligonucleotides , Introducing a synthetic support, a nucleotide reactant, and a synthetic reaction reagent into the reaction wells of the device; and deblocking, coupling, capping, and oxidizing the nuclear fenamic acid reactant to synthesize nucleate. 11_ The method of claim 10 in the scope of patent application, wherein the synthetic beat substance is a controlled porosity glass (c P G). 12. The method according to item i 0 of the patent application range, wherein the synthetic reaction reagent is imine phosphate. 69922-940131.DOC · 2 · This paper size is applicable to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 8 8 8 8 AB c D 1232867 6. Scope of patent application 13. If the scope of patent application is the 10th item The method, wherein the optimal reagent injection pressure parameter required for the synthetic oligonucleotide reaction is obtained by testing the device such as the scope of patent application No. 3, 5 or 8. 69922-940131.DOC ~ 3 ~ This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)