KR101677558B1 - column loading and frit removal equipment for synthesis and deprotection of oligonucleotide - Google Patents

column loading and frit removal equipment for synthesis and deprotection of oligonucleotide Download PDF

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KR101677558B1
KR101677558B1 KR1020150065901A KR20150065901A KR101677558B1 KR 101677558 B1 KR101677558 B1 KR 101677558B1 KR 1020150065901 A KR1020150065901 A KR 1020150065901A KR 20150065901 A KR20150065901 A KR 20150065901A KR 101677558 B1 KR101677558 B1 KR 101677558B1
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South Korea
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head frame
column
frit
plate
synthesis
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KR1020150065901A
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Korean (ko)
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채순기
송광용
정호진
이근우
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배재대학교 산학협력단
주식회사네오프로브
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0053—Details of the reactor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

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  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
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  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
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  • Genetics & Genomics (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Apparatus Associated With Microorganisms And Enzymes (AREA)
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Abstract

The present invention relates to a column mounting and frit removing apparatus for synthesizing and deprotecting oligonucleotides, and more particularly, to a method of inserting a large amount of column for oligonucleotide synthesis into a plate, To a column mounting and frit removing apparatus for oligonucleotide synthesis and deprotection capable of easily performing a process of removing a large amount of frit.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to column-loading and frit removal equipment for synthesis and deprotection of oligonucleotides,

The present invention relates to a column mounting and frit removing apparatus for synthesizing and deprotecting oligonucleotides, and more particularly, to a method of inserting a large amount of column for oligonucleotide synthesis into a plate, To a column mounting and frit removing apparatus for oligonucleotide synthesis and deprotection capable of easily performing a process of removing a large amount of frit.

Oligonucleotide is a short single-stranded DNA (deoxyribonucleic acid) or RNA (ribo-nucleic acid) consisting of 2'-deoxyribonucleotides (oligodeoxyribonucleotides) and is widely applied in genetic testing, research and scientific investigation. Generally, the oligonucleotide is synthesized by an automated device in the sequence specified by the user through chemical synthesis on a solid support. The synthesized oligonucleotide is polymerized by polymerase chain reaction (PCR), DNA sequencing, molecular hybridization, mutation, and library.

The method of synthesizing these oligonucleotides is the most commonly used 'phosphate triester' method which connects phosphodiester bonds forming the backbone of DNA structure using β-cyanoethyl phosphoramidite developed by Koster.

Specifically, the synthesis of the oligonucleotide is carried out in a cycle of 1) detritylation, 2) coupling, 3) capping, and 4) oxidation. The oligonucleotides can be synthesized by joining dA, dG, dT and dC in sequence according to the nucleotide sequence to be synthesized. When synthesis is complete, ammonium hydroxide is added to separate the oligonucleotide from the resin.

The synthesis of the oligonucleotides described above involves the use of a chemical modification base, which contains a considerable amount of bases or impurities that have not participated in the synthesis. Finally, oligonucleotides are synthesized by a purification process to remove them.

Related art is disclosed in Japanese Laid-Open Patent Application No. 2014-047169 ("oligonucleotide synthesizing apparatus and synthesizing method").

Conventional synthesizers synthesized oligonucleotides by using a well plate (typically 96-well deep well) and inserting a plurality of synthetic columns into a plate. Therefore, conventionally, since each column is inserted into the plate with its height adjusted, it takes a lot of time and labor.

Japanese Laid-Open Patent Application No. 2014-047169 ("oligonucleotide synthesizing apparatus and synthesizing method")

SUMMARY OF THE INVENTION The present invention has been made in order to solve the above-mentioned problems, and an object of the present invention is to provide an apparatus and a method for inserting a large number of columns at a uniform height by eliminating the inconvenience of inserting a column into a plate, And to provide a column mounting and frit removing apparatus for oligonucleotide synthesis and de-protection designed to remove the solid support chain frit at a time after completion of the synthesis.

The present invention relates to a column mounting and frit removing apparatus for the synthesis and deprotection of oligonucleotides and includes a receiving unit 110 placed on the bottom surface, A main body 100 including a strut 120 fixed to one side of the main body 110 in a vertical direction and a lifting part 130 connected to the strut 120 and vertically lifted in accordance with the operation of the lever 140, A rod 200 provided below the lifting part 130 and including a coupling part 210 to which the following head frame 300 is coupled at a lower part and a rod 200 installed on the supporting part 110, A lower jig 400 in which a plurality of columns for inserting a plurality of columns are inserted and an upper end thereof are coupled to the coupling portion 210. When the elevation portion 130 is lowered by the operation of the lever 140, The inserted column is pressed toward the bottom surface, and the head frame (300) and the head frame (300) is formed in a 'T' shape at an upper end of a vertically cut plane with respect to the ground, and the coupling part (210) has a hollow part formed therein, and the head frame (300) So that the upper end portion of the main body can be attached or detached.
The head frame 300 is provided with a flat head frame 310 having a lower flat plate shape and the flat head frame 310 is fixed to the upper surface of the column 300, And the head frame 300 is provided with a pin-shaped head frame 320 having a lower portion formed in a plate shape and a plurality of pins corresponding to the column formed on a lower side thereof, The head frame 320 simultaneously presses the column so that the fins are inserted into each column for frit removal in the plurality of columns.
In order to prevent movement and detachment of the head frame 300 after mounting the head frame 300 on the rod 200, the rod 200 is formed with a fixing part 220 protruding into the hollow part, And a groove 301 corresponding to the fixing portion 220 is formed on the lower jig 400. The lower jig 400 is formed with an upper portion recessed inwardly to fix and fix the plate, And a shape in which a step is formed and the width becomes narrower toward the inner side.

Conventionally, when a large amount of oligonucleotides are synthesized, a plurality of columns have to be inserted one by one in the plate, so that the process time has to be increased and the manpower to be added to the process has to be increased.

The device according to the present invention devises a device for synthesizing and deprotecting oligonucleotides, which is achieved by mounting a rod, a head frame and a lower jig to a widely used hand press. Therefore, it is possible to uniformly insert a large number of columns uniformly at the same time with a simple structure of the apparatus, and it is possible to easily remove a large amount of frit for deprotection after completion of oligonucleotide synthesis by replacing the head frame There is an effect that can be.

Therefore, not only the process time is shortened but also the work amount is remarkably reduced, which is advantageous in the overall cost saving effect.

1 is a perspective view illustrating operation of an apparatus according to an embodiment of the present invention;
2 is a perspective view illustrating operation of an apparatus according to another embodiment of the present invention;
FIG. 3 is a perspective view (head frame of FIG. 1) showing a rod and a head frame according to an embodiment of the present invention;
4 is a perspective view (head frame of FIG. 2) showing a rod and a head frame according to another embodiment of the present invention;
5 is a cross-sectional view of a rod according to an embodiment of the present invention;
6 is a schematic view illustrating a lower jig according to an embodiment of the present invention;
7 is a schematic cross-sectional view of a lower jig according to various embodiments of the present invention.

Hereinafter, the technical idea of the present invention will be described more specifically with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are included to provide a further understanding of the technical concept of the present invention, are incorporated in and constitute a part of the specification, and are not intended to limit the scope of the present invention.

The present invention relates to a column mount and frit stripper for the synthesis and deprotection of oligonucleotides, the device according to an embodiment of the present invention comprises A main body 100, a rod 200, a lower jig 400, and a head frame 300, each of which will be described with reference to Figs.

As shown, the main body 100 includes a support 110 that rests on a floor surface, a support 120 that is vertically fixed to one side of the support 110, And a lifting unit 130 which is lifted and lowered according to the operation of the lifting unit 130. At this time, the main body 100 is generally the same as the hand press structure used for various purposes such as punching, compression, cutting, and bending. If the lever 140 is pulled downward, the elevation part 130 is lowered. On the contrary, if the lever 140 is raised upward, the elevation part 130 can be designed to be designed in various forms other than the structure shown in FIG.

3 and 4, the rod 200 is provided below the elevating part 130 (provided in the lower space of the elevating part 130 in FIG. 1), and a head frame 300, which will be described later, And an engaging portion 210 to which the engaging portion 210 is coupled. The upper portion of the rod 200 is fixedly coupled to the lower portion of the elevation portion 130 of the main body 100 and the head frame 300 is mounted on the lower portion thereof.

In addition, the lower jig 400 is installed on the receiving part 110, and a synthetic plate P on which a plurality of columns C for reaction are inserted is placed on the upper part. At this time, the column (C) uses the one containing the frit (Frit) which is a solid support (CPG) inside to synthesize oligonucleotide (refer to FIG. 7).

The upper portion of the head frame 300 coupled to the rod 200 is coupled to the coupling portion 210 of the rod 200. When the lift portion 130 is lowered by the operation of the lever 140, P to press the column C toward the bottom surface. 1 (a), a plurality of columns C are inserted into the synthetic plate P and placed on the lower jig 400 in a state where the lever 140 is lifted, and then the lever 140 is moved downward The head frame 300 presses the plurality of columns C downward as the lifting unit 130 descends.

Conventionally, in order to mount the columns on the plate for synthesis, the column is inserted into the upper hole of the plate, and then inserted into the plate by tapping with a rubber hammer. This process consumed a lot of time and labor for column insertion, and it was difficult to uniformly insert a plurality of columns at the same height.

Accordingly, the present invention contemplates an apparatus having the above-described structure, and a conventional hand press is used as the main body 100 to constitute the apparatus. Generally, hand presses are mainly used in processes such as punching, compression, cutting, and bending as described above, and they are not used in the synthesis of chemical compounds or in the field of biotechnology as in the present invention. However, the present invention applies such a hand press to an oligonucleotide synthesis process, designing and coupling the rod 200 to be attachable to the hand press (body 100) And the structure of the lower jig 400 is newly designed so that the apparatus is designed to be optimized for the oligonucleotide synthesis process so that the process can be performed more quickly and conveniently.

Hereinafter, each configuration will be described in detail with reference to the drawings.

As described above, the coupling part 210 of the rod 200 has a hollow part formed therein so that the upper end of the head frame 300 can be mounted or dismounted, and the upper end of the head frame 300 is fitted to the hollow part Structure. Thus, as shown in FIGS. 3 to 5, the upper portion of the head frame 300 is formed in a 'T' shape in cross section perpendicular to the ground. That is, the hollow portion inside the rod 200 is also formed in a 'T' shape so that the head frame 300 can be fit to the coupling portion 210.

3 to 5, when the head frame 300 is mounted in the coupling portion 210, the fixing portion 220 protruding into the hollow portion of the rod 200 to prevent movement and detachment of the head frame 300, Can be formed. In addition, a groove 301 corresponding to the fixing portion 220 may be formed on the head frame 200 as well.

As shown in the figure, a pair of fixing portions 220 may be provided at the inlet side of the coupling portion 210 on which the head frame 300 is mounted, and may be formed below the coupling portion 210, respectively. 5, the fixing part 220 may be formed in the order of a ball 221, a spring 222, and a screw 223, and the ball 221 may protrude into the coupling part 210. Referring to FIG. The head frame 300 is smoothly inserted as the ball 221 on the spring 222 rotates when the head frame 300 is mounted on the coupling portion 210. When the ball 221 is completely inserted into the inside of the coupling portion 210, The ball 221 is fixed to the groove 301 formed in the head frame 200 by the elastic force. At this time, it is preferable that the groove 301 has a hemispherically depressed shape corresponding to the ball 221.

As shown in FIGS. 1 and 2, the rod 200 is provided inside the lifting part 130, and when the head frame 300 is inserted, the door can be closed to prevent detachment. In this case, the lower region of the head frame 300 is exposed to the lower side of the lifting unit 130 as shown in FIG.

On the other hand, the head frame 300 coupled to the rod 200 is divided into two types depending on the application, and if necessary, the appropriate head frame 300 is mounted and detached.

The head frame 300 shown in FIGS. 1 and 3 is a flat plate-like head frame 310 formed in a flat plate shape at the bottom, and an apparatus provided with a flat plate-shaped head frame 310 as shown in FIG. 1 (b) When the lever 140 is pulled downward, the plurality of columns C are simultaneously pressurized so as to be inserted into the synthetic plate P at a certain position. At this time, since the head frame 300 is formed in a flat plate shape, a plurality of columns can be simultaneously inserted into the synthetic plate P at a constant height.

As mentioned above, conventionally, there is a problem that the column C is inserted into the synthetic plate P at a non-uniform height. Specifically, after the column is inserted into the synthetic plate (P), oligonucleotides are synthesized. During the synthesis, the reagent is put into the column (C) and the reagent is withdrawn under vacuum by applying a vacuum . If the column C is inserted non-uniformly, there is a problem that a vacuum is leaked. In addition, when the reagent is inserted into the column during the synthesis process, the injection needle moves from the upper part of the column. If the height of the column is not constant, the injection needle is caught by the column. The present invention has the advantage that the above problem can be solved completely because the column head can be uniformly inserted by using the flat head frame 310.

After completion of synthesis of the oligonucleotide by a series of reactions, the frit in each of the plurality of columns (C) must be removed. As shown in Fig. 7, oligonucleotides are synthesized on frit (F) located in the column. Conventionally, the frit is detached by positioning a synthetic plate P in which a column C is inserted on a 96-well deep well and pressurizing it with a linear pin at the top Pushing it down into the well plate), which also increased processing time and required labor input.

In the present invention, in order to remove the frit in the plurality of columns C, the flat head frame 310 attached to the rod 200 is first detached and attached to the pin head 310 as shown in Figs. 2 (a) and 4 The frame 320 is mounted.

At this time, the pin head frame 320 has a structure in which a lower portion is formed in a plate shape, and a plurality of fins 321 corresponding to the column C are formed on the lower side. Therefore, after the pin head frame 320 is mounted, the lever 140 is pulled downward to simultaneously press the column C to insert each pin into each column C as shown in FIG. 2 (b) F) can be removed at one time.

6 and 7, the lower jig 400 has a structure in which an upper portion is recessed inwardly, a plate is placed and fixed, a step is formed in an edge region, and a width is narrower toward the inner side It is a losing form. At this time, multiple reactors M may be fixed to the lower jig 400 in addition to the plate. For this, an end 410 is formed on the lower jig 400. 7 (a) is a schematic view of a process of fixing the synthetic plate P to the lower jig 400 and mounting the column C. FIG. 7 (b) (F) by fixing the frit (F) to the frit (400). Conventionally, the synthesis plate (P) for inserting the column for synthesis and the multi-reactor (M) for removing the frit for deprotection are different in height and width. That is, the end 410 is formed in the lower jig 400 in order to use the composite plate P or the multiple reactors M of different sizes in a compatible manner. Therefore, the present invention is advantageous in that it can be used as it is without the hassle of newly manufacturing multiple reactors and synthetic plates that have been used in the past.

1 and 2, the plate P can be used by being fixed on the multiple reactors M and the lower jig 400 can be used not only in the composite plate P but also in the multiple reactors M Can be fixed, and when oligonucleotide synthesis is performed on the column inserted in the synthetic plate P, the head frame 300 can be replaced as shown in FIG. 2 to perform the frit removal immediately .

The multiple reactors used in this case are devices for synthesizing oligonucleotides in the column, and they are generally used in the synthesis of oligonucleotides, so a separate explanation will be omitted.

Conventionally, when a large amount of oligonucleotides are synthesized, a plurality of columns have to be inserted one by one in the plate, so that the process time has to be increased and the manpower to be added to the process has to be increased.

The device according to the present invention is a device for devising an apparatus for oligonucleotide synthesis and de-protection, and is implemented by mounting a rod 200, a head frame 300 and a lower jig 400 on a widely used hand press. Therefore, it is possible to uniformly insert a large number of columns uniformly at the same time with a simple structure of the apparatus. By replacing the head frame 300, a large amount of frit for deprotection after oligonucleotide synthesis is completed can be easily There is an effect that can be removed.

Therefore, not only the process time is shortened but also the work amount is remarkably reduced, which is advantageous in the overall cost saving effect.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

C: Column P: Synthetic plate
M: Multiple reactors
100: main body 110:
120: column 130:
140: Lever
200: rod 210:
220:
300: Head frame 310: Flat head frame
320: Pin Head Frame
400: lower jig 410: stage

Claims (6)

In a column mount and frit stripper for the synthesis and deprotection of oligonucleotides,
A support 120 which is vertically fixed to one side of the support part 110 and a support part 120 which is connected to the support part 120 and which is vertically moved in accordance with the operation of the lever 140 A main body 100 including a lifting portion 130;
A rod 200 provided below the lifting part 130 and including a coupling part 210 to which a head frame 300 described below is coupled;
A lower jig 400 installed on the receiving unit 110 and having a plate on which a plurality of columns for receiving a reaction are placed;
A head frame 300 coupled to an upper end of the coupling part 210 and configured to press the column inserted into the plate toward the bottom when the lifting part 130 is lowered by the operation of the lever 140; And
The head frame 300 is formed in a 'T' shape in its upper portion in a cross section cut vertically with respect to the ground,
Wherein the coupling part (210) is provided with a hollow part inside and the upper part of the head frame (300) can be attached or detached to the hollow part. The method for synthesizing and deprotecting oligonucleotides Column mounting and frit removing device.
The headlamp according to claim 1, wherein the head frame (300)
And a flat plate-like head frame 310 having a lower portion formed in a flat plate shape,
Wherein the flat head frame (310) simultaneously presses the column so that the plurality of columns are inserted into the plate at a certain time.
The headlamp according to claim 1, wherein the head frame (300)
And a pin-type head frame (320) having a lower portion formed in a plate shape and a lower portion formed with a plurality of pins corresponding to the column,
Characterized in that the pin head frame (320) presses the column simultaneously to insert the fins into each column for frit removal in the plurality of columns. ≪ RTI ID = 0.0 > And a frit removing device.
delete The method according to claim 1,
A fixing part 220 protruding into the hollow part of the rod 200 is formed on the rod 200 to prevent movement and detachment of the head frame 300 after the mounting of the head frame 300, And a groove (301) corresponding to the fixing part (220) is formed.
2. The apparatus of claim 1, wherein the lower jig (400)
Wherein the upper portion is formed as a recessed structure and the plate is placed and fixed, and a step is formed in the edge region and a width is narrowed toward the inner side. Column mounting and frit removing device.
KR1020150065901A 2015-05-12 2015-05-12 column loading and frit removal equipment for synthesis and deprotection of oligonucleotide KR101677558B1 (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100183488A1 (en) * 2004-02-12 2010-07-22 Chemistry & Technology For Genes, Inc. Devices and methods for the synthesis of nucleic acids
CN203622998U (en) * 2013-09-29 2014-06-04 王玉伟 Improved hand press

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100183488A1 (en) * 2004-02-12 2010-07-22 Chemistry & Technology For Genes, Inc. Devices and methods for the synthesis of nucleic acids
CN203622998U (en) * 2013-09-29 2014-06-04 王玉伟 Improved hand press

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