CN209352873U - A kind of industrialization DNA synthesis column plate - Google Patents

Abstract

The utility model relates to DNA to synthesize field, and in particular to a kind of industrialization DNA synthesis column plate, including CPG carrier, void column pipe and 96 hole hollow plates, diameter >=4mm of CPG carrier, thickness >=4mm of CPG carrier；It is provided with assembly position in void column pipe, assembles internal diameter >=4mm of position, assembles height >=4mm of position, assembly position is cube or cylindrical body, assembles the internal diameter gradient other than position and reduces；96 hole hollow plates, be uniformly equally spaced in plate body 96 holes, and each hole is equipped with assembly position, each hole is that cube or cylindrical cavity design, cylindrical cavity is transitioned by cone cavity again and assembles position, circular cone or cylindrical cavity design are thought in assembly position, until bottom end liquid outlet part.The industrialization DNA of the utility model synthesizes column plate, it is suitable for a large amount of, industrial of DNA to be combined to, DNA synthetic quantity can rise to 50umol, reach a milligram rank, and synthesize chain and be up to 120-200bp, aggregate velocity is fast, high efficiency, mutation rate can industrialize, high degree of automation down to 0.5 ‰ hereinafter, using simple process.

Description

A kind of industrialization DNA synthesis column plate

Technical field

The utility model relates to DNA to synthesize field, and in particular to a kind of industrialization DNA synthesis column plate.

Background technique

As synthetic biology, artificial life, functional genomics, protein science, the synthesis of target medicine, nucleotide interfere medicine Object, DNA encoding compound library, clinical diagnostics research fast development, need the industry of magnanimity DNA to be combined to, and target Drug, nucleotide interference medicament, the quick, industrial of DNA encoding compound library are combined to be increasingly becoming new drug development and screening It is popular.Higher functional requirement will be proposed to the synthesis column plate of the DNA CPG carrier synthesized.Currently, a kind of well known DNA is closed It is to synthesize column tube by 96 DNA to arrange at plate, CPG media-filled is suitably assembled in 96 synthesis column void column pipes In position, it is put into the synthesis that DNA is carried out on matched DNA synthesizer.The quality of sintetics quality, is heavily dependent on The research and development and production of the design and injection molding and CPG carrier of 96 hole hollow plates.Existing DNA synthesis column or plate, however it remains it is some not Foot is in particular in following aspects:

First, current DNA synthesis column plate is mainly conventional amount used DNA compounding design, such as≤200nmol.It is main to use It is synthesized in Standard PCR Primer.It is not suitable for >=200nmol, or even the DNA industry of umol or even mg rank is combined to.

Second, current design, the amount of reagent not optimized causes to waste so that product use cost is high, and It pollutes the environment.For CPG carrier families product, used reagent, prime cost is auxiliary reagent at present, such as TCA, ACN, iodine solution, tetrahydrofuran etc., usage amount cause greatly cost high, moreover, these synthetic agents are all to have to change It learns corrosive, very big damage can all be brought to human body and environment.How the use of synthetic agent is reduced to greatest extent Amount is the urgent need of the promotion and industry development to synthesis Development of Biology.

Third, the structure design of CPG carrier also has disadvantages that at present: first, the diameter and thickness of CPG carrier is too small, Due to being limited by existence conditions, commercially available CPG carrier maximum gauge still in 4mm hereinafter, height is substantially again smaller than 4mm, Overall volume≤50.24ul, the scale for increasing carrier in this one small volume is very limited system, so its content scale It can only just accomplish 200nmol or so.It is unable to satisfy demand of the industrialization DNA synthesis to amount；Second, CPG content can not increase, use Column plate is synthesized in the DNA of targeted drug, nucleotide interference medicament, the synthesis of DNA encoding compound library, CPG content is in 1umol- Between 10umol, even more great talent can meet the requirement of drug development and screening, however presently commercially available CPG carrier contains Amount is mostly in 200nmol hereinafter, the DNA product being synthesized much can not meet the demand of industrialization client, with 200nmol's Commercially available synthetic column causes the serious wasting of resources, increases research and production cost, is unfavorable for building a conservation-minded society.

4th, the degree of automation is not high, no matter loads or product uses, essentially manual operation, and operation is extremely inconvenient, Cost is increased, automation, large-scale production are unfavorable for.

5th, the structure design of void column pipe or 96 orifice plates also has disadvantages that, is mainly manifested in and does not account for hardware compatibility Property, the leakproofness of assembly precision, 96 orifice plates and equipment, 96 orifice plates liquid splashing prevention effect and anti fouling performance.The DNA synthesizer of mainstream at present There are Dr.Oligo192 series, the MM192 series of Bioaotumation company, TAG Copenhagen of Biolytic company A/S company Oligomaker192 series, 192 series of Thermo Fisher company, currently marketed a few money DNA synthesize column Or plate, the synthesizer of above several companies can not all be accomplished completely compatible or general.Due to 96 orifice plate design problems, with The sealing ring tightness of equipment is not very good, easy gas leakage, leads to DNA synthesis failure.Also, the design in hole is not perfect, hole Design between hole is also incomplete, and the reagent in adding hole is caused to be easy to sputter next, the liquid pearl accumulated, due to 96 It is smooth unobstructed between orifice plate hole on upper surface and hole, and be easy to be rolled into adjacent holes, cause synthesis mutation rate excessively high, DNA synthesis Failure.

Utility model content

Above-mentioned to solve the problems, such as, the utility model provides a kind of industrialization DNA synthesis column plate, is suitable for industrializing The synthesis of DNA, has that time saving province's reagent, at low cost, synthesis chain length, aggregate velocity be fast, mutation rate is down to 0.5 ‰ or less, environmental protection The advantages that.

A kind of industrialization DNA synthesis column plate, including CPG carrier and 96 hole hollow plates, diameter >=4mm of CPG carrier, thickness >= CPG content >=8mg in 4mm, CPG carrier synthesizes scale >=200nmol, it can be understood as, which is large-sized CPG Carrier can be realized biggish synthesis scale；The external dimensions of 96 hole hollow plates: a length of 120mm-130mm, width 80mm- 88mm, a height of 10mm-60mm；Uniformly be equally spaced 96 holes in plate body, is made of 96 void column pipes, each hole is equipped with Position is assembled, assembly position is cube or cylindrical body, assembles internal diameter >=4mm of position, assembles height >=4mm of position, in the assembly position It is filled with CPG carrier, the 96 each holes of hole hollow plate are that cube or cylindrical cavity design, then are transitioned into cylinder by cone cavity Body cavity assembles position, and the following are circular cones or cylindrical cavity to design for assembly position, until bottom end liquid outlet part, 96 hole hollow plate The bottom end point mouth of hollow plate is divided into flat mouth, angle or sharp-crested structure, and the top of 96 hole hollow plate is provided with number, 96 hole The edge of hollow plate is provided with sealed frame, and design has reservoir between Kong Yukong, and 96 hole hollow plate two sides are provided with positioning Hole, the location hole side two.

Preferably, 96 hole hollow plates are arranged by 96 void column pipes.

Preferably, the diameter of CPG carrier or side length are between 4mm-8.5mm.

Preferably, the thickness of CPG carrier is between 4mm-50mm.

Preferably, in CPG carrier CPG content between 8mg-650mg.

Preferably, CPG carrier synthesizes scale between 200nmol-50umol.

Preferably, the external dimensions of 96 hole hollow plates: a length of 120mm-130mm, width 80mm-88mm, a height of 10mm- 60mm.Plate surrounding frame width 0.1-3mm, bottom end liquid outlet part are flat mouth, angle or sharp-crested structure.

Preferably, the diameter or side length of column tube or orifice plate assembly position assemble position height in 4mm- between 4mm-8.3mm Between 52mm.

Preferably, sintetics can be with using, without additional purification process after directly cleaning cutting.

The utility model has the beneficial effects that the industrialization DNA of the utility model synthesizes column plate, drug development and screening are aimed at (200nmol-50umol) and design, preferred synthetic quantity be 1-10umol.Following advantage has been compared with commercially available CPG carrier:

1, CPG carrying capacity has more selectivity in CPG carrier

Presently commercially available 200nmol or more than DNA synthesize column, limited by volume, 500 angstroms of apertures, carrying capacity can only be used The CPG powder of 70-100umol/g synthesizes the production of column as the DNA of extensive DNA synthesis.Had using such CPG powder Apparent drawback, using the smaller CPG in aperture, carrying capacity is higher, the DNA chain that is synthesized it is long it is shorter, purity is lower, quality more Difference.Column plate is synthesized using the industrialization DNA of the utility model, due to the increase of the volume of CPG carrier, corresponding CPG powder contains Amount also can increase, so more large aperture (1000 angstroms or 2000 angstroms), the CPG powder of lower carrying capacity (30umol/g or less) can be used Industrialization DNA synthesis column plate is manufactured at end, synthesizes column with the DNA of such CPG powder production, possesses DNA synthesis chain length, pure Spend the remarkable advantages such as high (95% or more), high-quality (below mutation rate 0.5 ‰).

2, synthetic agent dosage is saved

According to traditional production technology, UHMW-PE sieve plate presss from both sides the form of CPG powder, generally requires decades of times column volume Amount of reagent is chemically reacted, and the industrialization DNA of the utility model is used to synthesize column plate, can be reduced amount of reagent to original 40%.Nearly 60% dosage is saved, cost control ability is apparent.

3, super amount DNA synthesis

The DNA synthetic vectors of diameter or side length 8.5mm thickness 50mm be current world's industry most, the super large of CPG carrier Type has been filled up domestic and international blank, has been at the leading level in the world.The industry of DNA is made to be combined to become because of its " big " Reality.Scale is synthesized suitable for 200noml-50umol or even milligram grade, it is dry that this greatly facilitates targeted drug, nucleotide Disturb the DNA technical grade synthesis client of drug, the synthesis of DNA encoding compound library.Reagent dosage is not only reduced, saves and is produced into This, has saved resource, has also protected environment.

4, high efficiency, save time

The CPG carrier of diameter 8.5mm thickness 50mm, CPG content are up to 650mg.The CPG content of superelevation, the CPG of super large Aperture, ultralow CPG carrying capacity can reduce the usage amount for participating in the synthetic agent of reaction and reaction time, especially to greatest extent It is will greatly to shorten the time of coupling reaction, accelerates the time of single loop, mentions entire production link efficiency substantially It rises.

5, extremely low mutation rate, product quality are relatively reliable

In DNA synthesis process, TCA (trichloroacetic acid) deprotecting regent to 3% is commonly used, which is strong acid solution, easily Cause primer mutagenesis or depurination phenomenon.How to reduce the usage amount of the reagent or reduces connecing for CPG carrier vector and the reagent The touching time is beneficial to reduce the mutation rate of synthetic product, improves product quality.Traditional CPG synthesizes column, generally requires tens of The TCA of times column volume, in two times, to complete the process of deprotection, the chain of synthesis is longer, and the number for contacting deprotecting regent is got over More, the time is longer, also more be easy to cause DNA mutation and depurination phenomenon.And the industrialization DNA of the utility model synthesizes column CPG powder is inlaid into PE powder and solidifies because it uses sintered filter core sieve plate technique by plate, and the carrier after solidification can accommodate reagent Volume be original 40% hereinafter, so the volume of required TCA is reduced to original 2/5ths, can complete entire Deprotection process not only increases combined coefficient, and reduces the mutation rate of DNA synthesis, makes mutation rate well below 2 ‰ International standard has reached 0.5 ‰ or less.While improving product purity, make the length of its sintetics reach 120bp with On.

6, the design optimized solves the problems, such as 96 pore plate by sealing, compatibility, reduces the mutation rate of synthesis process, makes Product quality is relatively reliable.

7, the 96 great novelty of hole hollow plate

Presently commercially available DNA synthesis column is single tube form, no matter in the production of CPG carrier and using upper, is required to list Pipe operation, needs to load one by one, not only low efficiency, but also easily pollute and human operational error.96 hole hollow plates 1 Appearance, simplify by the production of CPG carrier and using process, production technology, be DNA synthesis the extremely innovative hair in field It is bright, the development of industry is advanced, new drug development is improved and screens the industrialization level of cause.

8, automation, scale and mass DNA synthesis are solved the problems, such as

Industrialization DNA provided by the utility model synthesizes column plate, is the automation for aiming at new drug development and screening, scale It is combined to and designs in batches, DNA synthetic quantity is 200nmol-50umol, can effectively save synthetic agent dosage, reagent consumption Few, cost control is to minimum；Super amount pharmaceutical synthesis, synthetic quantity may be up to 650mg, synthesize chain length, be up to 150-200bp； Synthesis cycle is short, and aggregate velocity is fast, and high efficiency saves time；Extremely low mutation rate, be world's industry most, product quality is more Reliably, environmentally friendly, using simple process, automation, scale, mass and industrialization degree are high.

Detailed description of the invention

Fig. 1 is a kind of top surface structure schematic diagram of industrialization DNA synthesis column plate of the utility model.

Fig. 2 is a kind of bottom surface structure schematic diagram of industrialization DNA synthesis column plate of the utility model.

Fig. 3 is a kind of cross-sectional view of industrialization DNA synthesis column plate of the utility model.

Fig. 4 is a kind of structural schematic diagram of the void column pipe of industrialization DNA synthesis column plate of the utility model.

Fig. 5 is a kind of cross-sectional view of the void column pipe of industrialization DNA synthesis column plate of the utility model.

Fig. 6 is a kind of the first assembling schematic diagram of the CPG carrier of industrialization DNA synthesis column plate of the utility model.

Fig. 7 is a kind of second of assembling schematic diagram of the CPG carrier of industrialization DNA synthesis column plate of the utility model.

In figure: the hollow plate of the hole 1-96；2- void column pipe；3- assembles position；4-CPG carrier.

Specific embodiment

In order to make the purpose of the utility model, technical solutions and advantages more clearly understood, below in conjunction with attached drawing and implementation Example, the present invention will be further described in detail.It should be appreciated that specific embodiment described herein is only to explain this Utility model is not used to limit the utility model.

Embodiment 1

Such as Fig. 1-Fig. 7, a kind of industrialization DNA synthesis column plate provided by the utility model, including CPG carrier 4 and void column pipe Diameter >=4mm of 2, CPG carriers 4, thickness >=4mm, CPG content >=8mg in CPG carrier 4 synthesize scale >=200nmol；Void column It is provided with assembly position 3 in pipe 2, assembles internal diameter >=4mm of position 3, assembles height >=4mm of position 3, assembly position 3 is cube or cylinder Body assembles the internal diameter gradient other than position 3 and reduces, and CPG carrier 4, the bottom end point of the void column pipe 2 are filled in the assembly position Mouth is divided into flat mouth, angle or sharp-crested structure.Wherein, the diameter of CPG carrier 4 or side length are 4.1mm-8.5mm；CPG carrier 4 With a thickness of 4mm-50mm.Preferably, the diameter of CPG carrier 4 is 8mm, with a thickness of 4.5mm.Wherein, the quantity for assembling position 3 is 1 It is a；The internal diameter for assembling position 3 is 8mm；The height for assembling position 3 is 5mm.CPG content is 200nmol-10umol in CPG carrier 4, excellent Selection of land, CPG content is 1umol-5umol in CPG carrier 4.

It is embodied as follows:

DNA synthesizes column, synthesizes scale 1umol, is divided into the CPG carrier and its mating void column pipe of diameter 8mm thickness 4.5mm 2。

1, the design of void column pipe:

2 column length 52mm of void column pipe, column tube top are side length 8.1mm, and the cuboid cavity of height 20mm, cavity lower part is closed up Cylinder is formed, body diameter 8mm is highly 5mm, and 8mm cylinder assembly 3 lower part of position, which is gradually closed up, arrives 2mm, and lower part flat mouth is set Meter.

2, the designing and preparing of CPG carrier 4 (based on preparation 1000)

1) raw material: CPG carrying capacity is 30umol/g, and the CPG vector contg of every 1umol is about 33.34mg CPG；HDPE powder The molecular weight at end is 1,000,000.

2) raw material volume calculates: the CPG carrier bulk of every diameter 8mm thickness 4.5mm is 226.08ul, by 1000 Proportion, total volume are 226.08ml ≈ 226ml.

3) CPG carrier raw materials for sintering configures: weighing 33.34g CPG and is put into 500ml graduated cylinder, is slowly added HDPE powder and arrives 226ml scale, is mixed by inversion for several times, then with HDPE polishing to the position 226ml；It is mixed by inversion repeatedly spare.

4) die-filling tool: in the aluminium-making mould in the hole containing 100 diameter 8mm height 4.5mm, uniformly addition is mixed CPG and HDPE blended powder are shaken flat with vibration gauge, cover upper cover plate, locked.

5) it is sintered: 200 DEG C, being sintered 40min, water cooling takes out CPG carrier.

3, assemble: the CPG carrier 4 of diameter 8mm thickness 4.5mm is assembled to column tube by manual push rod or fully-automatic equipment 8mm assembles position 3.

Embodiment 2

Such as Fig. 1-Fig. 7, a kind of industrialization DNA synthesis column plate provided by the utility model, including the hole of CPG carrier 4 and 96 are empty Plate 1, diameter >=4mm of CPG carrier, thickness >=4mm, CPG content >=8mg in CPG carrier 4 synthesize scale >=200nmol；96 Hole hollow plate 1 is 127mm long, wide 85mm, high 52mm, and be uniformly equally spaced 96 holes in plate body, is made of 96 void column pipes 2, often A hole is equipped with assembly position 3, and assembly position 3 is cube or cylindrical body, assembles the internal diameter or side length >=4mm of position 3, assembles the height of position 3 Degree >=4mm, each hole of 96 hole hollow plate 1 are that cube or cylindrical cavity design, then are transitioned into cylindrical cavity by cone cavity Position 3 is assembled, assembles position 3 to design in circular cone or cylindrical cavity, until bottom end liquid outlet part can referring to Fig. 4 and Fig. 5 It is the upper end of void column pipe 2 in the hole that cube or cylindrical cavity design to be interpreted as, assembly position 3 passes through with the cube or cylindrical body The cavity of cone connects；The bottom end point mouth of 96 hole hollow plate, 1 hollow plate is divided into flat mouth, angle or sharp-crested structure.Wherein, 96 hole hollow plates synthesize column void column pipe 2 by 96 DNA and arrange.The diameter of CPG carrier is 4.1mm-8.5mm；CPG carrier With a thickness of 4mm-50mm.Preferably, the diameter of CPG carrier is 5.8mm, with a thickness of 13.5mm.Wherein, the quantity for assembling position 3 is 1 It is a；The internal diameter for assembling position 3 is 5.8mm；The height for assembling position 3 is 14mm.CPG content is 200nmol- in CPG carrier 10umol, it is preferable that CPG content is 10umol in CPG carrier.

It is embodied as follows:

The hole DNA96 hollow plate 1, synthesize scale 10umol, be divided into diameter 5.8mm, thickness 13.5mm CPG carrier and its match Cover 96 hole sky hard boards.

1, the design of 96 hole hollow plates

The high 52mm of hollow plate synthesizes column void column pipe 2 by 96 and is derived, and orifice plate top outer diameter is side length 8.1mm, height The cuboid cavity of 20mm, cavity lower part close up to form cylinder, body diameter 8.1mm, are highly 10mm, its underpart is gradually received The cylindrical assembly position 3 that mouth is 5.8mm to diameter, assembly 3 height 14mm of position, design, institute are borrowed using Luer-style in assembly 3 lower part of position The edge for stating 96 hole hollow plates is provided with sealed frame, and design has reservoir between Kong Yukong, is arranged in 96 hole hollow plate two sides There are location hole, the location hole side two.

2, the preparation of CPG carrier (by 1000 calculating of preparation)

1) raw material: CPG carrying capacity is 80umol/g, and the CPG vector contg of every 10umol is about 125mg CPG；UHMW-PE The molecular weight of powder is 3,000,000.

2) raw material volume calculates: the CPG carrier bulk of every diameter 5.8mm thickness 13.5mm is 356.4999ul, is pressed 1000 proportions, total volume are 356.4999ml ≈ 356.5ml.

3) CPG carrier raw materials for sintering configures: weighing 125g CPG and is put into 500ml graduated cylinder, is slowly added UHMW-PE powder and arrives 356.5ml scale, is mixed by inversion for several times, then with UHMW-PE polishing to the position 356.5ml；It is mixed by inversion repeatedly spare.

4) die-filling tool: in the aluminium-making mould in the hole containing 100 diameter 5.8mm height 13.5mm, uniformly addition mixing Good CPG and UHMW-PE blended powder is shaken flat with vibration gauge, covers upper cover plate, locked.

5) it is sintered: 200 DEG C, being sintered 45min, water cooling takes out CPG carrier.

3, assemble: the CPG carrier of diameter 5.8mm thickness 13.5mm is assembled to 96 holes by manual push rod or fully-automatic equipment 5.8mm in hollow plate assembles position 3.

In embodiment 1 and embodiment 2, the diameter and thickness of CPG carrier are all larger than presently commercially available all Related products, wrap Include external product, accomplished that similar product full-size is horizontal in volume, in industry in the world small product size most.Target The CPG support C PG of the DNA commercial synthesis client's synthesis synthesized to drug, nucleotide interference medicament, DNA encoding compound library Content is up to 650mg, and comparing with similar product has significant advantage.

The above is only the preferred embodiment of the utility model only, is not intended to limit the utility model, all at this Made any modification, equivalent replacement or improvement etc., should be included in the utility model within the spirit and principle of utility model Protection scope within.

Claims (8)

1. a kind of industrialization DNA synthesizes column plate, which is characterized in that including CPG carrier, void column pipe or 96 hole hollow plates, CPG carrier Diameter >=4mm, thickness >=4mm, CPG content >=8mg in CPG carrier synthesize scale >=200nmol；Dress is provided in void column pipe Coordination, assembles internal diameter >=4mm of position, assembles height >=4mm of position, and assembly position is cube or cylindrical body, assembles interior other than position Diameter gradient reduces, and is filled with CPG carrier in the assembly position, the bottom end point mouth of the void column pipe is divided into flat mouth, angle or point Mouth structure；

Uniformly be equally spaced 96 holes in the plate body of 96 hole hollow plate, is made of 96 void column pipes, each hole is equipped with dress Coordination, assembly position is cube or cylindrical body, assembles internal diameter >=4mm of position, assembles each of height >=4mm of position, 96 hole hollow plates Hole is that cube or cylindrical cavity design, then are transitioned into cylindrical cavity by cone cavity and assemble position, and the following are circles for assembly position Cone or cylindrical cavity design, until bottom end liquid outlet part, the bottom end point mouth of the 96 hole hard board hollow plate be divided into flat mouth, The top of angle or sharp-crested structure, 96 hole hollow plate is provided with number, and the edge of 96 hole hollow plate is provided with sealed frame, And design has reservoir between Kong Yukong, and 96 hole hollow plate two sides are provided with location hole, the location hole side two.

2. industrialization DNA according to claim 1 synthesizes column plate, which is characterized in that 96 hole hollow plates are arranged by 96 void column pipes It arranges.

3. industrialization DNA according to claim 1 synthesizes column plate, which is characterized in that the diameter or side length of CPG carrier exist Between 4mm-8.5mm.

4. industrialization DNA according to claim 1 synthesizes column plate, which is characterized in that the thickness of CPG carrier is in 4mm-50mm Between.

5. industrialization DNA according to claim 1 synthesizes column plate, which is characterized in that CPG content is in 8mg- in CPG carrier Between 650mg.

6. industrialization DNA according to claim 1 synthesizes column plate, which is characterized in that CPG carrier synthesis scale exists Between 200nmol-50umol.

7. industrialization DNA according to claim 1 synthesizes column plate, which is characterized in that the external dimensions of 96 hole hollow plates: a length of 120mm-130mm, width 80mm-88mm, a height of 10mm-60mm, the wide 0.1-3mm of plate frame sealed around, bottom end liquid outlet portion It is divided into flat mouth, angle or sharp-crested structure.

8. industrialization DNA according to claim 1 synthesizes column plate, which is characterized in that column tube or the diameter of orifice plate assembly position Or side length assembles position height between 4mm-52mm between 4mm-8.3mm.