JP2002536158A - Systems, methods and computer program products for automatic delivery of fluids or gases - Google Patents

Abstract

(57) Abstract: Systems, methods, and computer program products for automatic delivery of fluids or gases. The present invention includes a motor drive system coupled to a rotary valve having a plurality of inlet and outlet ports. A plurality of vessels containing fluid and / or gas are fluidly connected to an inlet port of the rotary valve. A distribution valve is provided that controls the flow of fluid and / or gas from each container to an inlet port on the rotary valve. The processor controls the distribution valve to allow or restrict the flow of fluid and / or gas to the inlet port, and controls the movement and alignment of the motor, thereby controlling the rotation of the rotary valve I do.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to fluid or gas delivery systems, and more particularly, to multi-port fluid or gas delivery systems for use in automated chemical processing equipment. More specifically, the present invention relates to automated chemical synthesis of oligonucleotides or peptides, and particularly to devices and methods for performing the synthesis of multiple different oligonucleotides or peptides in a simultaneous manner.

Related Art Many different types of processes require the delivery of fluids and / or gases at different intervals and at different volumes. For example, in many chemical reactions (eg, chemical synthesis), reactants are delivered to a reaction chamber in a predetermined amount and reacted, and then are discharged from the chamber. Examples of such chemical processes include: oligonucleotide synthesis (including derivatized and / or labeled nucleotides), peptide synthesis (derivatized amino acids and / or
Or containing labeled amino acids), protein synthesis, ordered sequential biopolymer synthesis (eg, conventional DNA, antisense DNA, RNA, peptides, aptamers, diversomers, and polysaccharides), and DNA or biomolecules Purification.

[0003] With respect to oligonucleotide synthesis, the objectives of the Human Genome project have resulted in current and new technologies that require dramatically increased numbers of oligonucleotides. Such techniques include: forward and reverse primers for the polymerase chain reaction (PCR), primers that work for DNA sequencing, linkers for molecular cloning. , Short oligonucleotide for DNA-DNA hybridization, polymorphism mapping, sequence tag site (STS)
Mapping, radiation hybrid mapping, allele specificity mechanism (ASO),
And more recently, detection of mutations using arrays of oligonucleotides on flat surfaces (CHIP arrays), where a single array can contain hundreds to thousands of oligonucleotides.

[0004] Existing commercial equipment for producing oligonucleotides is severely limited by five major factors: (1) the scale of synthesis, (2) the number of oligonucleotides that can be purified at one time. Number, (3) quality of standard synthesis, (4) consumption of reagents, and (4) absence of communication software compatible with high-throughput processing.

[0005] Typically, any of the biological techniques described above have trace amounts of (5
~ 50 picomoles) using oligonucleotides, but existing commercial instruments
Produces 1,000 to 10,000 times more than needed. This translates to higher costs for producing the oligonucleotide. This is because the reagents used in this synthesis are expensive. This cost is conveyed to the end user performing the assay and can be cost prohibitively expensive, which forces researchers to search for alternative techniques, which causes significant delays in time and results. obtain.

[0006] Various solid phase oligonucleotide synthesis techniques are known to those skilled in the art. Such techniques include phosphoramidites, phosphotriesters, phosphodiesters,
Phosphite and H-phosphorylation methods, etc., each of which is generally
It is known in the field of molecular biology. For example, the β-cyanoethyl phosphoramidite method is described in Caruzzers et al.
for Preparing Ploycleotides, U.S. Patent No. 4,458,066, which is incorporated herein by reference.

An oligonucleotide is a macromolecule comprising a sequence of two or more nucleotides,
Each of these nucleotides contains a sugar and a base. Each nucleotide is separated from adjacent nucleotides by an internucleotide linkage, which effectively serves to link the nucleotides together. One process for oligonucleotide synthesis can be separated into four major segments, all of which require the addition of a single nucleotide to nucleotides immobilized on a solid support. Oligonucleotides can be constructed by multiple repetitions of these four segments to obtain the desired length. Similarly, additional or other chemicals or reagents can be added to the process to produce derivatized oligonucleotides, including derivatized oligonucleotides and / or labeled nucleotides.

[0008] These four segments are detritylation, coupling, capping,
And oxidation. Typically, 3 'nucleotides are immobilized on a solid support.
During detritylation, a deprotecting agent is added to remove a protecting group called dimethoxytrityl (DMT) from the 5 'end of this immobilized nucleotide, thus removing 5
'Deprotect' or expose the hydroxyl group. As a result, the last nucleotide of the sequence has one hydroxyl, which readily accepts the next phosphoramidite.

[0009] In the coupling segment, an excess of the desired nucleotide (the second nucleotide identified in the oligonucleotide sequence), which has been reacted with a mild acid to activate the 3 'phosphate, is added. The 3 'phosphate of the second nucleotide binds to the hydroxyl oxygen of the last nucleotide of the sequence, thus providing a support-bound nucleotide. After the support-bound nucleotides have been formed, the excess nucleotides are drained from the container by a washing solution such as, for example, acetonitrile (ACN). This results in the binding of the second nucleotide to the first nucleotide.

[0010] The capping segment reacts the uncoupled nucleotides to inactivate their 5 'end, thus preventing them from participating in subsequent coupling reactions. In particular, a capping agent is added to prevent any unprotected hydroxyl from reacting with the phosphoramidite introduced at a later stage. A wash solution such as ACN is re-introduced to drain the capping agent.

[0011] An oxidized segment is then applied to stabilize internucleotide linkages between the coupled bases. In one embodiment, an oxidizing agent is added to convert trivalent phosphorus to pentavalent. The oxidant is then drained off with the cleaning solution.

[0012] All four of these segments are ligated together to create one cycle, where one cycle of synthesis results in the addition of one nucleotide to the nucleotides attached to the solid support. Upon completion of the synthesis, the synthesized oligonucleotide is removed from the solid support by treatment with a strong base (eg, ammonium hydroxide).

[0013] A number of different bases can be used to form an oligonucleotide. The four most common bases are adenine, cytosine, guanine, and thymine (ie, A, C, G, and T, respectively). However, derivatized nucleotides or labeled nucleotides can be used in the process for making various oligonucleotides. Internucleotide linkages are most commonly phosphates, which are substituted at non-bridging oxygen atoms by various substituents (most commonly sulfur or alkyl, ester, or amide groups). Can be done.

The synthesis of peptides and proteins involves many of the same steps as oligonucleotide synthesis. In peptide synthesis, the deprotected amino terminus of the support-bound amino acid reacts with the activated carboxyl terminus of the introduced amino acid, resulting in a peptide bond. The process of deprotecting the amino terminus, activating the carboxyl terminus, and performing the coupling reaction with the next amino acid is repeated until the desired amino acid sequence is completed.

A peptide is at least two amino acids or derivatized amino acids (eg,
Amino acids and amino acid derivatives, including: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, Arginine, histidine, and derivatives thereof). Such amino acids and derivatives are described in Lehninger, Biochemistry, Worth.
Publishers, New York, NY and Stryer, Bio
chemistry, 3rd edition, WH Freeman and Co. , New
It can be found in York, NY.

The peptide synthesis process is based on the point that during solid phase synthesis, the peptide chain is built up one amino acid at a time with the first amino acid attached to the support, and the amino acids added one by one Is similar to the oligonucleotide synthesis process described above, in that the peptide chain remains until completed. During the process of adding additional amino acids, many of the same segments as described above must be performed (ie, deblocking, coupling (combined with activation), and capping).

Upon deblocking, the temporary α-amino protecting group is removed using a reactant. The next protected amino acid is then coupled to the existing deblocked amino acid. Once the coupling is complete, excess amino acid solution and reaction by-products are removed by washing. Once the peptide is assembled, it can be cleaved from the support and any side chain protecting groups can be removed. Then
The peptide may be used without further isolation or may be purified before use.

[0018] These and other methods for producing oligonucleotides, peptides, proteins, etc. are time consuming and the materials used are expensive and require specific handling and disposal after use. Need.

It is desirable to increase the number of oligonucleotides, peptides, and proteins that can be produced at one time, and to do it efficiently. However, large-scale synthesis raises some concerns. On the one hand, a high degree of uniformity is desired. On the other hand, competitive concerns affect the efficient use of materials. For example, in oligonucleotide synthesis, efficient use of phosphoramidites and ACN is particularly desirable. An excess of phosphoramidite is required to ensure that as much of the resulting oligonucleotide reacts with the newly introduced phosphoramidite, but the phosphoramidite introduced into the vessel is The amount of amidite must not be too large and wasteful. It is also desirable to reduce the amount of ACN used, while still excreting or at least diluting the remaining phosphoramidite as much as possible. If the evacuation is inadequate, the phosphoramidites remaining in the vessel or in the various conduits leading to the reaction chamber may create a non-uniform array.

Furthermore, the efficiency can be increased by automation as the number of synthesis steps is repeated. For example, in automated nucleic acid synthesis equipment, various steps are performed by a reagent delivery system, which distributes a number of chemical reagents into a synthesis reaction chamber in a predetermined order in a cycle. Typically, the present generation of automated DNA sequential synthesis involves placing a derivatized solid support, such as a controlled pore glass (CPG), into individual reaction chambers to provide a stable anchor; Then, solid-phase synthesis is started. Using a series of complex valve arrangements and pumps connected to the reaction chamber, selected appropriate reagents are sequentially filtered through the chamber in a predetermined manner. Contact of the reagents with the polymer units pre-fixed to the CPG, which is held and supported in the chamber by the sample-supporting porous frit, triggers a reaction, which results in sequential growth there. In any case,
It is important to note that for the purposes herein, reagents are delivered to the reaction chamber via a number of valves.

While each reaction chamber of such an assembly is effective for rapid mass production of a sequence defined oligonucleotide population, current assemblies are limited. These reaction chambers are arranged in an automated apparatus, so that the chemicals are introduced into the reaction chamber in an automated manner, sequentially and in appropriate amounts.
It can be added. Currently known automated synthesizers can produce only a few oligonucleotides at a time, and are limited by the number of reaction chambers loaded into the machine. The number of reaction chambers is practically limited by the increased complexity of piping and valve placement networks as the number of reaction chambers increases. This is because currently known synthesizers provide a tightly plumbed network from several reagent supply reservoirs to each reaction chamber. The increased reaction chamber capacity is limited due to physical limitations of the valve arrangement. In addition, conventional synthesizers are generally not assembled to be integrated with automation or other robotic laboratory equipment, and must be manually loaded and unloaded with each individual reaction chamber by an operator. Rather, this increases the opportunity for human error.

A more important limitation of the synthesis process is that all reagents are injected through a common manifold channel. Thus, only one reagent or a combination thereof may be dispensed to the selected reaction chamber at the same time. Further, for each independent synthesis or reaction, the common manifold passages and associated valves must be flushed with a cleaning agent so that the remaining phosphoramidite or deblocking agent does not undesirably accumulate in the reaction chamber. . This approach is time consuming and increases operating costs.

Furthermore, the efficiency of the synthesis depends in large part on the integrity of the reagent. In fact, the effectiveness of the synthesis process is very sensitive to the purity of the reagent. Cross-contamination between different reagents often adversely affects the chemical integrity of the reagents, and thus the effectiveness of controlled chemical reactions involving the reagents. One cause of cross-contamination is valves, especially multi-port valves, which choose to deliver between different reagents. The valve has an unavoidable dead volume associated with switching between reagents. Thus, a design consideration for a fluid delivery system designed to handle several types of fluids in one system is the reduction of cross-contamination between the fluids. For this reason, previous instrument designs that required absolute control of port-to-port contamination and random selection of chemical reagents have made the use of multiport valves difficult to prevent such contamination. Avoided.

Purification of DNA or biomolecules involves flushing reagents through a reaction chamber for purification at the same time. In this application, it is preferred that reagent cross-contamination is not as great as a concern, but that large and / or multiple sample purification can be performed efficiently. Conventional purification systems use a robot to empty the contents of a reagent pipette into a container containing the substance to be purified. The reagent is then evacuated from the container using reduced pressure. Although purification using these systems can be largely performed with little human intervention, the overall throughput of these conventional systems is limited. Because the robot
This is because each reagent must be separately added to a plurality of containers. Fluid and / or
Or quickly and efficiently deliver gas to the reaction chamber in an automated manner,
It is preferable to have a fluid and / or gas delivery system that can increase the overall throughput.

SUMMARY OF THE INVENTION The present invention is designed to deliver fluids and gases to a container, tube, well or chamber (eg, a reaction chamber containing a solid support) for various purposes.
For example, using the present invention, fluids and / or gases can be delivered to a reaction chamber for: oligonucleotide synthesis (including derivatized and / or labeled nucleotides), protein synthesis (derivatized amino acids and And / or containing labeled amino acids), ordered synthesis of biopolymers (eg, ordinary DNA
, Antisense DNA, RNA, peptides, aptamers, diversomers, and polysaccharides), or purification of DNA or biomolecules. Further, as will be apparent to those skilled in the art, the present invention may be used to deliver fluids and / or gases in various other applications.

With respect to oligonucleotide synthesis, the present invention may be used to allow oligopolynucleotides to be synthesized in the picomolar to micromolar range. In addition, the system of the present invention provides for parallel processing, allows for the simultaneous synthesis of one oligonucleotide and 200 oligonucleotides at a time, and does not require post-synthesis purification, allowing for many biological assays. Direct usage in is possible,
Produces high quality oligonucleotides. The devices of the present invention can be adapted or reformatted to increase the number of different oligonucleotides made at one time.

An advantage of the present invention is that it results in lower costs per oligonucleotide or peptide due to reduced scale and waste of reagents, as well as the need to produce such oligonucleotides or peptides. To reduce the time taken. Further, the present invention allows for active, real-time quality control of the synthesis process with an integrated power monitoring system.

The entire software control system allows incoming customer orders to be automatically transferred to the host computer. This host computer controls the entire mechanical process and stores and analyzes the output data for display on a graphical user interface.

The advantages noted for oligonucleotide synthesis are also recognized when performing other applications using the invention, such as peptide synthesis, polypolysaccharide synthesis, DNA purification or RNA purification. . These advantages include running multiple reactions at the same time, lower costs due to less waste of reagents, real-time quality control, reduced contamination, and shorter cycle times. High throughput and automatic control.

The present invention provides a system for fluid or gas delivery that uses relatively little reagent material for equivalent yields and eliminates the need to flush the fluid pathway. The passages of the present invention are extremely short and smooth, have little or no measurable dead volume and / or backflow, and are designed to premix chemicals when needed. The present invention can deliver the exact amount of reagents (up to a fraction of a milliliter) to the reaction chamber or solid support in the exact order without spilling any waste, thereby saving time and reagents I can do it. In addition, automation of this process reduces labor costs in operation, provides excellent process and quality control, and enables the production of significant numbers of products or reactants daily.

The system of the present invention comprises a container for storing starting reagents, chemicals, or other fluids for the reaction. These vessels are preferably pressurized with an inert gas. Each container is in fluid communication with its distribution valve. Implementation of the present invention
It is expected to include the simultaneous synthesis of 1 to 200 or more oligonucleotides, peptides, and the like. Thus, this vessel can be used to supply more than one reaction chamber, and preferably more reaction chambers. Thus, the container is preferably connected to the manifold via a dispensing valve on the container such that chemicals can be simultaneously supplied to more than one reaction chamber, and preferably to many reaction chambers. I have.

A rotary valve assembly is preferably coupled to the manifold via tubing, but the rotary valve can be directly connected to a container containing starting reagents, chemicals, and the like. The positioning of the valve rotor within the rotary valve assembly is controlled by a computer controlled motor drive system. The reaction chamber is preferably connected to the rotary valve. An output monitor is located around the tubing after the reaction chamber to monitor waste. This is because waste liquid exits the reaction chamber. However, a monitor may monitor the process at any point or points in the system of the invention to monitor this process.
Can be deployed. Data from this monitor is fed back to the host computer.

[0033] The rotary valve assembly includes a stationary hollow body member having a plurality of inlet ports at a base thereof and a cylindrical valve rotor disposed within the body member. The valve rotor has at least one communication port that can be selectively positioned in alignment with a selected inlet port in the base of the body member. A passage in the valve rotor connects the communication port with the reaction chamber. An inner surface in the body member having a continuous smooth cylindrical surface and an outer surface of the valve rotor are provided.

A further feature of the rotary valve assembly is an O-ring, which can be located around each inlet port and held in a desired position by a recess in the base of the stationary housing. The O-ring may alternatively be located in the valve rotor. Slight pressure acts on the rotary body member, thereby uniformly compressing the O-ring at all points near its circumference in the axial direction, thus providing a sealed interstitial space . This O-ring assists in providing a functionally leak-tolerant valve. Other methods for providing a leak-proof valve functionally include using a valve rotor made of TEFLON® material, ceramic or glass. Such a design preferably does not involve an O-ring if used.

A further feature preferably comprises a check valve, which is arranged in a passage in the valve rotor such that the balls of the check valve alternate between a closed position and an open position. It has been incorporated. This check valve is
Adjacent to each inlet port is disposed in the passage to prevent backflow. Alternatively, the check valve may be located without the inlet port. As the valve rotor rotates through the position of the valve, the passage above the check valve is filled and / or flushed with fluid or gas. This check valve is
Minimizing backflow of fluid to the inlet reduces the entrainment ratio.

Another feature of the valve rotor of the present invention is that the volume and shape of its passages are designed to improve mixing of the fluid before entering the reaction chamber. Mixing is further achieved in a volute chamber, which is provided at the end of the passage and in front of the reaction chamber, which can be located at any point in the passage. The volume of this swirl chamber, for a particular chemical,
It can be adjusted by design so that complete mixing of the chemical occurs before entering the reaction chamber.

For example, in oligonucleotide synthesis, in one step of a typical synthesis cycle, phosphoramidites are selected and a small amount is pulsed into the passage of the valve rotor. This valve rotor then moves to the activator position,
A small amount of the activator is then pulsed into this passage. This system is
Alternating the phosphoramidite pulse and the activator pulse is such that the two fluids are mixed when the pulse is forced up or down the passage toward the reaction chamber. Continuous. When the fluids reach the swirl chamber, they are further mixed. This is because the conical contour of the swirl chamber causes these fluids to swirl around the swirl chamber before entering the reaction chamber. If chemical mixing is not desired, the piston flow (i.e., the flow of fluid such that little mixing occurs between several fluids moving through the passageway) can produce relatively long pulses of only a single fluid. It can be easily achieved by repeating delivery through the passage.

[0038] The motor drive system may include a stepper motor and an electronics package. The electronics package controls the drive circuit and can also monitor one or more position detectors. Alternatively, the motor drive system can include a servo motor, which is
An encoder for determining the angular position of the motor and / or a resolver for determining the exact position of the motor.

The output monitor of the present invention is located in the reaction chamber in this system to monitor waste liquid from the reaction chamber. Similar monitors can also be located at other locations in the system. The output monitor preferably includes an optical sensor and detector assembly, which is located on the opposite side of a portion of the tubing, which guides from the reaction chamber to waste disposal. This optical sensor preferably comprises two LEDs of different wavelengths. The output monitor further includes an LED driver for controlling illumination of the LED. Light from the LED is transmitted from the LD by a narrow slit located between the optical sensor and the tubing on one side, and a narrow slit located between the detector assembly and the tubing on the other side. It is possible to pass through the tubing to the detector. A signal processor processes the signal received by the detector assembly. The feedback loop sends this signal back to the host computer so that, if necessary, continuous monitoring of the effluent can be used to meet the instantaneous correction measures during the synthesis process. This monitor may also be useful for analyzing the type and quality of the product produced.

A level sensor on the reservoir can be provided to indicate to the host computer when the reservoir is empty or nearly empty. The LED is preferably located on a portion of the sidewall such that it is located within the curved portion of the sidewall. A detector is located on the opposite curved portion of the side wall, opposite the LED.

If liquid is present in the reservoir, light from the LED travels directly across the reservoir to the detector. However, if there is no liquid above the light from the LED, the light will be refracted. This is because the refractive index of this liquid is different from that of air (eg, without liquid). In this case, a relatively small amount of light from the LED reaches the detector. Thus, an output signal that one of the storage containers is empty or nearly empty is delivered from the detector to the host computer. This host computer may signal a user to refill an empty specific storage container, or may send a signal to the system to automatically refill storage containers from stock bottles.

This computer program controls the process of the invention using the following steps: input of the desired reaction (eg, the desired nucleotide sequence for oligonucleotide synthesis, the desired Receiving an amino acid sequence or a list of desired reagents for purification), an event matrix of events required to generate the desired sequence or reaction.
ix), rotating the valve rotor towards an objective based on the events in the event matrix, energizing the dispensing valve for a particular reagent for this event to deliver that reagent in the reaction chamber Performing a time delay on the dispensing valve, removing energy from the dispensing valve at the end of the time delay, and until the desired sequence is synthesized or the desired reaction is completed. Repeating these steps for each event in.

The present invention provides for relatively fast processing. Because the contamination level is very low, little or no flushing is required between valve rotor positions. Further, the present invention allows a chemical reaction or reaction to be controlled by a simple continuous control string from a computer. This saves time and uses relatively few reagents. This is because the amount of chemicals or reagents delivered to the rotary valve can be optimized such that only a desired minimum amount is used. Such valves are ideally suited for oligonucleotide and peptide synthesis. The reason for this is that minimal mixing results in high efficiency.

Although the present application focuses primarily on the use of the present invention for particular applications (eg, oligonucleotide synthesis), the fluid and gas delivery systems of the present invention may be used in various other processes. It will be clear to those skilled in the art. For example, the system of the invention can be used as a DNA or RNA purification system, as a biomolecule purification system (eg, for proteins, hormones, cofactors, etc.), or for biopolymer synthesis. Further, the system of the present invention can be used to generate nucleotide derivatives or labeled nucleotides or amino acid derivatives or labeled amino acids.

Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail below with reference to the accompanying drawings. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the following drawings and description of the invention.

(Detailed Description of Preferred Embodiment) (1. Overall System) FIG. 1 shows the overall configuration of a system 100 according to an embodiment of the present invention. With reference to FIG. 1, compressed gas cylinder 104 provides an inert gas under pressure through various paths of system 100. In one embodiment, the inert gas is helium. In an alternative embodiment, the inert gas may be argon or nitrogen.

The path of the system 100 is pressurized to facilitate the flow of liquid through the system 100. This inert gas is also used to keep the chemical dry. In one embodiment, the pressure of system 100 is maintained at about 7.5 psi on all paths. However, in some cases, depending on the density and / or volume of the supplied fluid, it may be advantageous to increase or decrease the pressure in some or all of the paths.

In one embodiment, the pressure in the passage carrying the cleaning solution (eg, ACN) can be greater than 7.5 psi. This is because a larger amount of washing solution is used in the synthesis process than other reagents are used. Increasing the pressure in this path reduces the time it takes to deliver the cleaning solution to the reaction chamber. On the other hand, phosphoramidites or activators (TET)
) Can be below 7.5 psi, preferably 1-4 psi, so that the amount of reagent delivered during a given time interval is reduced and this amount can be better controlled. .

In one embodiment, a computer controlled regulator 108 may be used to monitor and maintain the pressure in the path that provides the various fluids needed for the synthesis. As shown in FIG. 1, the system 100 includes three regulators. However, it will be apparent to those skilled in the art that more or less regulators may be used as long as the gas flow rate is controllable.

In one embodiment, the computer controlled regulator 110 may also be used to monitor and maintain the pressure in the manifold 132, which may be used as described in further detail below. Distribute fluids and gases to various rotary assembly valves.

The gas distribution manifold 112 is used to distribute the inert gas from the gas cylinder 104 to a computer controlled regulator 108 and a computer controlled regulator 110. Computer controlled vent valve 116
Is used to backflush chemicals to their storage containers 120 and 124 when the system is not in use, or to fill working containers from storage containers (container The line for remote filling is not shown in FIG. 1). Although FIG. 1 shows three vent valves 116, it will be apparent to those skilled in the art that fewer or more vent valves may be used depending on the arrangement of the system. For example, each container can be connected to its own vent valve 116. Alternatively, a single vent valve 116
Can be used for all storage containers.

The system 100 uses a container 120 for phosphoramidites, but other chemicals or reagents can be used as needed. As shown in the embodiment of FIG. 1, the first four containers 120 contain adenine (A), guanine (G), cytosine (C), and thymine (T). In FIG. 1, the common names X, Y and Z
The remaining three containers 120, referred to as, are reserved for other phosphoramidites or other reagents.

The system 100 uses a container 124 for additional chemicals used during the synthesis process. In the embodiment shown in FIG. 1, the containers 124 contain reagents (eg, CAP A and CAP B, which are the capping reagents used in the capping segment) and bind at their 5 ′ ends. To inactivate unreacted nucleotide reactions. The container 124
It contains reagents (eg, OXID 1 and OXID 2, which are used during the oxidized segment) and stabilizes the internucleotide bond between the bound base and TCA. TCA is used during the detritylation segment to remove protecting groups from the 5 'end of the nucleotide. The chemical ACN contained in the container 124 is also a wash solution that can be used during application of other reagents to flush the system.
These particular chemicals are shown for illustration only. It will be apparent to those skilled in the art of oligonucleotide or peptide synthesis that different chemicals and different reagents may be used depending on the particular application and synthesis process utilized.

For example, other chemicals can be added to produce derivative or labeled nucleotides, and / or oligonucleotides containing such derivatives and / or labeled nucleotides. As used herein, "nucleotide" refers to a base-sugar-phosphate linkage. The nucleotide is
A monomer unit of a nucleic acid sequence (DNA or RNA). The term nucleotide is
Deoxyribonucleotide triphosphates (eg, dATP, dCTP, dITP,
dGTP, dTTP, or derivatives thereof). The term nucleotide as used herein also refers to dideoxyribonucleotide triphosphate (ddN
TP) and their derivatives and ribonucleotide triphosphates (eg, AT
P, UTP, GTP, CTP, or derivatives thereof). Examples of ddNTPs are ddATP, ddCTP, ddGTP, ddITP and ddTTP
But not limited thereto.

Examples of nucleotides and derivatives thereof that can be made using the present invention include dUTP, dATP, dTTP, dCTP, dGTP, dITP, 7-deaza-dGTP, α-thio-dATP, α-thio- dTTP, α-thio-dGTP,
α-thio-dCTP, ddUTP, ddATP, ddTTP, ddCTP, dd
Examples include, but are not limited to, GTP, ddITP, 7-deaza-ddGTP, α-thio-ddATP, α-thio-ddTTP, α-thio-ddGTP, α-thio-ddCTP or derivatives thereof. The nucleotides can be unlabeled or they can be radioisotopes (eg, ³ H, ¹⁴ C, ³² P or ³⁵ S), vitamins (eg, biotin), fluorescent moieties (eg, fluorescein, rhodamine) , Texas Red or Phycoerythrin), chemiluminescent labels, dioxygenin, bioluminescent labels, enzyme labels, and the like, and may be detectably labeled by conjugation thereto.

Each container 120 and 124 is connected to the gas supply 10 via a regulator 108.
4 is connected to compress, so that the chemical is pressurized in this container. Each container 120 and 124 is also fluidly connected to its own dispensing valve 128. For ease of illustration, only one distribution valve 128 is shown in FIG. However, each dashed line extending from containers 120 and 124 is intended to be connected to its own dispensing valve 128. In one embodiment,
The distribution valve 128 is a solenoid valve. The distribution valve 128 is preferably made of a TEFLON® material (EI Du Pont de Nemo).
(available from URs), which has been evaluated for use with the types of reagents typically used during the synthesis process. However, it will be apparent to one skilled in the art that the distribution valve 128 can be made using other materials, depending on the particular reaction or process being performed.

The practice of the present invention is intended to include the simultaneous synthesis of several oligonucleotides. In one embodiment, DNA and RNA synthesis (eg, oligonucleotide synthesis) can be performed simultaneously in eight separate rotary valve assemblies. In another embodiment, the system of the present invention currently comprises 1-20
It is configured to produce 0 oligonucleotides. In yet another embodiment, the system is configured to produce 96 oligonucleotides simultaneously. In these cases, containers 120 and 124 may be used to supply more than one rotary valve assembly 138 (as described below).

Thus, as shown in FIG. 1, each vessel 120 and 124 is fluidly interconnected via its distribution valve 128 to a manifold 132, thereby resulting in The phosphoramidite or chemical may be supplied to more than one rotary valve assembly at a time. In one embodiment, the manifold 132 is made of stainless steel (eg, Si
lcsteel® (Restek Corporation, Be
llfonte, available from PA)). This silica coating is used to inert the steel when used with reagents typically used during the synthesis process. It will be apparent to those skilled in the art that other materials may be used for the manifold 132, depending on the fluid and / or gas passing through the manifold for the particular reaction being performed.

In the embodiment shown, the dispensing valve 128 is computer controlled, and each of the pressurized chemicals is dispensed to the appropriate rotary valve assembly 138 via tubing 140 for distribution valve 128 itself. To the manifold 132. For each example, it is shown that the manifold 132 is connected to only one inlet port 146 of the rotary valve assembly 138. However, an outlet from the manifold 132 (shown in dashed lines in FIG. 1) supplies either another inlet port 146 of the rotary valve assembly 138 and / or an inlet port on a further rotary valve assembly (not shown). Intended to be. In one embodiment, the manifold 1
The outlet from 32 is fluidly connected to eight or more different rotary valve assemblies 138 (preferably 12, 24, 48, 96, 192, 384, etc.).

In one embodiment, tubing 140 is made of TEFLON® with an inner diameter of 0.031 inches and an outer diameter of 0.062 inches, although other types and sizes of tubing may be used. , Can be used. Tubing 140
Is preferably transparent, so that eluate measurements can be made directly through tubing. Manifold 132 distributes the chemicals evenly to each rotary valve assembly 138.

In the example of system 100, the synthesis process is performed with a wash solution (eg, ACN).
, All distribution valves 128 are turned off. Then AC
For those rotary valve assemblies 138 that require N, the valve rotor 136 is rotated to the ACN position (shown in FIGS. 4 and 5 below,
And will be discussed). Next, a distribution valve 128 connected to the ACN container 124
Is turned on and allows pressurized ACN to flow from the container 124 through the manifold 132. For those valve rotors 136 at the ACN location, the ACN flows through the valve rotor 136 and the reaction chamber 160. For these rotating rotors 136 in different positions, ACN does not flow through the rotating rotor 136. Thus, each valve rotor 136 is independent of each other.

Referring to FIG. 4, each rotary valve assembly 138 is connected to a motor drive system 142, as described in further detail below. One embodiment of the motor drive system 142 is shown in further detail in FIG. 5 and includes a stepper motor 144 and an electronic package 148.
In one embodiment, the stepper motor 144 is a Warner Elec
trick Motors and Controls Division, An
n Arbor MI (as model # KML061S02E), 2
It is a Superior SLO-SYN (registered trademark) stepper motor of 00 steps. Electronic package 148 controls the drive circuit (not shown in FIG. 1) and the zero position detector (not shown in FIG. 1). Shaft 152 extends from stepper motor 144. The shaft 152 may have an extension added at one end, so that indexing operations into the motor area (indexi)
ng), a square drive shaft (not shown) is added to ensure accurate and reproducible alignment of the valve rotor 136 with the inlet port 146 of the rotary valve assembly 138. Motor drive system 142 is described in further detail below with respect to FIG.

In another embodiment, motor drive system 142 may include a servo motor with electrodes. Such a motor drive system is more complex and costly, but the valve rotor 13 of the rotary valve assembly 138
The manner in which such a motor drive system for controlling the position of the motor 6 is constructed will be apparent to those skilled in the art.

The rotary valve assembly 138 includes a valve rotor 136 and an inlet port 146. The valve rotor 136 is fixedly attached to one end of the shaft 152 so that it can be rotated to align with the inlet port 146. Rotary valve assembly 138 is connected to reaction chamber 160. In one embodiment, control pore glass (CPG) 162 is located in reaction chamber 160. Such a reaction chamber is a Perceptive column, which is an ABI / Perceptive of Foste.
Available from r City, CA. Another such reaction chamber is the So
This is a DNA Synthesis Column available from the lid Phase Sciences of Novato, CA. This support can be made of any material or plastic, glass, agarose, metal, nitrocellulose,
Acrylamide, silica, nylon, cellulose, diazocellulose, modified polystyrene, polyvinyl chloride, polyethylene, dextran, polyvinyl fluoride, sepharose, polyacrylamide, latex, starch, polyvinyl toluene, polysaccharide, acrylic polymer, hydroxyapatite
It will be apparent to those skilled in the art that it can be a solid or semi-solid support made of a combination of materials, including apitetes). In particular, the support matrix is made from a resin (eg, polystyrene, polyacrylamide, acrylamide-impregnated silica or porous glass. The form of such a support can vary from beads, particles, filters, columns, and the like. In another aspect, the reaction chamber may be replaced with any tube, container, or other container for collecting a fluid or gas supplied through the system 100.

The output from reaction chamber 160 is connected to output monitor 164 for measurement of trityl and reagents in the eluate. The measurements made by the output monitor
Output to a host computer (not shown in FIG. 1) via line 166.

The waste diverter valve 168 is connected to the halogenated waste receptacle 17.
Control the separation of waste into zero and non-halogenated waste receptacles 172. Separation of waste is desirable because disposal costs for halogenated and non-halogenated waste are significantly different.

(II. Rotary Valve Assembly) FIG. 2 is a side view of the rotary valve assembly 138, the motor drive system 142, and the reaction chamber 160 according to an embodiment of the present invention. Referring to FIG. 2, tubing 140 is shown connected to inlet port 146 of rotary valve assembly 138. Only two inlet ports 146 are shown in FIG.
It is shown connected to the tubing 140 inside. However, in one embodiment of the invention, each of the inlet ports 146 is connected via tubing 140 to a corresponding reagent manifold 132.

A. Structural Assembly Referring to FIG. 3, a partial cross-sectional view of the rotary valve assembly 138 (taken along lines 3-3 in FIG. 2) is shown. Reaction chamber 160 is located on top of rotary valve assembly 138 and includes a support column (not shown) and a solid support (not shown). In one embodiment, the solid support is CPG. However, it will be apparent to one skilled in the art that, as discussed above, various reaction chambers or supports may be used depending on the particular application.

The rotary valve assembly 138 includes a first part 306 and a second part 308
Is formed. The first portion 306 is a cylindrical continuous smooth inner surface 31.
0 is a concave stationary body. In one embodiment, first portion 306 is made from stainless steel or aluminum. The material for this part is not covered. This is because, in this embodiment, there is no direct contact with the reagent. In one embodiment, second portion 308 is a stainless steel clad with silica (eg, Silcosteel®, which is
k Corporation, available from Bellefonte, PA). This silica coating, when used with reagents typically used during the synthesis process, is used to inert the steel. It will be apparent to those skilled in the art that other materials may be used for the second portion 308, depending on the fluid and / or gas passing through the manifold for the particular reaction to be performed.

The second part 308 is fixed by positioning the pin 312 on the first part 306 and forms part of a stationary structure. The second portion 308 includes a smooth upper surface 314 and an opening 316. Side wall 318 of second portion 308 around opening 316 includes a shaft bearing 358. Shaft 152 from stepper motor 144 extends through opening 316 and slides into recess 320 in valve rotor 136 to engage valve rotor 136.
fit). Bearings 358 are provided to facilitate rotation of shaft 152 within opening 316.

The second part 308 further has an inlet port 146. Two inlet ports 14
Although only 6 is visible in the cross-section of FIG. 3, in one embodiment, the second portion 308 includes 20 inlet ports 146, where 20 are provided to indicate the "off" position of the valve. One of the inlet ports 146 is not fluidly connected to a corresponding port of the valve rotor 136. It will be apparent to those skilled in the art that any number of inlet ports can be formed on the rotary valve to accommodate various fluids or gases.

As shown in FIG. 3, one of the inlet ports 146 is substantially vertical, and the other inlet port 146 is disposed in the second portion 308 at an angle to vertical. You. In one embodiment, the inlet ports 146 alternate in this manner, so that all of the inlet ports are matched with respect to the rotary valve assembly. In one embodiment, there are 20 inlet ports 146 around the top surface 314. The distal end of the inlet port 146 is connected to the upper surface 31 of the second portion 308.
4 so that they are all present in an annular passage with respect to the central axis of the rotary valve assembly.

The communication port 326 is formed on the lower surface 328 of the valve rotor 136. As the valve rotor 136 is rotated by the shaft 152 of the stepper motor 144, the communication port 326 may be aligned with the inlet port 146 and thereby be in fluid communication with the inlet port 146. In one embodiment, the valve rotor 136 is made of stainless steel coated with silica (eg, Silcost
eel®, which is available from Restek Corporation, B
(available from Elfonte, PA). This silica coating, when used with reagents typically used during the synthesis process, is used to make the steel inert. It will be apparent to those skilled in the art that other materials may be used for the valve rotor 136, depending on the fluid and / or gas passing through the manifold for the particular reaction to be performed. For example, as discussed above, in an alternative embodiment, the valve rotor 136 includes a TEF
It can be made of LON® material or ceramic or glass.

The upper surface 314 of the second portion 308 is provided with a recess 330 with respect to the distal end of the inlet port 146. In one embodiment, the O-ring 332 is located in the recess 330 and is connected to the communication port 326 or the lower surface 3 of the valve rotor 136.
Provide a seal around inlet port 146 to prevent 28 contamination. Recess 3
The 30 act to accurately position the O-rings 332 so that they are concentric with the inlet port 146 in the second portion 308. In addition, the recesses 330 prevent displacement of the O-ring 332 from their aligned position during rotation of the valve rotor 136. O-ring 332 assists in reducing entrainment of liquid flowing into the reaction chamber by sealing around inlet port 146 so that valve rotor 136 can move from one inlet port 146 to another. When rotating to 146, reagents from this inlet port do not mix with reagents from other inlet ports. However, it will be apparent to those skilled in the art that the present invention may be practiced without a corresponding recess 330 for the O-ring 332 and the inlet port 146, as discussed in further detail below.

The second portion 308 also includes a recess 334 in the upper surface 314 along a peripheral edge of the shaft 152 of the stepper motor 144. The second portion 308 further comprises a second recess 336 in the upper surface 314, which recess
Form a concentric circle around all six. O-rings 338 and 340 are sealed at recesses 334 and 336, respectively, to provide leak-proof
of) providing a valve; In one embodiment, O-rings 332, 338
And 340 are Kalrez® 3018 materials (perfluoroelastomer moieties, currently Dupont Dow Elastomers,
L. L. C. , Newark, Delaware).

The valve rotor 136 is located within the first portion 306 and is provided with a smooth cylindrical outer surface 342. In one embodiment, the valve rotor 13
6 is disposed within the first portion 306 such that its smooth outer surface 342 does not directly contact the inner surface 310 of the first portion 306. Valve rotor 13
6, an O-ring 332 into each of their respective recesses.
Slightly compress 338 and 340. As will be apparent, this results in compression of the O-rings 332, 338 and 340 around their peripheral edges. In a preferred embodiment of the present invention, the compression around the peripheral edge of each O-ring 332, 338 and 340 is uniform.

The tubing 140 is connected to the inlet port 146 and the tubing 140
By selectively rotating the valve rotor 136 until is aligned with the appropriate inlet port 146, fluid and / or gas is selectively delivered to the valve rotor 136. An alternative embodiment of the system 100 (where the distribution valve 128 is connected to the inlet port 146 via a distribution plate and manifold block) is shown in FIG.
-23 are described in detail below.

The communication port 326 of the valve rotor 136 is connected to the path 348. Path 348 extends through valve rotor 136 from communication port 326 to reaction chamber 160. In one embodiment, the volume of path 348 is a volume that retains at least twice the volume of the fluid in the smallest pulse of fluid delivered through inlet port 146 by the system. For example, in one embodiment, the volume of pathway 348 is between 10-1000 μL. In another embodiment, the volume of pathway 348 is between 50-500 μL. In yet another embodiment having the smallest pulse of 38 μL of fluid, the volume of path 348 is between 76-100 μL.

In another embodiment, the volume of channel 348 is at least half the volume of the reaction chamber. For example, in one embodiment, the ratio of the volume of the path 348 to the volume of the reaction chamber 160 is in the range of 2: 1 to 1: 1. in this way,
In this example, if the volume of the reaction chamber 160 is 100 ml, the path 34
8, the volume is between 50-100 ml. This helps to ensure proper mixing before the fluid reaches the reaction chamber 160.

The check valve 344 is disposed on the path 348. The ball of the check valve 344 alternately contacts the communication port 326 and the plug 345. Plug 34
5 is disposed on the check valve 344 so as to limit the range of movement of the ball of the check valve 344. In one embodiment, the check valve 344
Are made from TEFRON® material. However, the ball of the check valve 344 can be made from other materials so that the ball
It will be apparent to those skilled in the art that it can be made as close as possible to a substantially complete sphere and provide a good seat for the communication port 326. Examples of other materials include: steel, stainless steel, sapphire, ceramic, ruby or synthetic ruby.

In the embodiment of FIG. 3, O-rings 332, 338, and 340 are shown in FIG.
As shown at 0, a small gap 2004 space is created between the valve rotor 136 and the second portion 308, where the second portion 308 is rotated through different valve positions. Sometimes, it can be filled with a cleaning fluid, such as ACN, and / or can be washed away. A check valve 344 is provided to prevent fluid backflow from path 348 to this interstitial space 2004 or to inlet port 146.

An exploded view of the intersection between the communication port 326 and the inlet port 146 is shown in FIG. As shown in FIG. 20, the size of the communication port 326 below the check valve 344 is minimized to reduce contamination. Further, the area 2008 between the sides of the O-ring 332 is minimized to reduce the area where any backflow from the communication port 326 can collect.

Returning to FIG. 3, a path 348 connects the communication port 326 and the outlet port 346. Outlet port 346 is in fluid communication with reaction chamber 160. Fluid or gas flowing through communication port 326 passes through check valve 344 and travels through path 348 to swirl chamber 350. The swirl chamber 350 is conical in shape and is also designed to hold a fluid volume that is at least twice the fluid volume of the minimum pulse of fluid delivered by the system through the inlet port 146. In the embodiment shown in FIG. 3, vortex chamber 350 is at one end of path 348. However, swirl chamber 350 may be located anywhere in path 348.

As fluid moves through path 348 and through swirl chamber 350, the shape of swirl chamber 350 causes the fluid in valve rotor 136 to swirl, and thereby through outlet port 346 Mix fluids before leaving. Instead of a swirl chamber 350 for mixing the fluid in the valve rotor 136,
Or, in addition, it will be apparent to those skilled in the art that commercially available static mixers may also be used.

In another embodiment, a check valve 344 may be located at each of the inlet ports 146. In such an embodiment, the inlet port acts like an injector, where the check valve is spring-loaded and opens only when the communication port of the valve rotor is aligned with the check valve. In this embodiment, no substantial backflow of fluid enters the inlet port 146, but the backflow is
8 to the interstitial space 2004 between the valve rotor 136 and the second portion 308. In this embodiment, the inlet port configuration shown in FIG. 18 is used, and any gap space between the valve rotor 136 and the second portion 308 is minimized, as discussed in more detail below. Therefore, it is preferable to avoid the use of O-rings, thereby minimizing contamination.

The first portion 306 is further provided with an opening 352 on its upper end.
Outlet port 346 is located in opening 352 and is in fluid communication with passage 348 and reaction chamber 160. As shown in FIG.
6 has a recess 354 adjacent to the swirl chamber 350 and at the beginning of the outlet port 346. An O-ring 356 (similar to the O-ring described above) is located in the recess 354 and the valve rotor 1 in the first portion 306
Prevents fluid from leaking into the region above the tip of 36.

The thrust bearing assembly 360 is provided at the upper end of the valve rotor 136. The thrust bearing assembly 360 includes a thrust washer 362 and a thrust bearing 364. The thrust bearing assembly 360 further comprises a wave washer 366 above the tip of the thrust washer 362. The wave washer 366 has a wavy shape, so that the “wave” applies a downward pressure on the thrust washer 362. The thrust bearing 364 is then attached to the valve rotor 136 and O-rings 332, 338, and 3 to provide, for example, a leak-proof valve.
Provides a downward pressure on 40. It will be apparent to those skilled in the art that springs or other types of similar devices may be used to pre-load the O-rings so that they are in close contact.

(B. Port Layout) Referring to FIG. 4, a cross-sectional view taken along line 4-4 in FIG. 2 is shown. In particular, FIG. 4 shows a top view of the upper surface 314 of the second portion 308. This plan view illustrates an example of the placement of oligonucleotide synthesis reagents with respect to the inlet port 146 of the rotary valve assembly 138 according to an embodiment of the present invention. The particular arrangement of reagents as shown in FIG. 4 allows for solid phase synthesis, resulting in oligonucleotides of high purity and high yield. As will be appreciated, the number and arrangement of reagents can vary depending on the use of the present invention. For example, when used for peptide synthesis, or nucleic acid or protein purification, the configuration of the port layout can be determined based on the type and sequence of the reactions.

Starting from the home position 404 and operating clockwise, the four tetrazole (TET1, TET2, TET3 and TET4) positions 408, 420, 432
And 444 indicate that they are phosphoramidite (A, G, C, T) positions 412,
416, 424 and 428, respectively, and three additional (X, Y
, Z) spaced further adjacent to locations 436, 440 and 448, respectively. The X, Y and Z positions are provided to allow for various base modifications. For example, positions X, Y and Z can be used for mixed bases, labeled nucleotides, nucleotide derivatives, nucleotides to which reporter molecules are linked, or spacers (eg, carbon or polyethylene glycol spacers).
Positions X, Y, and Z may also be used for deoxyuracil, deoxyinosine, phosphorothioate, and other 5 ′ modifications (eg, phosphorylation, biotin, fluorescence, rhodamine, primary amine or fluorescent dye (Life
Technologies, Inc. , Rockville, MD)). Still further, the X, Y and Z positions are 5 ′ modifiers (eg, amino modifiers C3, C6, C12, amino modifier 5, amino modifier C6 TFA, phosphorylation or thiol modifier C6), 5 '
Or 3 'modifiers (e.g., thiol modifier C6S-S, spacer 9, spacer C3), sequence modifiers (e.g., amino modifier C6 dT, amino modifier C)
2 dT, biotin-dT, or carboxy-dT), a labeling reagent (eg, psoralen C2, biotin, fluorescein, acridine, BioTEG, cholesteryl-TEG or DNP-TEG), or a 3 ′ modifier (eg, amino modifier C3) CPG, amino modifier C7 CPG, thiol modifier C3 S-S C
PG, 3 ′ phosphate CPG, BioTEG CPG, fluorescein CPG or acridine CPG) (these are Glen Research, Sterlin).
g, currently available from VA).

[0090] Tetrazoles are mild acid activators for phosphoramidites. In use, the valve rotor 136 moves back and forth between the selected phosphoramidite inlet port to be added and its adjacent activator port. These two fluids are pulsed with a short, timed pulse
6 are alternately introduced into path 348. As a result, mixing of the phosphoramidite with the activator occurs in path 348 before the fluid reaches reaction chamber 160.

[0091] Continuing clockwise, the auxiliary reagents (CAP B, CAP A, OXID 1, OX
ID 2 and TCA) with two acetonitriles (ACN 1 and ACN)
2) Arranged at 456, 460, 464, 468, and 472 adjacent positions 452 and 476, respectively. CAP B port and CAP A port are ports for the two-part capping reagent used after the coupling phase. Since the capping reagent is unstable when mixed, it is stored in two parts. In this way, the ports of CAP A and CAP B are connected by two valve rotors.
Place them next to each other so that they can be easily moved between the two ports. In use, these two fluids are subjected to CAP before the fluids reach the reaction chamber 160.
Short and timed pulses are alternately introduced into the path 348 of the valve rotor 136 so that mixing of A and CAP B occurs in path 348.

Position 464 is a port for OXID 1 (oxidizing reagent), which is used after the capping phase and at the end of each addition of phosphoramidite.
Position 468 (labeled OXID 2) may be another port for an oxidizing reagent, or position 468 may be used as a spare port for another type of reagent (eg, a sulfurizing reagent). obtain. The TCA (deblocking or detritylating reagent) is located at location 472, close to home location 404. This is because the detritylation phase occurs just before each new phosphoramidite is added. The placement of ACN 1 and ACN 2 at positions 452 and 476, when used in a synthesis cycle, allows the ACN to quickly purge reagents when needed.

Finally, the GAS is provided at a location 480 adjacent to the home location 404. The gas can be any gas, preferably an inert gas, and more preferably a gas from the group consisting of helium, nitrogen or argon. The gas from position 480 is used to evacuate the valve rotor 136 and reagents from the reaction chamber 160 and dry the solid support in the reaction chamber 160. Specifically, by drying the solid support during different steps in the synthesis process, the solid support may better accept the next gas introduced into the reaction chamber 160. When using CPG as a solid support, for example, drying CPG between steps
It allows the CPG to take advantage of the capillary action in the holes of this CPG to better receive the next fluid introduced into the reaction chamber 160. In addition, by using gas to exhaust the system, the contaminat
ion ratio). This is because dilution of the reagent due to undesired mixing of the reagent is reduced.

In one embodiment of the present invention, a step is provided for adding a base to a β-cyanoethyl phosphoramidite synthesis cycle. Detritylation is generally performed as the first phase of a synthesis cycle. The valve rotor 136 is in the home position 4
Start at 04. The valve rotor 136 then rotates counterclockwise to the deblocking or detritylating reagent (TCA) position 472. From position 472, the TCA is dispensed to the reaction chamber 160 via the valve rotor 136 in several, preferably nine, timed pulses, with a predetermined pause between each pulse.

The valve rotor 136 then rotates clockwise again to the gas distribution (GAS) position 480, and rotates clockwise to the adjacent wash (ACN 2) position 476 before distributing the gas. , And one pulse of ACN that is timed is distributed. As discussed above, gas is used at this stage to flush reagents from the support surface, if necessary, and to dry the support surface.

In the second phase of the synthesis process, the coupling phase, a base is added. In this example, phosphoramidite adenine is added. The valve rotor 136 rotates clockwise to the TET 1 position 408 and the activator tetrazole is dispensed in one timed short pulse. The valve rotor 136 then rotates clockwise to the A position 412, and the phosphoramidite adenine is dispensed in one timed short pulse. The valve rotor 136 then moves back and forth between positions 408 and 412. As a result, the phosphoramidite and activator are distributed in alternating pulses to the path 348 of the valve rotor 136. These fluids pass through path 348
As they move through the swirl chamber 350, these fluids mix together and then travel back and forth through the reaction chamber 160.

In the third phase of the synthesis process, the capping phase, the capping reagent is added. Thus, the valve rotor 136 is positioned at the CAP A position 460
Counter-clockwise, and one portion of the capping reagent is dispensed in short, timed pulses. Next, the valve rotor 136 operates as a CAP.
Rotating counter-clockwise to position B 456, the other portion of the capping reagent is dispensed in short, timed pulses. As explained above, this capping reagent is unstable when mixed, and is divided into two parts. In this manner, the valve rotor 136 is positioned such that the two portions of the capping reagent are dispensed into the path 348 of the valve rotor 136 in alternating pulses.
Switch back and forth between 0 and 456. As these fluids move through path 348 and swirl chamber 350, they mix together and then travel to and from reaction chamber 160.

In the fourth phase of the synthesis process, the oxidation phase, an oxidizing reagent is added.
Thus, valve rotor 136 rotates clockwise to OXID 1 position 464 and a predetermined amount of oxidizing reagent is dispensed. This oxidizing reagent adds a water molecule to the base added to the solid support. Since the other reagents are moisture sensitive, valve rotor 136 then rotates clockwise to gas distribution (GAS) position 480 and distributes the gas. As discussed above, this gas is used at this stage to flush reagents from the support surface when needed and to dry the support surface. In one embodiment, the valve rotor 136 is then
Return to home position 404 and check to make sure that the motor drive system has recorded that valve rotor 136 is home. If it fails, and the motor drive system does not record that the valve rotor is home, the system stops the sequence. In another embodiment, valve rotor 136 is instead rotated directly to TCA position 472 to prepare for the next phosphoramidite addition.

As shown in FIG. 4, the use of an arrangement of reagents with respect to the upper surface 314 allows for shorter cycle times due to the proximity of each of the reagent positions to one another.
However, various reagent arrangements with respect to the inlet port 146 of the valve rotor 136
It will be apparent to those skilled in the art of oligonucleotide or peptide synthesis that they may be used for the production of oligonucleotides or peptides.

In one embodiment of the invention, the cycle for adding one oligonucleotide to a stretch of nucleotides is 1.5 to 3 minutes. Thus, in this embodiment, a 25 base long oligonucleotide may comprise about 38
Can be synthesized in ~ 75 minutes. It will be apparent to those skilled in the art that it is possible to achieve a cycle time of 30-45 seconds with the present invention, depending on the steps used during the nucleotide addition.

A second embodiment of a top view of the upper surface 314 in a second position is shown in FIG. In this embodiment, an inlet port 1804 is provided. One inlet port 18
08 is connected to a cleaning solution (eg, ACN). A wash channel 1812 is connected to ports 1808 and extends between each port. In use, as the valve rotor 136 rotates between the inlet ports, the communication port 326 of the valve rotor 136 allows the cleaning solution to pass through the cleaning channel 1812, thereby reducing contamination.

Additionally, two outlet ports 1816 and 1820 are provided on the top surface of this alternative embodiment. If fluid collects in the interstitial space between the valve rotor and the second portion of the rotary valve, the cleaning solution may be flushed through the interstitial space. Outlet ports 1816 and 1820 are provided to serve as outlets for draining fluid from the interstitial space during a flush. In this embodiment, the TEF
LON® material (available from EI Du Pont de Nemours) may be used for the valve rotor and the stationary part of the valve rotor assembly. This is because this material is non-reactive and deserves to be used with reagents typically used in synthetic processes. As discussed above, it is preferred to use this embodiment in a rotary valve design that does not use an O-ring to minimize interstitial space.

C. Alternative Embodiments An alternative embodiment of the system of the present invention is shown in FIGS. Referring to FIG. 21, a stem 2100 is shown. System 2100 includes distribution plate 2
104 and a manifold block 2108. The distribution plate 2104 and the manifold block 2108 connect the distribution valve 128 to the rotary valve 138.
Is designed to be used in place of tubing 140 for coupling to.

One of the problems with using tubing to connect this distribution valve to the inlet port of a rotary valve, as would be the case for a system designed to synthesize 96 oligonucleotides simultaneously. Second, the tubing is cumbersome when implementing a multi-composition process. Further, the use of large amounts of tubing increases the likelihood that one or more leaks can occur. Thus, using tubing for the connection is time consuming and requires constant maintenance. In addition, because the tubing is made of a somewhat elastic material, the tubing tends to work like a spring. When the dispensing valve is on, the flow of pressurized fluid through the tubing causes the tubing to extend slightly. The tubing then returns to its original size when the dispensing valve is switched off, which causes excess fluid in the tubing to squirt from the end of the tubing into the inlet port. System 210
A zero reduces the likelihood of fluid leakage from the dispensing valve to the rotary valve and allows for better control of the exact volume of fluid delivered to the reaction chamber.

As shown in FIGS. 21-23, the distribution plate 2104 includes an inlet port 2112
Having. The distribution valve 128 is connected directly to the inlet port 2112 by screwing or other mounting means apparent to those skilled in the art. Motor drive system 142
Is mounted on the bottom of the distribution plate 2104 via bolts, screws and the like. Shaft 152 of each motor drive system 142 passes through holes 2116 in distribution plate 2104 and aligned holes 212 in manifold block 2108.
Stretch up through zero. In one embodiment, the distribution plate 2104 and the manifold block 2108 are E. coli. I. Du Pont de Nemour
It is made using TEFLON® material, which can be purchased from s. In another embodiment, distribution plate 2104 and manifold block 2108 are made of nickel-coated stainless steel, where the coating is Ref
Adapted using a diffusion bonding process available from rac Systems, Phoenix, Arizona.

The manifold block 2108 is mounted on the distribution plate 210 using bolts 2132.
4 is attached. It will be apparent to those skilled in the art that the manifold block 2108 can also be mounted using screws or some other similar securing mechanism. The manifold block 2108 is composed of layers of material (details are shown in FIG. 23),
Each layer corresponds to its own reagent. Upper layer 212 of each manifold block 2108
4 replaces the second part 308 of the rotary valve 138 as shown in FIG. As shown, entrance port 146 is formed in upper layer 2124. As discussed above, an O-ring (not shown) may be placed around the entrance port 146 to reduce the entrainment ratio.

As shown in FIG. 22, the rotary valve 138 is connected to the manifold block 2.
108 is located on top of the upper layer 2124. Specifically, the valve rotor 136 is mounted on the shaft 152 of the motor drive system 142 and is centered on the shaft 152. Then, the first part 30 of the rotary valve 138
6 is located around the valve rotor 136 and is attached to the upper layer 2124 by screws, bolts, or the like. Reaction chamber 160 is located on top of rotary valve 138.

In use, fluid or gas is supplied to the distribution plate 21 via the inlet port 2112.
Enter 04. The fluid or gas then flows through a channel (not shown) connecting the inlet port 2112 to the outlet port 2128 on the distribution plate 2104
It is distributed to each manifold block 2108 arranged on the distribution plate 2104. In one embodiment, an O-ring (not shown), similar to the O-rings 332, 338, and 340 discussed above, is disposed about the outlet port 2128 and between the distribution plate 2104 and the manifold block 2108. To match.
This O-ring allows for leak proof transfer of fluid from distribution plate 2104 to manifold block 2108.

[0109] Once the fluids or gases enter the manifold block 2108, each fluid or gas has a separate passage so that the two fluids or gases do not contact or interact. . As shown in FIG. 22, the fluid or gas first rises up a vertical channel 2204 in the manifold block 2108 to reach a layer of the block dedicated to the particular fluid or gas. An example of one layer 2308 of the manifold block 2108 is shown in FIG.

Once the fluid or gas reaches layer 2308 in manifold block 2108, the fluid or gas flows around layer 2308 in horizontal channel 2304. The horizontal channel 2304 is in fluid communication with another vertical channel 2312, which is in communication with a specific inlet port 146 of the upper layer 2124 (see FIG. 21). Each inlet port 146 has a corresponding vertical channel 2312 that moves through the manifold block 2108. Horizontal channel 2304 in each layer
Care must be taken when designing a to ensure that reagent contamination does not occur and does not overlap with vertical channel 2312 or 2204. In one embodiment, the horizontal channels 2304 in the manifold block 2108 are located around the outermost of this layer, as shown in FIG. In each successive layer, the horizontal channel is located slightly inside the horizontal channel of the layer below that horizontal channel so that no overlap occurs. Each fluid or gas has another layer 2308. There may be multiple layers 2308 depending on the number of fluids or gases to be supplied to inlet port 146.

In the embodiment shown in FIGS. 21-23, the manifold block 2108
Designed to house eight rotary valves and a motor. The distribution plate 2104 is then designed to accommodate six manifold blocks 2108 or 48 rotary valves. As will be apparent to those skilled in the art, distribution plate 2104 may be designed to accommodate a number of rotary valves. In one embodiment, the two distribution plates 2104 are positioned adjacent to each other, such that integration of the 96 rotary valves can occur simultaneously.

III. Motor Drive System Referring to FIG. 5, a motor drive system 142 is shown. As discussed above in Section II, motor drive system 142 is used to rotate valve-rotor 136 to its indicated position.

In one embodiment, stepper motor 144 is also used to rotate valve rotor 136. Stepper motors are attractive for use in this application. Because, without relying on the gear train,
This is because it can be used as a direct controller. In one embodiment, stepper motor 144 has 200 steppings per revolution.

While stepper motors are essentially simple devices, they still require external means to determine the zeroth or “home” reference. Stepper motors typically have four windings. This must be energized when the motor armature rotates, either in pairs or alone. The unique excitation pattern for each stepping must repeat every four steppings when the motor is turned. The rotational position of this motor is monitored by keeping track of the number of steps and their direction.

Typically, the driver electronics for the motor is supplied either as a computer compatible card that can operate three or four motors, or another system for determining a zero reference for the motor.

In this embodiment, as shown in FIG. 5, the driver electronics 504 and the zero reference system 508 are mounted on the rear end of the stepper motor 144 and replace the conventional computer card or the conventional motor zero reference system. Used for In particular, driver electronics 504 is mounted on circuit board 506. In one embodiment, the motor drive system 142 is controlled directly from the host computer using as little as 5 binary bits of data. Four bits are used for motor excitation and one bit is used to report back to the zero reference position. This motor drive system 1
Using the digital input / output (IO) attached to the host computer
) The card can be used to easily operate over 100 stepper motors simultaneously. In one embodiment, the optical isolation of the motor circuitry of the driver electronics 504 reduces the susceptibility of the computer electronics to be damaged by other external forces, usually associated with transient high voltage and driving high power inductive loads. May be preferred. In one embodiment, the optical isolation is located on a separate circuit board separate from the driver electronics 504.

With currently available computers (eg, PCs), it is possible for the host computer to generate motor pulse excitation patterns directly for multiple motors. In one embodiment, between one to 96 or more stepper motors, one motor for each rotary valve assembly is operated simultaneously by means of only one Pentium PC computer operating at 233 MHZ.

The motor drive system 142 of the present invention uses a stepper motor 144 having a shaft 152 available at both ends of the stepper motor 144. The first end 512 of the shaft 152 is connected to the stepper motor 144 by the valve rotor 1.
Used to connect to 36. First end 512 may be directly coupled to valve rotor 136, or first end 512 may be coupled to extension shaft 516, as shown in FIG. The extension shaft 516 may then be coupled to the valve rotor 136. The second end 520 is used to mount the zero position reference system 508, as described in further detail below. It will be apparent to those skilled in the art that a stepper motor having a shaft only available at one end of the motor may also be used, where the driver electronics 504 and the zero position reference system 508 are the same as the valve rotor 136 Connected to the end of the shaft.

The driver electronics 504 is connected to a host computer (not shown) by connector 548 and preferably through the optical isolator described above. The driver electronics 504 controls the stepper motor 144 such that the position of the stepper motor 144 is monitored and controlled by the host computer sending a transistor-transistor logic (TTL) level signal to the driver electronics 504. Is done. this is,
By taking advantage of the host computer's ability to generate motor excitation signals, maintain a track of motor position, and read position sensors, the zero position reference system 508 can be integrated with driver electronics 504 as part of the motor drive system 142. Allows us to prepare together.

A. Zero Reference System The zero reference system 508 consists of a light emitting diode (LED) 524 mounted on a fixed plate 528. A rotating aperture disk 532 is mounted on the second end 520 of the shaft 152. A silicon detector 536 and an operational amplifier (OA) 540 are fixedly mounted on a circuit board having driver electronics 504.

To accurately identify a unique motor position from the 200 positions, it is essential that the zero position reference system 508 be approximately accurate for one portion of the 400. Silicon detector 536 has a rectangular sensing element (not shown).
In one embodiment, the rectangular sensing element measures approximately 0.008 inches x
Up to 0.240 inches. Opening disk 532 has a radial opening 544 having a width that matches the size of the sensing element of detector 536. As such, in the above embodiment, the width of the radial opening 544 is approximately 0.008 inches.

It will be apparent to one of skill in the art that one or more detectors 536 may be used to detect more than one motor position. For example, additional radial apertures and detectors may be added to the aperture disc so that the zero position reference system 508 determines when the motor is in the "home" and mid-way positions.
Can be identified.

The aperture disk 532 rotates in proximity to the sensing element of the silicon detector 536, mounting the source LED 524 below the aperture disk 532. In one embodiment, the LEDs 524 are mounted approximately 0.5 inches below the opening disk 532. By incorporating a thin sensing element mounted radially to the shaft 152 relative to the shaft 152, the largest signal is available when the opening 544 of the opening disk 532 exposes light from the LED 524. .

An operational amplifier 540 is connected to the silicon detector 536 and used to amplify the signal from the detector 536 so that the signal can be sent back to the host computer. In one embodiment, operational amplifier 540 includes M
micropoewr Single Suppley Rail to Rail
It is an amplifier (OP191). This particular amplifier is preferred. Because the amplifier may use the same power supply used to operate the stepper motor 144. As such, in this embodiment, the zero reference system 508 is powered by the same supply voltage of 5-12 volts as the excitation power supply provided to the stepper motor 144. Further, the motor drive system 142 can make position measurements using the shaft 152 zero position reference system 508 after the shaft 152 of the stepper motor 144 has stopped rotating, so that electrical interference due to motor excitation can be reduced. Less than.

B. Driver Electronics Driver electronics 504 is provided to drive the motor and develop the pulses used to ramp up the stepping speed. The host computer sends an excitation pattern signal to the driver electronics to indicate where the motor should move (i.e., one step clockwise or one step counterclockwise) and update the position information of the motor. In one embodiment, the stepper motor 144 has 20 different positions, each position being described using 10-step increments (eg, the first position, typically the “home” position is 200; The second position is up to 390, such as 210). Each position corresponds to an inlet valve 146 of the rotary valve assembly 138.

In one embodiment, the driver electronics 504 is comprised of four output Darlington transistors, with diode clamps to protect the output transistor from transient reverse voltages when the transistor is turned off.
The excitation pulse width and timing are determined directly by the host computer,
Therefore, to adapt the electronics to the computer, stepper motor 1
Minimize the amount of electronics that must be mounted on 44.

Each stepper motor 144 is connected to a host computer by an external optical isolation board and a digital IO card attached to the computer. In one embodiment, six 96-bit IO cards are used to control 96 stepper motors.

In one embodiment, the anti-backlash feature is also incorporated into a computer program that drives the stepper motor 144, as discussed in detail below, so that the rotation of the valve rotor 136 starts at the beginning of the rotation. Regardless of the direction, always end in the same direction. For example, the computer program is designed to cause the stepper motor 144 to rotate the valve rotor 136 past the desired position one step and then back. This anti-backlash feature preferably overcomes any drag on rotation of valve rotor 136 due to O-rings 332, 338 and 340.

(IV. Output Monitor) Referring to FIG. 6, an output monitor 600 is shown. The output monitor 600 is a dual wavelength spectrophotometer used with the synthesis system 100 (shown in FIG. 1) to monitor the efficiency of the reaction being performed by the system 100. In one embodiment, the output monitor 600 monitors the amount of trityl present in the effluent for the oligonucleotide synthesis process. The output monitor 600 can also be used to monitor the amount of various reagents or chemicals present in the effluent resulting from the reaction.

As is well known in the art, an important measure of the efficiency of the process during the synthesis includes measuring the amount of trityl present in the effluent. The use of monitoring reagents, such as trityl-based groups, to monitor yields has been described by Horn et al. In US Pat.
, 800,166. This patent is incorporated herein by reference.

The nature of the spectrum of trityl is that basically the extinction of blue light that can be provided by a blue light emitting diode (LED) is a functional relationship to the amount of trityl present in the effluent. In the present invention, the trityl monitoring process is based on tubing 1 where the effluent containing trityl is typically used in the manufacturing process.
This is done while within 40.

Another measure of the efficiency of the synthesis process is to measure the amount of reagents or chemicals in the effluent. If there is a significant increase in the amount of a particular reagent or chemical in the effluent, this is an indicator that the reaction with the reagent or chemical is complete.
With this type of feedback, it is possible to optimize the amount of reagents or chemicals delivered to the reaction chamber, thereby ensuring that a complete reaction takes place and reducing waste of reagents or chemicals Become.

The dual-wavelength spectrophotometer of the output monitor 600 includes an optical sensor 604, an LED driver 608, and a signal processor 612.

A. Optical Sensor Referring to FIGS. 6 and 7, the optical sensor 604 is composed of two parts.
It has a common circular cross section with a space for placing tubing 140 between the two parts. In the first part, the blue LED 616 and the red LED 620
Attached adjacent to each other in a row along one side of tubing 140. L
Light emanating from EDs 616 and 620 passes through a narrow slit 704 (shown in FIG. 7) parallel to and centered on axis 708 of tubing 140. The narrow width 706 of the slit 704 is selected to match the inner diameter 710 of the tubing 140. The length of the slit 704 corresponds to the combined diameter of the two LEDs 616 and 620.

A detection assembly 624 including a silicon diode photodetector 628 with an internal amplifier (not shown) is located behind the comparison slit 712 in the second part of the optical sensor 604 and directly opposite the illumination slit 704. You.

The LEDs 616 and 620 are turned on alternately at a rate of 1 KHz. LE
The optical path length between D 616 and 620 and the detection assembly 624 optimizes the transmission of LED light through the center of the tubing 140 using aligned slits 704 and 712 and the location of the LEDs 616 and 620, Is designed to minimize the transmission of light around the effluent.

B. LED Driver The LED driver 608 consists of an analog square wave transmitter that is counted down and decoded to provide a series of signals. First, a square wave signal with a low source impedance of 1 KHz capable of driving 96 LEDs simultaneously is provided.

The LEDs 616 and 620 are connected in parallel with opposite polarities so that they alternately turn on during half a square wave cycle. A variable series resistor for each diode is included so that the light output can be matched to a standard value. Also, since it is desirable to monitor the combined light transmission signal only during the latter half cycle of each irradiation period, a set of measurement gates is obtained by decoding the fundamental oscillation signal and the combined split square wave.

C. Signal Processor Referring to FIG. 6, the signal from silicon detector 628 is amplified by an internal amplifier (not shown) to a value of +10 volts for both red and blue emissions. Matching values are achieved by using a clear effluent and adjusting individual light levels using a series potentiometer.

In an example of a trityl measurement, as the trityl concentration increases, the blue signal weakens and the red signal remains essentially constant. These two signals are separated using a synchronous demodulator and filter 632 consisting of two analog switches (not shown) and a differential amplifier (not shown).

Next, the differential output is demodulated and a low-pass filter (10 Hz cutoff) 6
It is filtered at 32 and passed to an analog / digital converter card 636 attached to a digital computer 640. The signal passed to the analog / digital converter card 636 is a non-linear function of trityl concentration. This is also true, even if the measurements are made in a quartz cuvette, where a simple exponential is sufficient to perform a linear approximation.

In one embodiment, the measurement is complicated by the presence of a stray light path around the trityl solution on the tubing 140 wall. Thus, the data is linearized using an empirical formula created by correcting the voltage vs. concentration curve plotted using an EXCEL spreadsheet. This algorithm is satisfactory even with multiple channels.

In the described system, a red LED 620 is used to perform the reference measurement. By removing the red signal, it is possible to eliminate uncorrected noise and sensor drift.

In an embodiment of a trityl measurement, a computer 640 can display the measurement to a user in numerical and / or graphical form, thereby achieving continuous monitoring of the efficiency of the synthesis process. Further, based on the trityl measurement, adjustments to the synthesis process can be made during the process, increasing efficiency and reducing unnecessary waste of reagents.

In one embodiment, the measured signal from the output monitor indicates the shift of the assumed baseline reference when the assumed baseline reference moves from one transparent reagent to the other. Show. For example, if both reagents are colorless and look the same, but the ACN is replaced by TCA, a significant shift is seen as the monitor output voltage.

If instead of taking a measurement through tubing (having a circular cross section), the same measurement is taken through a rectangular quartz cuvette, the measured value and reference line through the quartz cuvette using TCA or ACN are coincident .

FIG. 19 shows an example of an output measurement of different reagents in the effluent resulting from an oligonucleotide synthesis process using the system 100 of the present invention. In particular, FIG.
Shows a graph with a curve 1904. Curve 1904 is a curve showing the effluent during the phosphoramidite addition. Curve 1904 has several peaks representing different segments of the synthesis process. The first peak 1908 represents trityl in the effluent. The second peak 1912 represents the binding segment where additional base is added. The third peak 1916 represents the capping reagent,
Here, the capping reagent is added to the reagent chamber. The fourth peak 1920 represents an oxidized segment where an oxidizing reagent is added to the reaction chamber.

FIG. 19 also shows the shift of the reference line reference when a new reagent is introduced into the reaction chamber. For example, a wash solution (AC) used before the detritylation segment
The reference line of N) is shown as a reference line 1924. The lower reference line for the detritylation reagent (TCA) is shown as reference line 1928. Once the reaction is complete, more reagent is present in the effluent and remains in the reaction chamber. This shifts the reference line. As such, continuous monitoring of the effluent indicates when the reaction with a particular reagent or a particular chemical has been completed. If reagents or chemicals are still added to the reaction chamber after the reaction is completed, the time intervals of the reagents or chemicals need to be adjusted to avoid waste. Similarly, if the supply of a reagent or chemical is cut off during the reaction, the time interval of the reagent or chemical must be adjusted to ensure a complete reaction.

When performing measurements via tubing, as described in the present invention, one logic of why the output voltage changes for different reagents is that the measurement vessel (ie, tubing) is annular, lens-like. And that the reagents have different refractive indices. Changes in the refractive index caused by changing the reagent correspond to changes in the effective focal length of the cylindrical lens formed by the inner diameter of the tubing. These changes in focal length can easily change the distribution of light transmitted from the LED to the detector, resulting in a change in the measured reference line as shown in FIG.

In the present invention, as various reagents are passed through the reaction chamber, it is possible for the output monitor to detect the washout characteristics of the system by changes in the measured baseline for different reagents. Typically, a trityl monitor is used for cleaning (A
CN) Measure only the trityl output during the cycle. An advantage of the power monitor of the present invention is that it can detect how much of the effluent remaining in the reaction chamber is wasted reagent. Since the reagents used in the synthesis are expensive, it is important to use only the same amount of reagents as those required for the synthesis process.

In another embodiment, the reagents are colored with different colors and / or dyes, or different concentrations of the same color and / or dye, depending on the number of LEDs used. As such, each reagent flowing through the tubing absorbs light from the LED separately. In this way, the output monitor can be used to specifically identify which reagent was added to the array at any given time. This output can be used to confirm that the correct reagents have been added in the correct order to obtain the final product profile.

By using the output monitor feedback of the present invention, the amount of reagents or chemicals added to the reaction chamber during a particular reaction can be adjusted to reduce reagent or chemical waste. For example, results from the output monitor may be fed back to the host computer to adjust the timing or pressure of system events upon addition of reagents or chemicals. This allows the amount of reagent or chemical delivered to the reaction chamber to be controlled. This optimizes the use of reagents and chemicals to avoid waste and ensure a complete reaction.

In alternative embodiments, two or more separate output monitors can be used. For example, a first output monitor for measuring the amount of a particular reagent in the effluent is located immediately adjacent to the outlet from the reaction chamber 160. This arrangement allows near real-time feedback of data to the host computer, optimizing the synthesis process. A second output monitor for measuring the trityl concentration is located a specific distance from the outlet of the reaction chamber 160. This arrangement away from the reaction chamber is preferred for trityl measurement, as in the detritylation, a minute pulse of the reagent TCA is supplied to the reaction chamber 160 with a minute pulse between each pulse. Using a small pulse, the amount of TCA used for detritylation is better controlled. It is preferred that the fluid flowing through the tubing on the trityl monitoring side has a steady flow when integrating over the time required to calculate the efficiency as described in detail below. As such, the long steady pressure of the wash solution created by the detritylation segment pushes the trityl past the output monitor at a constant rate.

V. Level Sensor System In one embodiment of the present invention, storage vessels 120 and 124 may be provided with a level sensor system 1700 shown in FIG. Level sensor system 1700 indicates to the host computer when one or more storage containers are empty or near empty. FIG. 17 shows a cross-sectional view of one of the storage containers 120, 124 so that the storage container sidewall 1704 is visible. LED 1708 is disposed on one portion of sidewall 1704 such that LED 1708 is disposed within the curvature of the sidewall. A silicon detector 1712 is located opposite the LED 1708 on the opposite curved portion of the side wall 1704 as shown in FIG.

The LEDs 1708 are powered by a 5 volt power supply. Both LED 1708 and silicon detector 1712 are grounded as shown in FIG. Further, the output from silicon detector 1712 is sent to an analog-to-digital converter (not shown) and then to the host computer of system 100.

The LED 1708 and the detector 1712 are located near the lower part of the periphery of the storage container. If there is a fluid in the storage container, the light from the LED 1708 propagates directly across the storage container to the silicon detector 1712, as shown by the dotted arrow in FIG. However, if there is no fluid above the light from LED 1708, the light will be refracted because the refractive index of the fluid will be different from the refractive index of air (eg, no fluid). In this case, most of the light from the LED 1708 is
Do not reach 2. As such, an output signal indicating that one of the storage containers is empty or nearly empty is sent from the silicon detector 1712 to the host computer.

[0157] In one embodiment, this causes the host computer to alert the user to refill a particular empty storage container. However, in another embodiment, each of the smaller storage containers is fluidly coupled to a corresponding larger stock bottle. When the host computer receives a signal indicating that a particular storage container is empty or near empty, the host computer may send a signal to automatically refill a smaller storage container from the stock bottle. In one embodiment, the signal is sent to a valve or pump that automatically refills the storage container.

Many of the reagents used in the synthesis process degrade over time. As such, it is preferable to use up all of the older reagents in the smaller container before refilling the smaller container. In addition, automatic refilling is preferred because the reagents are typically substantially clear fluids and human error is likely to occur when the storage containers are accidentally filled with the wrong reagents or chemicals. In embodiments where the container is automatically refilled by the system, the only human involvement, and thus the potential for human error, is in the initial connection of the larger stock bottle to the smaller storage container.
From this point on, all refills occur without human intervention.

VI. Process of the Invention The system 100 controls the reaction through a set of rotary and distribution valves that regulate the flow of a large volume of fluid or gas. Any given fluid or gas is selected by the rotary valve and the volume dispensed is measured by operating the dispensing valve at a specified time. This time is determined based on the volume of fluid or gas delivered to the reaction chamber and the pressure of the fluid or gas in the fluid container.

Further, the calculation of the time to operate the distribution valve takes into account the number of channels being supplied at one time (ie the number of reaction chambers). If many channels are provided by distribution valves, it will take longer for the fluid or gas to reach all intake ports. Thus, the amount of flow is adjusted by the number of channels being supplied. In one embodiment, a look-up table is created that tells the computer how much time is needed based on the number of channels being served.

[0161] Simultaneous start-up in all reaction chambers, and thus adjustment of all valves (dispensing and rotary) capable of achieving simultaneous reactions, allows entry of sequences into a sequence listing and sequences of sequences for the host computer. This is done by using an event matrix table that translates the instructions into In one embodiment, these instructions are
Generated as a comma separated file by a soft (R) Excel spreadsheet. Examples of a series of sequence listings are shown in FIGS. 8A, 8B and 8C. An example of an event matrix is shown in FIG.

A. Sequence Listing Referring to FIGS. 8A-8C, the desired sequences for an exemplary oligonucleotide synthesis are inserted in Table 800 and spaced, provided in row 804. 1
In an embodiment, the arrangement of table 800 is provided by the customer in the form of a computer file that is read by a host computer. In the embodiment shown in FIG. 8A, there are eight rows 804. Each row 804 is numbered corresponding to the number of channels to the left of the row. Each channel number corresponds to a different array created. In this example, there are eight different arrangements that can be entered in table 800. In other embodiments, 96 or more different sequences may be entered at once.

In addition to filling in the desired sequence, the user can also select the terminal base (en
dbase) should be shown in Table 800 if it should be detritylated. If detritylation occurs, this allows additional bases to be added to the sequence of the next synthesis. If detritylation does not occur, the resulting sequence is less susceptible to further synthesis. Therefore, as shown in FIG. 8A, the term “DM
"T" or "NO DMT" is used to indicate whether detritylation or non-detritylation occurs at the end of the synthesis process.

Next, as shown in Table 808, the array is transformed. The first transformation simply reverses the order of the items in the sequence, so that the sequences are delivered to the reaction chamber and appear in order. For example, in the first row 804 of table 800, the array is "G
ACCTGGTCAG ". In the corresponding first row 812 of table 808, the array has been inverted to read "GACTGTCCCAG". Furthermore, in this first transformation, the DMT instructions are assigned numbers. In this example, Equation 4 is used to indicate a detritylation or DMT instruction. Equation 5 is used to indicate a non-detritylation or NO DMT instruction. This number is added directly after rearranging the sequence.

In this example, there are 40 positions provided for input of the sequence. After inputting the sequence and detritylation instructions, the remaining positions in the table are "spaces (spac
e) ". In one embodiment, those positions marked with a capital letter S are filled with instructions to bring the valve rotor to the home position.

Table 808 is again translated as the instructions are shown in a format readable by the host computer. This conversion is shown in Table 816 of FIG. 8C.
In table 816, each instruction from table 808 is converted to a number by referring to event matrix 904, as described below with reference to FIG.

B. Event Matrix As shown in FIG. 9, the steps for adding each base during the exemplary oligonucleotide synthesis process are shown in the event matrix 904 as a series of events. In one embodiment, the event matrix 904 is provided using a Microsoft® Excel spreadsheet. Row 908 shows each event in the event matrix. In one embodiment, there are a total of 54 events or rows. Although an event matrix has been described for oligonucleotide synthesis, it is clear that the event matrix can be adapted for use in various reactions that require the delivery of fluid and / or gas sequences.

Column 908 lists the number of events for each event. Adjacent column 912 lists the volume (in milliliters) of each fluid or gas delivered during a particular event. The measurement of this volume is based on the pressure of the fluid container or gas and the time interval during which the distribution valve corresponding to the particular fluid or gas is open. This time interval for each event is shown in column 9 in milliseconds.
As shown in FIG. If no fluid or gas is delivered during an event, row 9
The row corresponding to 12 indicates a "WAIT" event, and the corresponding distribution valve position indicates an "OFF" position, as described in detail below.

Columns 920 and 924 show instructions for Home or “H” locations. Column 920 relates to commands sent to valve rotor 136. As shown in FIG. 9, when the home position is selected, the valve rotor 136 moves to “Ho”.
Receive an instruction to return to the "me" position. Column 924 relates to commands sent to distribution valve 128. Next, when the home position is selected, the valve rotor 136 is selected.
There is no fluid or gas flowing to. Accordingly, distribution valve 128 receives a command to maintain the "off" state.

The next column, 924, 928, 932, 936, 940, 944, 948, 952
, 956, 960, 964, 968, 972, 976 and 980 indicate the position of the rotary valve and the solenoid valve status corresponding to each event required to add a particular phosphoramidite to the array. For example, column 924 lists the rotary valve position for each event used to add adenine to the sequence being synthesized. Column 928 lists requests for status of distribution valve 128 for each event due to the need for a particular phosphoramidite cycle. If more than one array is being synthesized at the same time, each channel corresponding to the array sends a list of the array of events from a particular column to the processor, and the computer program will allow the appropriate set of distribution valves to be utilized. Combine events so as to eliminate duplication.

When an event matrix is created, the positions of various rotary valves are described by reagent abbreviations (mnemonic) (for example, TCA or ACN1).
Some reagents may be available at more than one rotary valve location. In this case, the numerical subscripts are used to specify different positions.

As described above, certain reagent containers may provide reagent to more than one inlet port of a rotary valve assembly. In this case, all intake ports for reagents are controlled by a single distribution valve. Thus, as shown in row 9 of column 928, the command for the rotary valve commands the rotary valve to move to the ACN2 position. However, since there is only one state on the distribution valve for ACN, the distribution valve state shows only the ACN state.

Columns 984 and 988 show the rotary valve position and solenoid valve status relative to the detritylation step or DMT. As shown in column 992,
The event matrix 904 uses "R" for return and "B" for bottom, and "B" for bottom, and uses "R" for bottom. The file can be automatically read by DNA synthesis software. Next, the modification of the synthesis cycle is performed in an Excel spreadsheet, which in turn creates an event matrix file separated by commas.

As shown in FIG. 9, each instruction sequence in the event matrix 904 (ie, 916-988) is given an ascending column number, starts at 1 and ends at 19. The number of these columns is used to convert chart 808 into the chart shown in FIG. 8C.

Next, referring back to the example of FIG. 8C, each item in the array is replaced with the number of rotary valve rows corresponding to that particular item. For example, the first base in row 812 of table 808 is "G". The row of the rotary valve corresponding to the event matrix 904 is row 8. The first entry in the corresponding row 820 of table 816 is the number “8”. Each base is converted to a numerical equivalent.

If there is “4” indicating detritylation at the end of the sequence, “4” indicates the event matrix 9
04 with “18” corresponding to column 804. If there is a "5" at the end of the sequence that does not indicate detritylation, the "5" is replaced with a "2" corresponding to column 920, instructing the rotary valve to return to the "Home" position. Similarly, all of the “S” instructions in Table 808 are “2” in Table 816 indicating the location of “Home”.
]. The instructions in Table 816 are then sent to the host computer that uses these instructions to control motor drive system 142 and distribution valve 128 throughout the synthesis process.

(VII. Method of Performing a Reaction Using the Present Invention) A host computer that controls a reaction is programmed using a computer program that executes a process. In one embodiment, the computer program is Microsoft® Windows® 95 or Microsoft® Visual B running under NT.
written in asic. Visual Bass from Microsoft (R)
IC provides a relatively easy way to get user-friendly interactive graphics with the ability to communicate with motors and distribution valves (eg, solenoid valves) via plug-in digital and analog input / output cards I do. It will be apparent to one skilled in the art of computer programming that various programming languages (eg, programming languages such as C ++) and various computer systems may be used to perform the methods of the present invention.

[0178] Computer programs use an event matrix to coordinate operations on the various channels, which may be adding different fluids or gases simultaneously. Each channel has an array (seq) of items for reactions assigned to each channel.
has its own rotary valve that is used to select the fluid or gas needed to perform the change. Fluid or gas is supplied to the inlet port of the rotary valve assembly by a manifold that simultaneously supplies all of the rotary valves with a particular fluid or gas while controlling all rotary valves. For a particular fluid or gas flowing into the reaction chamber, the rotary valve needs to be in a fluid or gas dedicated position and the corresponding dispensing valve needs to be activated.

The data in the event matrix 904 is converted into a conversion table by a computer program, where the first column contains the time interval in milliseconds assigned to a given row, and the second column Gives the step position on the stepper motor for the rotary valve position corresponding to the abbreviation entry. The position value of this step is expressed as an integer between 200 and 400, as described above. This number represents the new position used in the rotary valve control software that commands the rotary valve to a new position. In the case of a rotary valve having 20 inlet positions, there is a port at each tenth step position (eg, 210, 220, etc.).

The third and fourth columns are used to contain status information for the corresponding distribution valve. Since two 8-bit words (ports) are required to control 16 valves, two columns are used for this information. Accordingly, the computer program creates two columns in the conversion table from the original distribution valve columns in the event matrix 904. The digital value is assigned to the desired distribution valve state for each channel and placed in the table. In the example of FIG. 9, a row represents each event for addition of a specific base. This converted table fully describes the process steps required to add a single base.

FIGS. 10A, 10B, and 10C show a flowchart 1000 of steps used by a computer program to control a synthesis process or reaction using the present invention. The program begins at step 1002. As shown in step 1004, the system 100 calibrates the valve rotor 136 of the rotary valve 138 and in step 1006 checks to see if the valve rotor 136 is in the "Home" position. If the valve rotor is not in the "Home" position, the counter is incremented and the system returns to step 1004 to recalibrate the rotary valve. After three failed attempts to calibrate the rotary valve, as tested in step 1008, the process is aborted as shown in step 1010 and the process is terminated as shown in step 1012.

When the valve rotor 136 is in the “Home” position, the system 100
In step 1014, the monitoring data is initialized, in step 1016, the anti-backflash flag is initialized to zero, and in step 1018, the program stop flag is set to zero. Next, the system
As described above, as shown in step 1020, the event matrix 904 is ready to be read and transformed, and as shown in step 1022, the desired array from table 816 is read and displayed. As described above, before reading the sequences from Table 816, they have been converted from the original form, as shown in Table 800.

The original sequence can be entered manually by the user, or imported from another computer file, and provided to the user, as shown in FIG.
It can be displayed using a graphical user interface (GUI).

As shown in GUI 1100 of FIG. 11, a set of text windows 1104 in which sequences can be entered and / or displayed can be provided. In this embodiment, each window is about 75 characters wide, but an array with up to 200 items can be entered. One skilled in the art will recognize that the system
It can be clearly programmed to accommodate an array having any number of items.

At the lower left side of the GUI 1100, the text window 1108 automatically displays the step 1
020 shows the current event matrix data file loaded into 020. This is a Microsoft (R) EXCEL file dedicated to the specific operation described above, and the default input is an event generated for some other purpose, such as PURGE or some form of system test. It is desirable to change to a matrix data file. The file designer can be edited in the window as needed.

If the window is full, the array will automatically scroll to the left to make room for more input. The sequence can be entered in either the upper or lower case letter. Unique and different arrays can be entered in successive windows, and the arrays need not all be the same length.

The system 100 then activates the analog-to-digital card for output monitoring, the digital interface card for the motor drive system and the distribution valve for reading data, as shown in step 1024. Step 102
As shown in FIG. 6, the system 1026 activates an output monitoring picture box during synthesis and submits the output data to the user.

[0188] When the activation of the system 100 is completed, the computer program is prepared and the process is started, as shown in step 1028. This process is
This is described in detail in the flowcharts shown in FIGS. 10B and 10C.

The first step in the process is to invoke the first event as shown in step 1030. The system then shuts off all dispensing or solenoid valves in step 1032, causing a 100 ms delay as shown in step 1034, and then, based on commands from the event matrix, as shown in step 1036. Rotate the rotary valve to the position of the rotary valve.

The program communicates to the rotary valve by establishing a new position for each valve rotor in the new position array, as needed. A subroutine called "set rotary valve" compares the data in the new position array with the data in the current position array and generates a "delta" value equal to the difference between the new position and the current position.

In order to facilitate movement to a new position with the shortest route, if the difference is greater than half a turn, a complete turn is added to the above current position. If the difference exceeds half a turn in the opposite direction, one complete turn is returned from the current position.

As described above, the stepper motor 144 is used to move the valve rotor 136 from the current position to a desired new position. In one embodiment, the stepper motor 144 has four windings and performs two hundred steps per revolution. A voltage is applied to the motor windings in pairs with an exciting pattern that repeats every four steps. The excitation pattern required for any particular phase direction (here, two windings need to be energized) performs a division modulo 4 on the position values. Can be identified. 4
The reminder modulo is used as an index to look up a table that converts the reminder modulo 4 to the corresponding excitation pattern.

Communication of the excitation pattern to the stepper motor 144 is achieved using 4-bit binary data (half of an 8-bit digital I / O port). Each 8-bit port controls two motors. Communication to the high order bit motor is obtained by multiplying the calculated digital value by 16 and adding the result to the value to the low order bit motor assigned to the same port.

The control algorithm firstly states that if the motor is not already in the desired new position,
The motor determines which route needs to be traveled to the desired new location. If movement is required, the new position is changed by one step in the desired direction. The program then performs a modulo 4 reminder for the changed new position and sends the corresponding execution pattern to the motor. This moves the motor to the new changed position. This process is repeated for all motors in the system, then a short 5-10 ms time delay is performed (motor run pulse width), allowing the motor to move to the new current position.
As all motors begin to move at the same time, the time delay can be adjusted dynamically, improving the starting characteristics of the motor.

The system then tests to see if all valve rotors have reached their destination, as shown in step 1038. If one or more rotary valves are still moving toward the new position, the system returns through steps 1032-1036 until all valves have reached their respective destinations. If the valve rotor stops rotating, the system proceeds to step 10
The position of the rotary valve is updated by the computer as shown at 40. Next, all the motor excitation is stopped, and the next setting for the movement of the valve rotor is performed. In this regard, the anti-backflush described above is used,
Rotate all motors one step clockwise, followed by one step counterclockwise.

When controlling a stepper motor, there are three possible commands, one step clockwise, one step counterclockwise, and stop in place.
ce). As a result, the motor is preferably calibrated to a known position at program start, as shown in step 1004,
If the motor is directed to various new positions, as discussed at 40, note how it was directed. In the present invention, a zero reference detector 508 is mounted on each motor 144 that identifies a "HOME" position. This position is the OFF position for the rotary valve, and all other positions are referenced to this position. The "HOME" position is also calibrated to be one of the 50 positions encountered when the motor makes one complete revolution (the modulo 4 reminder is 0).

At the beginning of the program, all motors are rotated one step at a time until the zero position reference sensor 508 is established and each motor reaches the “HOME” position.

In one embodiment of the invention, the valve rotor 136 has 20 designated positions, one of which the motor reaches every 10 steps. Using the algorithm 96 described above, the stepper motor can be controlled with about 50 lines of source code, plus the code used to activate various digital I / O card environments. In one embodiment, the source code is provided by Microsoft (
(Registered trademark) Visual Basic. But C
It will be apparent to those skilled in the computer programming arts that various other computer languages, such as ++ and other programming languages, may be used. In another embodiment, about 30 lines of additional code are used in a motor position calibration subroutine named Rotary Valve Calibration.

When the rotary valve position is updated, the appropriate dispensing valve is energized based on the state command in the event matrix to be converted, as shown in step 1042. As described above in one embodiment, the distribution valve is controlled by two 8-bit ports. One port gets its assignment from the column of the event matrix to be converted, which immediately follows the corresponding rotary valve column. The second port gets the assignment from the next column of the transformed event matrix (not shown in event matrix 904). Additional distribution valves can be controlled by expanding the number of columns in the event matrix to be transformed.

When applying voltage to the distributed valve, the system initiates a time interval delay for a particular event based on the time interval information in the event matrix, as shown in step 1044. When the time interval delay is completed for the event, as shown in step 1046, the distribution valve is turned off, as shown in step 1048.

In one embodiment, the time interval may be set as “trityl” during the synthesis process. This means that a trityl measurement should be taken during this time interval. In such a case, at step 1050, the system calls the next event and queries whether the time interval for this event is set as "Trityl". If so, the system performs a trityl measurement and displays the trityl data to the user, as shown in step 1052. The trityl data may be shown in a graphical format for each channel performing the synthesis process, or may be displayed to the user in numbers or other formats.
It will be clear to those skilled in the art. An example is illustrated in the GUI 1200 shown in FIG.

Based on the trityl measurement, the system calculates and displays the efficiency data as shown in step 1054. In one embodiment, efficiency data is calculated using a second trityl integral as a reference base. During the first detritylation, a second trityl measurement is used as a reference base, since excess trityl is present in the eluate. In such a case, the first trityl measurement will not be an accurate estimate of efficiency. This trityl measurement integration is calculated and compared to the reference-based trityl integration to determine the efficiency using the following equation:

[0203]

(Equation 1) Here, nl is a number obtained by subtracting 2 from the trityl integration. Two is subtracted because the reference integral is based on the second trityl integral.

Next, the system tests to see if there is another event at step 1056. If there are no other events, the program ends at step 1058. However, if there is another event, the program returns to the point marked X in FIG. 10B as shown in step 1060 and continues to step 1032 and so on.

Returning to FIG. 11, the save trickle button 1112 opens another GUI 1300 as shown in FIG. GUI 1300 provides windows and buttons to save the selected trityl trace to a disk file. G
UI 1300 has two text windows. In the two text windows, instructions are given for saving the trityl file. The data entered into the channel number window 1304 selects a particular channel (ie, a DNA sequence) where the trityl trace is to be stored. The data input to the window disk file name window 1308 indicates the file name and position of the above-mentioned trityl trace. The file is easily read like an Excel spreadsheet. The data, including the analog trityl trace, traces the corrected trityl integral for each trace, time, data and event matrix identifier.

After entering the desired set of arrays, the user may click the START button 1116 at the bottom center of the screen to initialize the start of the synthesis process. After system initialization, the screen changes to another GUI where the monitoring screen is presented.

The STOP button 1120 can be used to end the program by setting the event counter and array counter to end values. By this subroutine, the rotary valve is set at the home position and the solenoid valve is set at the OFF state.

The stepper motor is rotated to the home position and stopped by the RValve Cal button 1124. The home position is identified by a zero position reference detector which detects the rotational position of the aperture disk by light from the LED lighting device and the silicon detector. As each motor reaches the home position, the text window 1104 corresponding to each channel turns green. If one of the motors does not reach the home position, the text window 1104 corresponding to that motor remains red.

Two windows 1132 and 11 located below the RV Set button 1128
There are 36. Press the RV Set button 1128 to select Channel Number (
Channel and rotary valve position (position) can be set. Next, the RV Set button 1128 causes the rotary valve to reach the requested position. If the required location is reached, the location window will be red, and if the required location is not the home location, location window 1136 will be green.

There are two windows 1144 and 1148 located below the SV “On” button 1140. With the SV “On” button 1140, the solenoid valve state can be set manually. The SV Valve window 1144 shows which valve is selected and the SV Time window 1148 shows the time interval for valve activation in milliseconds. If no time is given, the valve will switch to SV "OF
It remains active until the "F" button 1152 is clicked. If the valve continues to operate, the SV Time window 1148 will be green and will remain green during the time interval when the valve is active. When the valve stops, the SV Time window returns to normal color.

The “0” motor “ON” button 1156 causes motor excitation for 0 degree phase excitation for the motor. This excitation is used to mechanically set the aperture disk to one of the 0 degree phase excitations. This excitement is 10
It stays on for a second and then shuts off automatically to avoid motor burnout.

The RV idle button 1160 can be used to cause the rotary valve to enter an idle state. The O-ring of the rotary valve assembly tends to stick if the rotary valve remains inactive for an extended period of time. The RV idle button 1160 can be used to prevent the O-ring from locking due to the rotary valve movement pattern. In one embodiment, the pattern moves to the home position, moves one step clockwise from the home position,
It consists of a valve rotor that returns to the home position, moves one step counterclockwise from the home position, and then returns to the home position. This pattern is repeated on a system that stops for 10 seconds between each step.

[0213] The lead trityl A / D button 1168 is connected to the trityl channel window 117.
2 and A / D value window 1176. The user can set the channel number in the trityl channel window 1172 and then press the read trityl A / D button 1168 to obtain a trityl read for the selected channel. The raw data for the trityl measurement (ie, the trityl voltage) for the selected channel is displayed in the A / D value window 117.
6 is displayed. This serves as a check to ensure that the output from the output monitor has successfully reached the host computer. In one embodiment, the output monitor takes 10 seconds to read. These reads are used to adjust the potentiometer on the output monitor circuit board and calibrate the zero measurement. This is especially useful when the user changes the tubing of the system (eg, when the system requires recalibration).

A DecVal button 1180 and corresponding text window 1184 are
As shown in FIG. Using the DecVal button 1180, the system may read the binary value equivalent to the decimal number where the monitor is at the home position. The result of this test is shown in text window 1184.

[0215] The RV test button 1188 is also shown in FIG. This button can be used to test the rotary valve in any case by manually sending all buttons to the home position. As described above, if some of the motors do not reach the home position, the corresponding text window 1104 is red. For those motors that reach the home position, the corresponding text window 1104 is green, as indicated by the zero position reference sensor.

The form 3 button 1164 calls another GUI 1200 as shown in FIG. The GUI 1200 includes one trityl waveform window 1 for each channel.
Display 204 sets. Each window 1204 has two text boxes. That is, one box 1 for the associated rotary valve position
208, as well as another box 1212 for the current efficient calculations for this channel. Efficient calculations are performed on the corrected concentration integration performed on each trityl trace.
(gration) change.

The stop button 1216 terminates the synthesis and returns the rotary valve to the home position by using all closed solenoid valves. The pause button 1220 is
Sets a software flag that causes the program to pause at the end of the creation of the current basic form. The rotary valve is sent to the home position using all closed solenoid valves. Resume button 1224 may be used to restart a composition interrupted by a pause. The delay between pausing and resuming is over an hour. If the delay exceeds one hour, the program automatically ends as if the STOP button 1216 was pressed.

The form 1 button 1228 returns the user to a GUI 100 where data can be saved or a new batch of DNA sequences can be started.

The Sol A window 1232 displays the current low bit solenoid valve status as a decimal number. The Sol B window 1236 displays the current high bit solenoid valve status as a decimal number. Interval window 1240
Displays the time interval in milliseconds for the current event.

[0220] The event length window 1244 displays the number of events in the current event matrix. The event # window 1248 displays the number of events of the current event.

The sequence length window 1252 displays the maximum sequence length currently used by some of the channels, and the sequence number (Seq #) window 1256 displays the sequence number for the current base being generated.

VIII Environment The present invention may be implemented using hardware, software, or a combination thereof, and may be implemented in a computer system or other processing system. Indeed, in one embodiment, the invention relates to one or more computer systems capable of performing the functions described herein. An example of a computer system 1400 is shown in FIG. The computer system 1400 includes one or more processors, such as a processor 1404. Processor 1404 is connected to a communication infrastructure 1406 (eg, a communication bus, crossover, or network). Various software embodiments are described with respect to this exemplary computer system. After reading this specification, it will become apparent to a person skilled in the relevant art how to improve the invention using other computer systems and / or computer architectures.

[0223] Computer system 1400 may include a display interface 1402 for transferring graphics, text, and other data from communication infrastructure 1406 (or from a frame buffer (not shown)) for display on display unit 1430. Can be provided.

The computer system 1400 may also include a main memory 1408, preferably a Land Access Memory (RAM), and may include a secondary memory 1410. The secondary memory 1410 is, for example, a hard disk drive 1412 corresponding to a floppy (registered trademark) disk drive, a magnetic tape drive, an optical disk drive, or the like.
And / or may include an erasable storage drive 1414. Erasable storage drive 1414 reads from or writes to erasable storage unit 1418 in known manner. The erasable storage unit 1418 corresponds to a floppy (registered trademark) disk, a magnetic tape, an optical disk, and the like, and these are read and written by an erasable storage drive 1414. It is understood that the erasable storage unit 1418 comprises a computer usable storage medium having computer software and / or data stored thereon.

In alternative embodiments, secondary memory 1410 may include other similar means that allow a computer program or other instructions to be loaded into computer system 1400. For example, such means may include an erasable storage unit 1422 and an interface 1420. Such examples include program cartridges and cartridge interfaces (eg, as found in video game devices), erasable memory chips (EPROM or PROM).
(Eg, ROM) and associated sockets and other erasable storage units 1422 and interfaces 1420 that allow software and data to be transmitted from the erasable storage unit 1422 to the computer system 1400. .

The computer system 1400 also includes a communication interface 1424. Communication interface 1424 enables software and data to be transmitted between computer system 1400 and external devices. Examples of communication interface 1424 include a modem, a network interface, an Ethernet card, a communication port, a PCMCIA slot and card, and the like. The software and data transmitted via the communication interface 1424 may include electrical signals,
Signal 14, which may be an electromagnetic, optical, or other signal
28 format. These signals 1428 are provided for communicating with an interface 1424 via a communication path (ie, channel) 1426.
This channel 1426 carries a signal 1428 and may be a wire or cable,
It can be implemented using fiber optics, telephone lines, mobile phone lines, RF lines, and other communication channels.

As used herein, the terms “computer program media” and “computer usable media” are used, and generally refer to erasable storage drive 1414, hard disk installed on hard disk drive 1412, and signal 1
It is called a medium like 428. These computer program products are means for providing software to computer system 1400. The invention relates to such computer products.

A computer program (also called computer control logic) is stored in main memory 1408 and / or secondary memory 1410. A computer program may also be received via communication interface 1424. When such a computer program is executed, the computer system 1
400 enable performing the functions of the invention described herein.
In particular, the computer programs, when executed, enable processor 1404 to perform the functions of the present invention. Accordingly, such a computer program refers to the controller of the computer system 1400.

In embodiments where the present invention is implemented using software,
Using an erasable storage drive 1414, hard disk drive 1412, or communication interface 1424, software may be stored on a computer program product and loaded into computer system 1400. When executed by the processor 1404, control logic (software) causes the processor 1404 to perform the functions of the invention described herein.

In another embodiment, the invention relates to, for example, an application specific integrated circuit (
ASIC) and is preferentially implemented in hardware. Implementation of hardware state equipment so as to perform the functions described herein will be apparent to those skilled in the relevant art.

In yet another embodiment, the present invention is implemented using a combination of both hardware and software.

IX Examples In two embodiments of the invention as used for oligonucleotide synthesis, optimization of the event matrix 904 is performed by synthesizing the first oligonucleotide on this device. To cleave and deprotect the oligonucleotide product from the solid support. These oligonucleotides are then dried, resuspended in water and quantified using a spectrophotometer before being analyzed by high pressure liquid chromatography (HPLC). The results are shown in FIG. 15A, FIG. 15B, FIG. 16A and FIG.

The ideal efficiency of the synthesis using the β-cyanoethyl phosphoramidite method is 99.8%
It is. The present invention is capable of producing an oligonucleotide having a binding efficiency of 99.7% to 99.8%. Conventional synthesizers typically result in lower coupling efficiencies (eg, 98.5% to 99.5%).

Graphs 1504 and 1604 show the results of the HPLC analysis of the oligonucleotide, where the X-axis is minutes and the Y-axis is measured at the concentration of the absorbance unit measured by an ultraviolet (UV) detector. You. Tables 1508 and 1608 list the quantitative results of the HPLC analysis of the oligonucleotide. Columns 1512 and 1612 show the retention time between minutes for each peak appearing on the UV detector. Columns 1516 and 1616 are graphs 1504 and 1 respectively.
The height of each peak on 604 is shown. Columns 1520 and 1620 show the percent area under the peak on each graph based on integrating the area under the curve. Similarly, columns 1524 and 1624 show the percentage height of each peak on graphs 1504 and 1604, respectively.

This result is advantageous for oligonucleotides synthesized using the present invention. This is because the large peak corresponding to the oligonucleotide shown as peak 1528 in FIG. 15A and peak 1628 in FIG. 16A is much larger than the other peaks on the graph. As shown in Table 1508, peak 1528
The percent area under is 95.81%, and the percent area under the residual peak in FIG. 15A does not exceed 1%. Similarly, the percent area under the peak 1628 is 86.16%, and the percent area under the residual peak in FIG.
% Does not exceed.

(X Conclusion) Although the invention has been described and described in some details for purposes of clarity of understanding, it has implications for the scope of the invention or any particular embodiment. Without giving, equivalents may be practiced by modifying or altering the present invention within a wide range and equivalents of conditions, compositions, and other parameters,
It will be apparent to one skilled in the art that such modifications or alterations are intended to be included within the scope of the appended claims.

All publications, patents, and patent applications cited herein can be presented at the level of one of ordinary skill in the art to which this invention pertains, as if each individual publication, patent, and patent application was filed. The applications are hereby incorporated by reference to the same extent as if specifically and individually indicated to be incorporated by reference.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example, and not limitation. It will be apparent to those skilled in the art that various modifications in form and detail can be made herein without departing from the spirit and scope of the invention. This refers to technologies and terminology (te
rm) is true. Accordingly, the invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

The features and advantages of the present invention will become more apparent from the detailed description given above when taken in conjunction with the drawings. In the drawings, like reference numbers indicate identical or functionally similar elements. In addition, the leftmost digit of the reference number is
Identify the drawing in which the reference number first appears.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing an embodiment of the delivery system of the present invention.

FIG. 2 is a side view of a first embodiment of the rotary valve assembly, stepper motor and reaction chamber of the present invention.

FIG. 3 is a cross-sectional view of the rotary valve assembly and reaction chamber taken along line 3-3 of FIG.

FIG. 4 is a cross-sectional view of the rotary valve assembly taken along line 4-4 of FIG.

FIG. 5 is a partial cross-sectional view of the motor drive system of the present invention.

FIG. 6 is a block diagram of an output monitor according to the present invention.

FIG. 7 is a diagram of a part of the output monitor of FIG. 6;

8A-8C are sequence listings used in an exemplary synthesis process of the invention.

FIG. 9 is an event matrix used in an exemplary synthesis process of the present invention.

FIG. 10A is a high level flowchart of a method for performing a reaction according to the present invention.

FIG. 10B is a high level flowchart of a method for performing a reaction according to the present invention.

FIG. 10C is a high-level flowchart of a method for performing a reaction in accordance with the present invention.

FIG. 11 is a graphical user interface (GUI) for use in loading sequences in the present invention.

FIG. 12 is a GUI for use in monitoring the output of the system of the present invention.

FIG. 13 shows a GUI for use in saving data from the output monitor of the present invention.
It is.

FIG. 14 is an example of a computer system used to implement the present invention.

FIGS. 15A and 15B show an exemplary graph and a table of the results, respectively, of the HPLC analysis of a 17-mer oligonucleotide synthesized according to the present invention.

FIGS. 16A and 16B show an exemplary graph and a table of the results of HPLC analysis, respectively, of a 30-mer oligonucleotide synthesized according to the present invention.

FIG. 17 shows a level sensor system according to the present invention.

FIG. 18 is a cross-sectional view of the second embodiment of the rotary valve assembly taken along line 4-4 of FIG.

FIG. 19 shows the continuous output from the output monitor during the synthesis process using the present invention.

FIG. 20 shows an exploded view of a portion of the rotary valve assembly of the present invention.

FIG. 21 shows a perspective view of an alternative embodiment of the system of the present invention.

FIG. 22 shows a partial side cross-sectional view of the alternative embodiment system shown in FIG. 21.

FIG. 23 shows a partial top cross-sectional view of the system of the alternative embodiment shown in FIG. 21.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 1/10 C07K 1/10 G01N 37/00 101 G01N 37/00 101 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ) , BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, N, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP , KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Schett, William H. United States Virginia 22101, McLean, Claymore Court 6626 (72) Inventors Ally, Abdall H. United States Maryland 20878, Gaithersburg, Darnesstown Road 13905 F-term (reference) 4C057 AA30 4G068 AA02 AA04 AB01 AB11 AC17 AD40 AD45 AD47 AE03 AF07 AF09 4G075 AA01 BA10 BB05 BB10 CA63 DA02 ED08 FB02 FB04 FB06 4H045 FA

Claims (112)

[Claims] 1. A fluid or gas delivery system, comprising: a rotary valve having a stationary housing and a valve rotor,
A rotary valve, wherein the stationary housing comprises a plurality of inlet ports formed therein, and wherein the valve rotor is disposed for rotation within the stationary housing; and a check valve disposed on the rotary valve. A delivery system comprising: 2. The delivery system of claim 1, wherein the valve rotor has a passage formed therein such that the check valve is located within the passage. . 3. The delivery system of claim 2, wherein the check valve is located at one end of the passage adjacent the inlet port. 4. The delivery system according to claim 2, further comprising: a vortex chamber located at one end of the passage. 5. The delivery system of claim 4, wherein the swirl chamber has a conical shape and is configured to allow mixing of fluids therein. 6. The delivery system of claim 2, wherein the passage is configured to allow mixing of a fluid therein. 7. The delivery system of claim 6, wherein the volume of the passage is at least twice the volume of a minimum pulse of fluid delivered to the passage. 8. The delivery system of claim 6, further comprising a reaction chamber disposed in fluid communication with the rotary valve, such that the ratio of the volume of the passage to the volume of the reaction chamber is reduced. , The delivery system being in the range of 2: 1 to 1: 1. 9. The delivery system according to claim 1, wherein: a motor is disposed at one end of the motor.
A delivery system, further comprising: a motor. 10. The delivery system according to claim 9, wherein said motor is a stepper motor. 11. The delivery system of claim 1, wherein the check valve comprises a plurality of check valves, wherein each of the check valves is one of the plurality of inlet ports. Delivery system. 12. The delivery system according to claim 1, further comprising: a processor that controls rotation of the valve rotor of the rotary valve. 13. The delivery system of claim 1, wherein a fluid or gas enters the valve rotor from its lower surface and exits from its upper surface of the valve rotor. 14. The delivery system of claim 1, further comprising: a plurality of distribution valves for controlling distribution of a fluid or gas to the plurality of inlet ports on the rotary valve. Delivery system. 15. The delivery system according to claim 14, wherein said plurality of distribution valves are solenoid valves. 16. The delivery system according to claim 14, wherein: a processor, wherein the processor enables or restricts a flow of the fluid or gas to the plurality of inlet ports. A delivery system for controlling the plurality of dispensing valves. 17. The delivery system of claim 1, wherein said system is used to perform a synthesis process. 18. The delivery system according to claim 1, wherein the system comprises a plurality of rotary valves. 19. The delivery system of claim 18, wherein said plurality of rotary valves comprises between 2 and 96 rotary valves. 20. The delivery system according to claim 1, further comprising: at least one container containing the fluid or gas for supplying the system. 21. The delivery system of claim 1, wherein: a plurality of containers, each containing a fluid or gas, wherein the plurality of containers comprises:
A plurality of containers in fluid communication with the plurality of inlet ports of the rotary valve. 22. The delivery system of claim 1, further comprising: a source of pressurized gas for pressurizing the system. 23. The delivery system of claim 1, wherein the fluid comprises at least the following: a nucleotide, a nucleotide derivative, a labeled nucleotide, a wash solution, a capping reagent, an oxidizing agent, and a deblocking reagent. A delivery system selected from the group consisting of one. 24. The delivery system of claim 1, further comprising: a monitor for measuring the efficiency of the delivery system. 25. The delivery system of claim 1, further comprising: a monitor for measuring output quality of the delivery system. 26. The delivery system of claim 1, further comprising: a graphical user interface to assist in controlling the delivery system. 27. The delivery system of claim 24, comprising: a graphical user interface connected to the monitor, wherein the user can view efficiency data generated by the monitor; A delivery system, further comprising: a graphical user interface for using the efficiency data to control the synthesis process. 28. The delivery system of claim 1, further comprising: a reaction chamber fluidly connected to the rotary valve. 29. The delivery system of claim 28, wherein a support surface is located within said reaction chamber. 30. The support surface of claim 29, wherein the support surface is control pore glass.
A delivery system according to claim 1. 31. A fluid or gas delivery system for performing a synthesis process, comprising: a plurality of rotary valves; and a manifold disposed between the fluid or gas supply and the plurality of rotary valves. Wherein the manifold has an inlet and a plurality of outlets, and wherein the plurality of outlets are fluidly connected to an inlet port of each of the plurality of rotary valves. . 32. The delivery system of claim 31, further comprising: a plurality of distribution valves disposed between the fluid or gas supply and the manifold. 33. The delivery system of claim 31, wherein said plurality of rotary valves comprises between 2 and 96 rotary valves. 34. A valve for performing a synthesis process in a reaction chamber, comprising: a passage through which a fluid or gas flows; and a check valve disposed within the passage. Wherein the passage is in fluid communication with the reaction chamber. 35. A fluid or gas delivery system for performing a synthesis process, comprising: a passage, through which the fluid or gas flows, a vortex chamber, wherein the chamber comprises: A delivery system comprising: a vortex chamber disposed at one end of the passage and configured to allow mixing therein; and a reaction chamber disposed in fluid communication with the passage. 36. The delivery system according to claim 35, further comprising: a check valve disposed in the passage. 37. A system for performing a plurality of reactions simultaneously, comprising: a plurality of motors: a plurality of rotary valves each having a plurality of inlet ports and an outlet port, wherein the plurality of rotary valves have a plurality of inlet ports and an outlet port. A plurality of rotary valves, each of which has a valve rotor arranged to rotate therewith, wherein each of the valve rotors is connected to one of the plurality of motors. A manifold disposed in fluid communication with at least one inlet port of each of the plurality of rotary valves for distributing to the inlet port:
And a processor that controls movement and alignment of the valve rotor. 38. The system of claim 37, wherein: a plurality of dispensing valves, wherein the plurality of dispensing valves are connected to the plurality of inlet ports on the rotary valve. Or a plurality of distribution valves for controlling the flow of gas. 39. The system of claim 38, wherein said processor further controls said plurality of distribution valves to allow flow of said fluid or gas to said plurality of inlet ports. Or restricting, system. 40. The system of claim 37, further comprising: a source of pressurized gas for pressurizing the fluid in the system. 41. The system of claim 37, wherein: a plurality of reaction chambers, each of the reaction chambers being in fluid communication with the outlet port of one of the plurality of rotary valves. The system further comprising: a plurality of reaction chambers disposed. 42. The system of claim 37, wherein: a plurality of output monitors, each of said monitors for measuring an efficiency of said synthesis process in a respective reaction chamber. A plurality of output monitors disposed at an outlet of a plurality of reaction chambers, wherein the output monitor causes the system to terminate or alter the reaction. 43. The system of claim 37, wherein each of the valve rotors has a passage formed therein and a check valve disposed within the passage. 44. A rotary valve, comprising: a stationary housing having a plurality of inlet ports formed therein; and a valve rotor positioned for rotation within the stationary housing. And
Wherein the valve rotor has at least one connection port formed therein, aligned with the inlet port of the stationary housing, and an outlet port formed on the opposite side of the valve rotor. A rotary valve, comprising: a valve rotor. 45. A rotary valve, comprising: a stationary housing having a plurality of inlet ports formed therein; and a valve rotor, wherein the valve rotor comprises the stationary housing. Disposed within the stationary housing so as to be rotatable therein, wherein the rotary valve has a passage formed therein such that the passage allows mixing of the fluid therein. A rotary valve, comprising: a valve rotor. 46. The rotary valve of claim 45, wherein the volume of the passage is at least twice the volume of a minimum pulse of fluid delivered to the passage. 47. The rotary valve of claim 45, wherein the rotary valve is in fluid communication with a reaction chamber, wherein the ratio of the volume of the passage to the volume of the reaction chamber is 2: 1. A rotary valve that is in the range of １1: 1. 48. The rotary valve according to claim 45, further comprising: a plurality of O-rings disposed around the inlet port. 49. The rotary valve according to claim 45, further comprising: a check valve disposed in the passage of the valve rotor. 50. A rotary valve comprising: a stationary housing having a plurality of inlet ports formed therein; a valve rotor disposed within the stationary housing, such that the valve rotor is there. A rotary valve, comprising: a valve rotor: and a check valve disposed in the rotary valve. 51. The rotary valve according to claim 50, wherein said check valve is located within said valve rotor. 52. The rotary valve according to claim 50, wherein:
The check valve includes a plurality of check valves, each check valve comprising:
A rotary valve disposed at one of the plurality of inlet ports. 53. The rotary valve according to claim 50, wherein:
A rotary valve, wherein the valve rotor has a passage formed therein, and wherein the check valve is disposed within the passage. 54. The rotary valve according to claim 53, wherein said check valve is located at one end of said passage adjacent said plurality of inlet ports. 55. A method for automatic fluid delivery, comprising the steps of: (a) adding two or more fluids sequentially to a mixing chamber such that the fluids are mixed within the mixing chamber; Obtaining: and (b) delivering the fluid to a reaction chamber by adding an additional fluid or gas to the mixing chamber, wherein the reaction chamber is fluidly connected to the mixing chamber. A method comprising: 56. The method of claim 55, wherein the automated fluid delivery is for performing a synthesis process. 57. The method of claim 55, wherein said step (a)
) Comprises: (i) introducing a short pulse of a first reagent into the mixing chamber; (ii) introducing a short pulse of a second reagent into the mixing chamber; and (iii) a predetermined amount of Until the first and second reagents are introduced into the mixing chamber
Repeating steps (i) and (ii). 58. The method of claim 55, wherein said mixing chamber comprises a passage. 59. The method of claim 55, wherein the mixing chamber comprises: a passage: and a vortex chamber disposed at one end of the passage. 60. The method of claim 55, wherein the volume of the mixing chamber is at least twice the volume of a minimum pulse of reagent delivered to the mixing chamber. 61. A method for performing a reaction, comprising: (a) generating an event matrix for performing the reaction, wherein the event matrix has a plurality of events. (B) rotating a rotary valve to a first destination based on a first numerical value in the event matrix for a first event of the plurality of events; Activating a distribution valve based on a second value in the event matrix for the first event after reaching one destination: (d) a third value in the event matrix for the first event. Performing a time delay for the distribution valve based on: (e) deactivating the distribution valve when the time delay is completed; and (f) steps (b)-(e). Enough repetition, step of performing the reaction provided by the event matrix includes, a method. 62. The method of claim 61, wherein said event matrix is generated based on a nucleotide sequence. 63. The method of claim 61, wherein said event matrix is generated based on an amino acid sequence. 64. The method of claim 61, wherein said event matrix is based on a reaction to make an oligonucleotide. 65. The method of claim 61, wherein said event matrix is based on a reaction to make a peptide. 66. The method of claim 61, further comprising: (h) deactivating the distribution valve prior to step (b). 67. The method of claim 61, further comprising: (h) monitoring the efficiency of the reaction. 68. The method of claim 67, further comprising: (i) modifying the event matrix based on a result of the monitoring step. 69. The method of claim 67, further comprising: (i) interrupting the reaction based on the results of the monitoring step. 70. The method of claim 61, wherein said reaction comprises oligonucleotide synthesis. 71. The method of claim 61, wherein said reaction comprises peptide synthesis. 72. A computer program product comprising a computer usable medium having computer program logic recorded in a computer to enable a processor in a computer system to perform a reaction. The computer program logic comprises: generating means for enabling the processor to generate an event matrix for a desired response, wherein the event matrix has a plurality of events; Rotating means for enabling the processor to rotate the rotary valve to the destination based on the values in the event matrix for each of the events of the event; based on the values in the event matrix for each event To enable the processor to operate the distribution valve. Activation means for enabling the processor to perform a time delay on the dispensing valve based on a value in the event matrix for each event; expiration of the time delay Control means for allowing the processor to deactivate the dispensing valve if the processor program product has been activated. 73. The computer program product of claim 72, wherein the control means further stops the actuation of the distribution valve prior to rotation of the rotary valve. 74. The computer program product of claim 72, comprising: calculating means for enabling the processor to calculate efficiency data for the reaction; and wherein the processor comprises the efficiency data. Display means for enabling the display of
A computer program product, further comprising: 75. A method for monitoring the synthesis of a peptide or oligonucleotide comprising the steps of: (a) performing a synthesis process to produce a desired sequence; Monitoring the synthesis process to determine. 76. The method of claim 75, wherein said monitoring determines an amino acid sequence of the synthesized peptide. 77. The method of claim 75, wherein said monitoring determines a nucleic acid sequence of the synthesized oligonucleotide. 78. The method of claim 75, wherein a dye is added to one or more reagents used during the synthesis process. 79. A method for performing a reaction in a synthesis system, comprising: (a) introducing a short pulse of a first reagent into the system; Introducing a short pulse of a second reagent; and (c) until a predetermined amount of said first reagent and said second reagent pass through said system.
Repeating the steps (a) and (b). 80. The method according to claim 7, wherein the first reagent and the second reagent are the same.
10. The method according to 9. 81. The method of claim 80, wherein said first reagent and said second reagent are detritylation reagents. 82. The method of claim 79, wherein said first reagent is a first portion of a capping reagent and said second reagent is a second portion of said capping reagent. 83. The method of claim 79, wherein said first reagent is a phosphoramidite and said second reagent is an activator. 84. A method for aligning reagents around ports of a rotary valve to perform a synthesis process, comprising the steps of: (a) activating a port adjacent to each port comprising a phosphoramidite. Placing a beta; (b) placing a detritylation reagent proximate a port comprising a wash solution; (c) proximate a second port of the two-port capping reagent; Locating a port; and (d) locating a wash solution proximate one of the first port and the second port of the capping reagent. . 85. The method of claim 84, further comprising: (e) locating a port for the gas proximate the home port location. 86. A device at a port around a rotary valve for performing a synthesis process, comprising: a port for an activator in proximity to each port comprising a phosphoramidite; a port in proximity to a port comprising a wash solution. A first port of a two-port capping reagent proximate a second port of the two-port capping reagent; and a proximate one of the first port and the second port of the capping reagent. An at least one of a port for a washing solution to be applied. 87. The apparatus of claim 86, further comprising: a port for gas proximate to the home port location. 88. A monitor for use in monitoring a reaction, comprising: a light sensor, wherein the light sensor is mounted on both sides of a tube used for the reaction. A first LED of a wavelength, a second LED of a second wavelength, and a detector assembly mounted on the opposite side of the tube, the first wavelength and the second
A light sensor having different wavelengths; an LED driver for controlling illumination of the first LED and the second LED; a first narrow slit disposed between the tube and the first LED and the second LED; A second narrow slit disposed between a tube and the detector assembly and further diametrically opposed to the first narrow slit; and a signal processor. 89. The monitor of claim 88, wherein said LED driver turns on said LED alternately. 90. The first LED is a blue LED and the second LE
89. The monitor of claim 88, wherein D is a red LED. 91. The monitor of claim 88, wherein the first narrow slit and the second narrow slit are parallel and concentrated on an axis of the tube, and wherein the first narrow slit and the second narrow slit The width of a second narrow slit is selected to fit the inside diameter of the tube, and the length of the first narrow slit and the second narrow slit match the first LED and the second LED Monitor equal to diameter. 92. The monitor of claim 88, wherein the detector assembly comprises: a silicon diode photodetector; and an internal amplifier. 93. The monitor of claim 88, wherein the LED driver comprises: an analog square wave oscillator that provides a low power impedance square wave signal driving the first LED and the second LED; A plurality of variable series resistors that enable the output from the first LED and the second LED to meet standard values. 94. The monitor according to claim 88, wherein the first LED and the second LED are mutually connected with a series resistor having opposite polarities such that the first LED and the second LED are alternately turned on during a half cycle of the square wave. Monitors connected in parallel. 95. The monitor of claim 88, wherein the signal processor comprises: a synchronous demodulator for separating the output signals from the first LED and the second LED, producing a differential output. A low-pass filter for filtering the differential output; and converting the filtered differential output to digital data for processing;
An analog-to-digital converter card, wherein the digital data is linearized using an empirical formula developed by correcting a voltage versus concentration curve.
A monitor with a converter card. 96. A method for monitoring a reaction online, comprising: (a) adding a predetermined amount of fluid to a system; (b) monitoring output data from the system; And (c) providing the output data to a processor, the processor comprising:
Analyze the output data to determine whether the reaction with the fluid has ended, and if necessary, actively adjust the amount of the fluid provided to the system based on the output data. Performing the method. 97. The following steps: (d) calculating efficiency data from the output data; and (e) terminating the reaction if the output data exhibits an efficiency below a predetermined level. 97. The method of claim 96, further comprising: 98. A method for online monitoring of a reaction in a reaction chamber during a synthesis process in a synthesis system, comprising: (a) inputting a desired sequence into the system; (b) A) adding nucleotides or amino acids or their derivatives to the reaction chamber; c) monitoring output data from the system; and d) determining whether the correct nucleotides or amino acids have been added. Comparing the desired sequence against the output data. 99. A motor drive system, comprising: a computer controlled motor having a shaft extending from the motor; and converting signals from the processor to signals readable by the motor. A motor drive system, comprising: driver electronics attached to the motor to drive the motor. 100. The motor drive system of claim 99, further comprising: a zero position reference system mounted on the shaft of the motor. 101. The motor drive system according to claim 99, wherein said computer controlled motor is a servo motor with an encoder. 102. The motor drive system according to claim 99, wherein said computer controlled motor is a servo motor with a resolver. 103. The motor drive system according to claim 99, wherein said computer controlled motor is a stepper motor. 104. The motor drive system of claim 99, wherein the motor drive system is connected to a system for performing a synthesis process. 105. A motor drive system, comprising: a motor having a shaft extending from the motor, the shaft comprising:
A motor having a first end extending from an upper surface of the motor and a second end extending from a lower surface of the motor; a zero position reference system attached to the second end of the shaft; and a processor. A driver electronics mounted on the lower surface of the stepper motor for converting the signal from the motor into a signal readable by a stepper motor. 106. The motor drive system according to claim 105, wherein the zero position reference system comprises: an LED; a silicon detector having a detection element; and a circle attached to the second end of the shaft. A rotating aperture disk having an opening, wherein the LED is located on one side of the circular opening of the rotating aperture disk and the silicon detector is located on the other side of the circular opening. A motorized drive system, comprising: a rotating aperture disk mounted and diametrically opposed to the LED. 107. The motor drive system according to claim 106, wherein said zero position reference system further comprises an operational amplifier. 108. The motor drive system according to claim 106, wherein the width of the circular opening is equal to the width of the detection element. 109. The motor drive system according to claim 106, wherein the detector element has the highest signal available when the circular aperture of the aperture disc does not cover light from the LED. The motor drive system is mounted with a major axis that is radial to the shaft. 110. A level sensor for a bottle containing liquid, comprising: an LED disposed around a curve on the outer surface of the bottle; and a LED around another curve on the outer surface of the bottle. A detector disposed on the opposite side of the LED, wherein the detector is configured to detect a first one of the light emitted from the LED when a fluid level in the bottle is above the detector and the LED. And when the fluid level in the bottle is below the detector and the LED,
A detector for detecting a second amount of light emanating from the LED. 111. The level sensor of claim 110, wherein the signal from the detection assembly provides a signal to an alarm when the fluid level in the bottle is below the LED and the detector. A level sensor that is sent to the processor for sending. 112. The delivery system of claim 1, wherein the delivery system is used to perform a reaction, the reaction comprising: oligonucleotide synthesis, peptide synthesis, polysaccharide synthesis, protein synthesis, At least one of protein purification, peptide purification, DNA purification, RNA purification, and biomolecule purification
A delivery system selected from the group consisting of: