Fixed solid phase synthesizer pipe-line systemTechnical Field
The utility model belongs to the technical field of gene synthesis supporting equipment, and particularly relates to a pipeline system of a fixed solid phase synthesizer.
Background
With the development of synthetic biology, the demand for synthetic primers is increasing; with the requirement of intelligent manufacturing, integration, platformization and modularization are current trends. To cope with the more diverse needs of customers, better service customers; simple production process and high product efficiency. Providing another synthesis mode for synthesis; enriches the existing product line.
The existing oligonucleotide solid phase synthesis synthesizer can be divided into a mobile platform synthesizer and a rotary platform synthesizer according to the structure constitution; the device can be divided into a microfluidic liquid adding module at the front end and a reaction bin at the rear end according to the process flow and a gas-liquid treatment module; our development is based on the high integration multi-channel gas-liquid processing device that rotary platform used.
At present, a solid-phase phosphoramidite triester method is basically adopted for primer synthesis. The solid phase phosphoramidite triester method for synthesizing DNA fragment has the characteristics of high efficiency, rapid coupling and relatively stable initial reactant. The method is to complete the synthesis of DNA chain on solid carrier, and the synthesis direction is from 3' end, and adjacent nucleotides are connected via 3 '. Fwdarw.5 ' phosphodiester bond. The specific reaction steps are as follows:
firstly, deprotecting a protecting group (Deblocking), reacting an active group protected on a solid phase carrier (CPG, resin and the like) with trichloroacetic acid, and removing a protecting group DMT of a 5 '-hydroxyl group of the active group to obtain a free 5' -hydroxyl group;
second, activation (Activation) of mixing phosphoramidite protected nucleotide monomers with tetrazole activator and feeding the mixture into a synthesis column to form a phosphoramidite tetrazole active intermediate (the 3 '-end of which is activated but the 5' -end of which is still protected by DMT), wherein the intermediate is subjected to condensation reaction with deprotected nucleotides on GPG;
thirdly, connecting (Coupling), when the phosphoramidite tetrazole active intermediate meets the nucleotide with the deprotected group on CPG, carrying out affinity reaction with the 5' -hydroxyl, condensing and removing tetrazole, and extending the synthesized oligonucleotide chain forward by one base;
fourth, capping (Capping), after the condensation reaction, to prevent the unreacted 5' -hydroxyl group attached to CPG from being extended in the subsequent cyclic reaction, the terminal hydroxyl group is usually blocked by acetylation, typically an acetylating agent is formed by mixing acetic anhydride with N-methylimidazole or the like;
fifth, oxidation (Oxidation), the nucleotide monomer is connected with the oligonucleotide connected on CPG through the phosphoester bond during condensation reaction, the phosphoester bond is unstable and easy to be hydrolyzed by acid and alkali, at this time, the phosphorothioate is converted into phosphotriester by using tetrahydrofuran solution of iodine, and the stable oligonucleotide is obtained.
Through the above five steps, one deoxynucleotide is attached to the nucleotide of the solid support. The trichloroacetic acid is then used to remove the protecting group DMT on its 5' -hydroxyl, and the above steps are repeated until all the bases to be synthesized have been attached. The color of the TCA processing stage can be observed in the synthesis process to determine the synthesis efficiency.
DNA synthesizers based on the above methods have a variety of main differences in the synthesis yield, the difference in reagent consumption and the amount of time for a single cycle. In addition, when different monomers are synthesized, the synthesis is carried out in a reaction column of a synthesizer, a plurality of different types of deoxynucleotides are synthesized one by one to form single-stranded nucleotides (or called primers), various required monomers and common reagents are conveyed into the synthesis column through a pipeline to react, for example, after the monomer A is synthesized, the monomer A is contained in the pipeline, and when the monomer T is synthesized, the synthesis of the monomer T is influenced by the A mixed in the pipeline; or when different monomers are synthesized and switched, the pipeline is filled with air, and the air contains moisture, belongs to impurities and affects the quality of the finally synthesized single-stranded nucleotide.
The utility model aims to provide a pipeline system of a fixed solid phase synthesizer, which can solve the problems.
Disclosure of Invention
The utility model mainly solves the technical problem of providing a fixed solid phase synthesizer pipeline system, which can not pollute each other in liquid adding, is flexible in liquid adding, reduces impurities existing in the primer synthesizing process and ensures the quality of synthesized primers.
In order to solve the technical problems, the utility model adopts a technical scheme that: the utility model provides a fixed solid phase synthesizer pipeline system which comprises a TCA pipeline subsystem, a monomer pipeline subsystem, an active agent pipeline subsystem, an oxidant pipeline subsystem, a cap pipeline subsystem, an acetonitrile pipeline subsystem, a switching valve and a two-way valve;
the TCA pipeline subsystem, the monomer pipeline subsystem, the active agent pipeline subsystem, the oxidant pipeline subsystem and the cap pipeline subsystem respectively comprise a reagent bottle, a pipeline and a three-way two-position valve, one end of the pipeline is communicated with the reagent bottle, the three-way two-position valve is positioned in the pipeline, the other end of the pipeline is connected with a switching valve, the switching valve is connected with a synthesis column through a main pipe, and the synthesis column is connected with a waste liquid tank through a waste liquid pipeline;
the acetonitrile pipe subsystem comprises a reagent bottle and a flushing pipeline, one end of the flushing pipeline is communicated with the reagent bottle, the other end of the flushing pipeline is divided into a plurality of branch pipelines, and each branch pipeline is communicated with one three-way two-position valve.
Further, the switching valve comprises a left switching valve and a right switching valve, the two-way valve comprises a left two-way valve and a right two-way valve, the TCA pipeline subsystem and the monomer pipeline subsystem are respectively communicated with the left switching valve, and the active agent pipeline subsystem, the oxidant pipeline subsystem and the cap pipeline subsystem are respectively communicated with the right switching valve;
the acetonitrile pipe subsystem is simultaneously communicated with the left switching valve and the right switching valve;
the main pipe comprises a left main pipe and a right main pipe, the left two-way valve is located in the left main pipe, the left switching valve is communicated with the synthetic column through the left main pipe, the right two-way valve is located in the right main pipe, and the right switching valve is communicated with the synthetic column through the right main pipe.
Further, the monomer pipe subsystem comprises a monomer A pipe subsystem, a monomer G pipe subsystem, a monomer C pipe subsystem and a monomer T pipe subsystem.
Further, the cap line subsystem includes a cap a line subsystem and a cap B line subsystem.
Further, the two-way valve is a two-way two-position valve.
Further, the switching valve is a multiple-in-one-out valve.
Further, the three-way two-position valve is a three-way two-position electromagnetic valve.
The beneficial effects of the utility model are as follows:
the utility model carries out reasonable and ingenious design on the pipeline system, various monomers, oxidants, active agents and the like used in primer synthesis are all synthesized by adopting independent subsystems and finally entering a synthesis column through a switching valve and a two-way valve, in addition, a flushing reagent (namely acetonitrile) pipeline subsystem is communicated with the subsystems and used for flushing different reagents or residual substances in a pipeline when the monomers are switched, so that the purity of the next substance entering the pipeline is ensured, and the pipeline is filled with acetonitrile when the different reagents are switched, so that air cannot exist in the pipeline, moisture is completely eradicated, and the purity and quality of primer synthesis are ensured.
The foregoing description is only an overview of the present utility model, and is intended to provide a better understanding of the present utility model, as it is embodied in the following description, with reference to the preferred embodiments of the present utility model and the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of the principles of the present utility model;
the reference numerals are as follows:
the TCA pipeline subsystem 1, a reagent bottle 11, a pipeline 12 and a three-way two-position valve 13;
a monomer A pipeline subsystem 2, a monomer G pipeline subsystem 3, a monomer C pipeline subsystem 4, a monomer T pipeline subsystem 5, an active agent pipeline subsystem 6, an oxidant pipeline subsystem 7, a cap A pipeline subsystem 8 and a cap B pipeline subsystem 9;
acetonitrile pipe subsystem 10, reagent bottle 101, flushing pipeline 102 and branch pipeline 103;
a left switching valve 20, a right switching valve 30, a left two-way valve 40, a right two-way valve 50, a synthesis column 60, a waste liquid line 70, a waste liquid tank 80, a left main pipe 90, and a right main pipe 100.
Detailed Description
The following specific embodiments of the utility model are described in order to provide those skilled in the art with an understanding of the present disclosure. The utility model may be embodied in other different forms, i.e., modified and changed without departing from the scope of the utility model.
Examples: the fixed solid phase synthesizer pipeline system comprises a TCA pipeline subsystem 1, a monomer pipeline subsystem, an active agent pipeline subsystem 6, an oxidant pipeline subsystem 7, a cap pipeline subsystem, an acetonitrile pipeline subsystem 10, a switching valve and a two-way valve as shown in figure 1;
the TCA pipeline subsystem, the monomer pipeline subsystem, the active agent pipeline subsystem, the oxidant pipeline subsystem and the cap pipeline subsystem respectively comprise a reagent bottle 11, a pipeline 12 and a three-way two-position valve 13, one end of the pipeline is communicated with the reagent bottle, the three-way two-position valve is positioned in the pipeline, the other end of the pipeline is connected with a switching valve, the switching valve is connected with a synthetic column 60 through a main pipe, and the synthetic column is connected with a waste liquid tank 80 through a waste liquid pipeline 70;
the acetonitrile pipe subsystem comprises a reagent bottle 101 and a flushing pipeline 102, one end of the flushing pipeline is communicated with the reagent bottle, the other end of the flushing pipeline is divided into a plurality of branch pipelines 103, and each branch pipeline is communicated with one three-way two-position valve.
The switching valve comprises a left switching valve 20 and a right switching valve 30, the two-way valve comprises a left two-way valve 40 and a right two-way valve 50, the TCA pipeline subsystem and the monomer pipeline subsystem are respectively communicated with the left switching valve, and the active agent pipeline subsystem, the oxidant pipeline subsystem and the cap pipeline subsystem are respectively communicated with the right switching valve;
the acetonitrile pipe subsystem is simultaneously communicated with the left switching valve and the right switching valve;
the main pipe comprises a left main pipe 90 and a right main pipe 100, the left two-way valve is located in the left main pipe, the left switching valve is communicated with the synthetic column through the left main pipe, the right two-way valve is located in the right main pipe, and the right switching valve is communicated with the synthetic column through the right main pipe.
The monomer pipe subsystem comprises a monomer A pipe subsystem 2, a monomer G pipe subsystem 3, a monomer C pipe subsystem 4 and a monomer T pipe subsystem 5.
The cap line subsystem includes a cap a line subsystem 8 and a cap B line subsystem 9.
In this embodiment, the two-way valve is a two-way two-position valve.
In this embodiment, the switching valve is a multiple-in-one-out valve. In this embodiment, the switching valve is an amide valve, which is corrosion-resistant, and when one inlet is opened, the other inlets are closed, and the outlet is opened, and the switching valve itself is not known in the art, so that the principle is not repeated.
In this embodiment, the three-way two-position valve is a three-way two-position electromagnetic valve.
In this embodiment, the three-way two-position valve, the left two-way valve, the right two-way valve, the left switching valve and the right switching valve are all electrically connected with a controller of the synthesizer. The controller is used for automatically controlling the switching of the three-way two-position valve, the switching of different pipeline systems of the switching valve and the switching of the two-way valves. The corresponding reagent in the reagent bottle is pressed into the pipeline by the pressure difference through the pressure stabilizing system, and the micro-milliliter level liquid injection can be accurately realized through the pressure stabilizing system.
The working principle and the working process of the utility model are as follows:
in the solid phase phosphoramidite triester method, reagents are required to be added according to different sequences; finishing the synthesis of the primer;
the following systems need to realize different sequential liquid feeding, and the following functions are needed to be achieved;
first, liquid feeding accuracy
Secondly, the liquid adding can not pollute each other;
thirdly, adding the required reagent at any time according to the liquid adding flexibility;
the utility model can completely achieve the functions by reasonably and skillfully designing the pipeline system, and ensures the synthesis quality, purity and the like of the primer.
The method comprises the following steps:
taking TCA injection as an example:
the three-way two-position valve of the normal TCA pipeline subsystem is communicated with the branch pipeline of the acetonitrile pipeline subsystem, so that the whole pipeline is ensured to be full of acetonitrile;
when TCA needs to be added, a three-way two-position valve of a TCA pipeline subsystem is switched to be communicated with a pipeline of the TCA pipeline subsystem, then quantitative TCA injection is carried out, at the moment, the pipeline of the TCA pipeline subsystem is communicated with a left switching valve, the left switching valve is simultaneously communicated with a left main pipe, meanwhile, the left switching valve is not communicated with other pipelines (specifically referred to as pipelines of all monomer pipeline subsystems), the left two-way valve of the left pipeline is in an open state, the corresponding actions of the valves are controlled by a controller, the TCA injection is controlled to be injected into a synthesis column all the time, after the injection amount of TCA meets the requirement, the three-way two-position valve of the TCA pipeline subsystem is controlled to be communicated with a branch pipeline of an acetonitrile pipeline subsystem, and then acetonitrile is used for slowly injecting TCA reagent remained in the pipeline into the synthesis column (reactor); then, the operation is completed; after judging that the step is finished; the acetonitrile liquid can clean the pipeline and prepare the pipeline for the next liquid beating; similarly, the liquid beating of other reagents or monomers is also carried out according to the steps, the synthesis of the primer is sequentially realized, and the waste liquid flows back into the waste liquid box under the action of gravity;
because the power of various reagents depends on the pressure at the reagent bottle during liquid pumping (the effect of the other pressure stabilizing system of the synthesizer is not protected by the utility model and is not repeated), the three-way two-position valve of each subsystem is communicated with the pipeline of the system or the branch pipeline of the acetonitrile pipeline subsystem, for example, when TCA is pumped in, the three-way two-position valve of the TCA pipeline subsystem is switched to be communicated with the pipeline of the TCA pipeline subsystem, and other subsystems are all switched to be communicated with the branch pipeline of the acetonitrile pipeline subsystem; therefore, by arranging the multiple-inlet-one-outlet switching valve, when one inlet is opened, the other inlets are closed, so that the pressure difference generated by the pipeline can be prevented from countercurrent flow of TCA (TCA at this time) along the pipelines of other subsystems, and other reagents or acetonitrile and the like are polluted.
In this embodiment, the two-way valve can be in a closed state after the liquid is pumped, and pressure in the pipeline is ensured without pressure relief.
In this example, the reagent displacing the common reagent line is flushed by letting acetonitrile be the common reagent; the mutual non-cross contamination is realized;
flexibility of filling, now all reagents are in the same filling priority position; one or more mixed reagents can be added at any time; meets the synthesis requirement.
The foregoing description is only illustrative of the present utility model and is not intended to limit the scope of the utility model, and all equivalent structures made by the description of the utility model and the accompanying drawings, or direct or indirect application in other related technical fields, are included in the scope of the utility model.