CN107557356A - A kind of small multi-channel library construction instrument and the method for building library - Google Patents
A kind of small multi-channel library construction instrument and the method for building library Download PDFInfo
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- CN107557356A CN107557356A CN201710792769.5A CN201710792769A CN107557356A CN 107557356 A CN107557356 A CN 107557356A CN 201710792769 A CN201710792769 A CN 201710792769A CN 107557356 A CN107557356 A CN 107557356A
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Abstract
The present invention provides a kind of small multi-channel library construction instrument and the method for building library, more particularly to gene sequencing technology field, including frame, the frame is provided with the first supporting plate and controller, first supporting plate, which is provided with, to be used to deposit reagent strip and the rack for test tube and sample adding device of pipette tips, the rack for test tube side is provided with waste liquid box, the rack for test tube bottom is movably equipped with heater and magnetic separating device respectively, the heater and the magnetic separating device are connected to the first transmission mechanism and the second transmission mechanism, the top of the rack for test tube is provided with the sample adding device, the sample adding device connects the 3rd transmission mechanism, first transmission mechanism, second transmission mechanism and the 3rd transmission mechanism are electrically connected the controller.Of the invention thoroughly solves the problems, such as to prepare sample by hand time-consuming low with flux, and the system whole flow process is automatically performed by one-stop work station, avoids malfunctioning and is more suitable for the standardization in laboratory.
Description
Technical field
The invention belongs to gene sequencing technology field, and in particular to a kind of small multi-channel library construction instrument and structure library
Method.
Background technology
Gene sequencing market developed rapidly in recent years, especially two generation sequencing technologies high fluxs, high-accuracy, low cost etc.
Advantage allows it to be more widely used.The industrial chain of two generations sequencing can be divided into the instrument and equipment end of upstream, middle reaches sequencing clothes
The analysis of the sequencing data in business and downstream.External two generation sequencer manufacturers mainly have Illumina, Life technologies
With Roche etc..Illumina, which is occupied entirely, to be shown to 2013 degree of year whole world sequencing instrument market survey results according to Genomeweb
Ball be sequenced instrument market 71% share, Life technologies be then number two get 16% the market share, Roche and
PacBio is then with 10% and 3% point of row third and fourth.
The flow of two generations sequencing is sample preparation (DNA is purified and library construction), sequencing and data analysis.Wherein be sequenced by
Sequenator automatically completes, and sample preparation needs substantial amounts of manual operations to complete, and not only takes but also flux is very low, and one day only
4 sample preparations can be completed.In addition, it is higher to prepare requirement of the sample to technical staff by hand, due to the costliness of sequencing reagent,
Do not allow to malfunction and waste.
Therefore it is badly in need of a kind of small multi-channel library construction instrument that can solve the problem that existing issue and the method for building library.
The content of the invention
It is an object of the invention to provide a kind of small multi-channel library construction instrument and the method for building library, thoroughly solve
The problem of sample is time-consuming low with flux is prepared by hand, and the system whole flow process is automatically performed by one-stop work station, avoids malfunctioning
And it is more suitable for the standardization in laboratory.
The invention provides following technical scheme：
A kind of small multi-channel library construction instrument, including frame, the frame are provided with the first supporting plate and controller, institute
State the first supporting plate and be provided with and be used to deposit reagent strip and the rack for test tube and sample adding device of pipette tips, the rack for test tube side is provided with useless
Liquid box, the rack for test tube bottom are movably equipped with heater and magnetic separating device, the heater and the Magneto separate respectively
Device is connected to the first transmission mechanism and the second transmission mechanism, and the top of the rack for test tube is provided with the sample adding device, institute
State sample adding device and connect the 3rd transmission mechanism, first transmission mechanism, second transmission mechanism and the 3rd driver
Structure is electrically connected the controller.The number of first supporting plate is 1,2 or multiple, also imply that can have it is multiple anti-
Work station is answered in same machine, the heater controls for system temperature, and the magnetic separating device is easy to magnetic field to control
System, moved on to when needing and separating magnetic bead and supernatant at sample well, magnetic bead is separated with supernatant, when needing mixing magnetic bead and solution
When, the magnetic separating device is removed, is easy to magnetic bead to be evenly distributed in solution, the sample adding device is mainly used in reagent
Addition, reagent and magnetic bead mixing and magnetic bead and supernatant separation when siphon away supernatant.First transmission mechanism, described
Two transmission mechanisms and the 3rd transmission mechanism respectively move the heater, the magnetic separating device and the sample adding device
To the opening position of needs, the controller controls first transmission mechanism, second transmission mechanism and the 3rd transmission
The effect of mechanism.Reagent strip and pipette tips can be placed on the rack for test tube with alternate, the rack for test tube can also be divided into reagent strip
Rest area and pipette tips rest area.
Preferably, the barcode reader for scanning kit information is movably equipped with the rack for test tube, the bar code is read
Read device and connect the 4th transmission mechanism, the 4th transmission mechanism is electrically connected the controller.The barcode reader, which is used to scan, to be tried
Agent box information, it is easy to kit used in determination whether correct.
Preferably, it is the 4th transmission mechanism includes being sequentially connected the second motor, the second motor gear, second same
Walk band and the second driven gear and the be parallel to each other with second timing belt the set along the frame length direction the 3rd
Line slideway, the barcode reader are connected with second timing belt and moved on the 3rd line slideway.
Preferably, the sample adding device includes sample-adding pump, sample-adding needle assemblies and elevating mechanism fixed plate, the lift
Structure fixed plate is connected in first supporting plate between the sample-adding pump and the sample-adding needle assemblies by flexible pipe, described
3rd transmission mechanism is to be connected with the sample-adding needle assemblies and drive the sample-adding needle assemblies in the elevating mechanism fixed plate
The 3rd linear electric motors to move up and down, the rack for test tube bottom, which is provided with, drives the rack for test tube to make along the frame width
The Y-direction transmission mechanism that moves forward and backward and the rack for test tube is driven to make the X of side-to-side movement to driver along the frame length direction
Structure.The sample-adding pump drives the sample-adding needle assemblies to inhale the function that tapping body completes sample-adding or removal waste fluid.The sample needle group
Part is arranged to move up and down, and the rack for test tube can be with X to the left and right or Y-direction moves forward and backward and coordinates sample adding device work.Institute
The number for stating sample needle and sample-adding pump can be 8, can once make 8 samples.The sample-adding pump is placed on institute per tetrad
State the both sides of frame.
Preferably, be provided with the second supporting plate between the rack for test tube and first supporting plate, first supporting plate and
Y-direction transmission mechanism and X are respectively equipped with second supporting plate to transmission mechanism, the Y-direction transmission mechanism and the X to transmission
Mechanism is connected with the rack for test tube and second supporting plate respectively, and the Y-direction transmission mechanism and the X are electric to transmission mechanism
The even controller.The X is to transmission mechanism by driving second supporting plate to move so as to drive the rack for test tube to move.
Because the rack for test tube X is less than the frequency moved of the rack for test tube Y-direction to motion during whole operation, described by the Y-direction
Transmission mechanism is located at the X on transmission mechanism.The X must be used when pipette tips are only changed during whole operation to biography
Motivation structure.
Preferably, the Y-direction transmission mechanism includes the first timing belt, is located at first motor at the first timing belt both ends
The first guide rail that gear and the first driven gear, the first motor and two are parallel to each other, first guide rail with it is described
First timing belt is parallel, the bottom surface of the rack for test tube be provided with the first straight line bearing that is used for crossing first guide rail and with it is described
The connected timing belt tabletting of first timing belt, second supporting plate are provided with first motor gear, described first driven
Gear and first guide rail, the second supporting plate bottom are provided with second straight line bearing, and the X includes being all provided with to transmission mechanism
In the 4th linear electric motors in first supporting plate and two the second guide rails being parallel to each other, second guide rail crosses described
Second straight line bearing, the 4th linear electric motors are connected with second supporting plate, and first supporting plate is led to provided with strip
Hole, first motor are connected through the long strip through hole with first motor gear.First motor is set
Space is saved under first supporting plate, while saves the material of fixed first driven gear.
Preferably, the heater includes several for inserting the incubation groove of test tube, for fixing the incubation groove
Incubation groove fixed plate and for incubate groove heating adding thermal resistance or heating cushion, first transmission mechanism include several
On first straight line guide rail, several first sliding blocks intersected with first straight line guide rail activity and the driving heater
The first straight line motor of lower motion, first sliding block and the first straight line motor are incubated in groove fixed plate located at described,
The first straight line motor is connected with the controller, and short transverse of the first straight line guide rail along the frame is located at described
The side of rack for test tube.The incubation groove insertion test tube carries out closed type air heating, is advantageous to be rapidly achieved required temperature saving
Material, compared to adding water to carry out heating in the incubation groove incubation trough inner water can be avoided to spill appearance of coming or be evaporated
Fragile machine influences experiment effect.The incubation groove is made up of metal material, can include aluminium or stainless steel.
Preferably, the magnetic separating device includes magnetic field fixed plate and the strong magnet being located in the magnetic field fixed plate, institute
State the second transmission mechanism include 2 second straight line guide rails, 2 the second sliding blocks intersected movable with the second straight line guide rail and
Second straight line motor, second sliding block are located at the both ends of the magnetic field fixed plate, and the second straight line motor is located at the magnetic
In the fixed plate of field and the controller that is electrically connected, width of the second straight line guide rail along the frame are located at the rack for test tube
Both sides.Can also be that magnet fixed plate is provided between the magnetic field fixed plate and the strong magnet, that is, the magnet is consolidated
The both ends of fixed board are connected with the magnetic field fixed plate and the strong magnet respectively, avoid magnet from producing the second straight line motor
Influence, the position that the strong magnet is distributed in the magnet fixed plate is corresponding with the position of the test tube hole of the rack for test tube.
Preferably, the strong magnet is L-type.It is easy to fully contacting for the test tube and the strong magnet.
The method in small multi-channel library construction instrument structure library is as follows：Tried using DNA purification kits and library construction
Two kinds of kits of agent box, and two kinds of kits use all in the form of reagent strip.
The first step：DNA is purified, and DNA sample to be purified is put into the sample well of purification kit, is opened small-sized more logical
Road library construction instrument, instrument starts self-test, and after self-test is qualified, the reagent strip equipped with DNA sample and pipette tips are put into the test tube
In frame, start to purify after the barcode reader reading reagent box validation of information, make magnetic bead and DNA samples by adding magnetic bead successively
Product blending incubation 5 minutes, first time Magnetic Isolation, which remove supernatant, adds 80% ethanol cleaning DNA sample for the first time makes 80% ethanol
1-2 minutes are mixed with magnetic bead, second of Magnetic Isolation removes supernatant, second of addition 80% ethanol cleaning DNA sample makes 80% second
Alcohol mixes 1-2 minutes with magnetic bead, third time Magnetic Isolation removes supernatant, magnetic bead is dried 5 minutes plus elution buffer delays elution
Fliud flushing and magnetic bead blending incubation 5 minutes, Magnetic Isolation simultaneously keep 2-3 minutes and transfer supernatant into fresh sample pipe, wherein in magnetic
Pearl and DNA sample blending incubation 5 minutes, 80% ethanol mix with magnetic bead and elution buffer and magnetic bead mixing are to pass through institute
Stating sample adding device, ceaselessly pressure-vaccum DNA sample makes it mix what is realized, naturally it is also possible to by the X to transmission mechanism or institute
Y-direction transmission mechanism is stated so that the rack for test tube is moved to mix DNA sample, it is to fill the Magnetic Isolation that Magnetic Isolation, which removes supernatant,
Put move on to the sample well bottom adsorb magnetic bead again with the sample adding device remove waste liquid, pass through the Y-direction transmission mechanism move
Move the rack for test tube to move on to waste liquid in the waste liquid hole of reagent strip, pipette tips are dropped into the waste liquid box, the use of new pipette tips are logical
Cross the X drives the sample needle to move up and down realization to the transmission mechanism movement rack for test tube and the 3rd transmission mechanism
, it is to move the rack for test tube and institute by the Y-direction transmission mechanism to add magnetic bead and 80% ethanol and elution buffer
Stating the 3rd transmission mechanism drives the sample needle to move up and down realization, and it is by drying at room temperature or the heating that magnetic bead, which is dried,
Device realizes heat drying.
Second step：The DNA after purification of acquisition is processed into DNA small fragments, digestion or ultrasonic wave are used outside instrument
The mode for interrupting DNA is realized；
3rd step：Library is built, the heating devices heat is reached 30 degree, successively by End repairing reagents
30 degrees Celsius incubate 30 minutes, A-tailing reagents incubate 30 degrees Celsius of 30 minutes and 30 degrees Celsius of Ligation reagents incubations
15 minutes, incubate be required for the mixed liquor purifying of acquisition, purification process and the purification process of the first step after terminating every time
It is identical, finally obtain the DNA direct Sequencings of purifying；Or successively by 30 degrees Celsius of 30 points of incubations of End repairing reagents
30 degrees Celsius of clock and Ligation reagents incubate 15 minutes, incubate be required for purifying the mixed liquor of acquisition after terminating every time, pure
Change method is identical with the purification process of the first step, finally obtains the DNA direct Sequencings of purifying.
The beneficial effects of the invention are as follows：Thoroughly solve the problems, such as that the manual sample for preparing is time-consuming low with flux, the system is whole
Individual flow is automatically performed by one-stop work station, is avoided malfunctioning and is more suitable for the standardization in laboratory.
Brief description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
Apply example to be used to explain the present invention together, be not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is axle side of the present invention schematic diagram；
Fig. 2 is explosive view of the present invention；
Fig. 3 is rack for test tube schematic diagram；
Fig. 4 is the construction of gene library flow chart of DNA sample after purification.
Wherein in figure mark for：1st, frame；2nd, the first supporting plate；21st, long strip through hole；3rd, the second supporting plate；31st, second is straight
Bobbin is held；3rd, controller；4th, rack for test tube；41st, timing belt tabletting；42nd, first straight line bearing；5th, sample adding device；51st, sample-adding pump；
52nd, needle assemblies are loaded；53rd, elevating mechanism fixed plate；6th, waste liquid box；7th, heater；71st, groove is incubated；72nd, groove is incubated to fix
Plate；8th, magnetic separating device；81st, magnetic field fixed plate；82nd, magnet fixed plate；83rd, strong magnet；9th, the first transmission mechanism；91st, first
Line slideway；92nd, the first sliding block；93rd, first straight line motor；10th, the second transmission mechanism；101st, second straight line guide rail；102nd,
Two sliding blocks；103rd, second straight line motor；11st, the 3rd transmission mechanism；111st, the 3rd linear electric motors；12nd, barcode reader；13rd,
Four transmission mechanisms；131st, the second motor；132nd, the second motor gear；133rd, the second timing belt；134th, the second driven gear；
135th, the 3rd line slideway；14th, X is to transmission mechanism；141st, the 4th linear electric motors；142nd, the second guide rail；15th, Y-direction transmission mechanism；
151st, the first motor；152nd, the first motor gear；153rd, the first timing belt；154th, the first driven gear；155th, first lead
Rail.
Embodiment
As Figure 1-3, a kind of small multi-channel library construction instrument, including frame 1, frame 1 are provided with the first supporting plate 2
With controller 3, the first supporting plate 2, which is provided with, to be used to deposit reagent strip and the rack for test tube 4 and sample adding device 5 of pipette tips, rack for test tube 4 one
Side is provided with waste liquid box 6, and the bottom of rack for test tube 4 is movably equipped with heater 7 and magnetic separating device 8, heater 7 and Magneto separate respectively
Device 8 is connected to the first transmission mechanism 9 and the second transmission mechanism 10, and the top of rack for test tube 4 is provided with sample adding device 5, sample-adding
Device 5 connects the 3rd transmission mechanism 11, and the first transmission mechanism 9, the second transmission mechanism 10 and the 3rd transmission mechanism 11 are electrically connected control
Device 3 processed.
Specifically, the barcode reader 12 for scanning kit information, barcode reader 12 are movably equipped with rack for test tube 4
Connect the 4th transmission mechanism 13, the 4th transmission mechanism 13 is electrically connected controller 3.Barcode reader 12 is used to scan kit information,
It is easy to kit used in determination whether correct.The second motor 131 that 4th transmission mechanism 13 includes being sequentially connected, the
Two motor gears 132, the second timing belt 133 and the second driven gear 134 and it is parallel to each other with second timing belt 133
The 3rd line slideway 135 set along the length direction of frame 1, barcode reader 12 are connected and the 3rd with the second timing belt 133
Moved on line slideway 135.
Specifically, sample adding device 5 includes sample-adding pump 51, sample-adding needle assemblies 52 and elevating mechanism fixed plate 53, lift
Structure fixed plate 53 is connected, the 3rd driver in the first supporting plate 2 between sample-adding pump 51 and sample-adding needle assemblies 52 by flexible pipe
Structure 11 is be connected and drive to be loaded needle assemblies 52 move up and down in elevating mechanism fixed plate 53 the with sample-adding needle assemblies 52
Three linear electric motors 111, the bottom of rack for test tube 4 are provided with the Y-direction driver that driving rack for test tube 4 is made to move forward and backward along the width of frame 1
Structure 15 and driving rack for test tube 4 make the X of side-to-side movement to transmission mechanism 14 along the length direction of frame 1.Sample-adding pump 51 drives sample needle
Component 52 inhales the function that tapping body completes sample-adding or waste suction liquid.Sample-adding needle assemblies 52 are arranged to move up and down, and rack for test tube 4 can be with
X is to the left and right or Y-direction moves forward and backward and coordinates sample adding device 5 to work.The number of sample needle and sample-adding pump 51 can be 8, once
8 samples can be made.Sample-adding pump 51 is placed on the both sides of frame 1 per tetrad.It is provided between the supporting plate 2 of rack for test tube 4 and first
Y-direction transmission mechanism 15 and X are respectively equipped with to transmission mechanism 14, Y in second supporting plate 3, the first supporting plate 2 and the second supporting plate 3
To transmission mechanism 15 include the first timing belt 153, the first motor gear 152 for being located at the both ends of the first timing belt 153 and first from
Moving gear 154, the first motor 151 and two the first guide rails 155 being parallel to each other, the first guide rail 155 are synchronous with first
Band 153 is parallel, the bottom surface of rack for test tube 4 be provided with the first straight line bearing 42 that is used for crossing the first guide rail 155 and with the first timing belt
153 connected timing belt tablettings 41, the second supporting plate 3 are provided with the first motor gear 152, the first driven gear 154 and first
Guide rail 155, the bottom of the second supporting plate 3 are provided with second straight line bearing 31, and X includes being located at the first supporting plate 2 to transmission mechanism 14
On the 4th linear electric motors 141 and two the second guide rails 142 being parallel to each other, the second guide rail 142 cross second straight line bearing 31,
4th linear electric motors 141 are connected with the second supporting plate 3, by driving the second supporting plate 3 to move so as to drive rack for test tube 4 to move.
Second supporting plate 3 is provided with long strip through hole 21, and the first motor 151 passes through long strip through hole 21 and the phase of the first motor gear 152
Even.First motor 151 saves the material for fixing the first driven gear 154 located at 2 times saving spaces of the first supporting plate
Material.
Specifically, heater 7 includes the temperature for incubating groove 71, groove 71 being incubated for fixing that several are used to insert test tube
Educate groove fixed plate 72 and adding thermal resistance or heating cushion for incubating the heating of groove 71, the first transmission mechanism 9 include several the
One line slideway 91, several first sliding blocks 92 intersected with the activity of first straight line guide rail 91 and driving heating device are about 7
Located at incubating in groove fixed plate 72, first is straight for the first straight line motor 93 of motion, the first sliding block 92 and first straight line motor 93
Line motor 93 is connected with controller 3, and short transverse of the first straight line guide rail 91 along frame 1 is located at the side of rack for test tube 4.Incubate groove
71 insertion test tubes carry out closed type air heating, are advantageous to be rapidly achieved required temperature saving material, compared in incubation groove 71
Adding water to carry out heating can avoid incubating water in groove 71 and spill either to be evaporated to be easily damaged machine or influenceing to test effect
Fruit.Incubate groove 71 to be made up of metal material, aluminium or stainless steel can be included.
Specifically, magnetic separating device 8 includes the strong magnet 83 of magnetic field fixed plate 81, magnet fixed plate 82 and L-type, magnet is solid
The both ends of fixed board 82 are connected with magnetic field fixed plate 81 and strong magnet 83 respectively, and the second transmission mechanism 10 includes 2 second straight line guide rails
101st, 2 the second sliding blocks 102 intersect with the activity of second straight line guide rail 101 and magnetic separating device 8 is driven along the width side of frame 1
The second straight line motor 103 moved forwards, backwards, the second sliding block 102 are located at the both ends of magnetic field fixed plate 81, second straight line motor 103
In magnetic field fixed plate 81 and the controller 3 that is electrically connected, width of the second straight line guide rail 101 along frame 1 are located at rack for test tube 4
Both sides.The position that strong magnet 83 is distributed in magnet fixed plate 82 is corresponding with the position of the test tube hole of rack for test tube 4.
As shown in figure 4, the method in small multi-channel library construction instrument structure library is as follows：Using DNA purification kits with
Two kinds of kits of library construction Kit, and two kinds of kits use all in the form of reagent strip.
The first step：DNA is purified, and DNA sample to be purified is put into the sample well of purification kit, is opened small-sized more logical
Road library construction instrument, instrument starts self-test, and after self-test is qualified, the reagent strip equipped with DNA sample and pipette tips are put into rack for test tube 4
In, start to purify after the reading reagent box validation of information of barcode reader 12, magnetic bead is mixed with DNA sample by adding magnetic bead successively
It is even be incubated 5 minutes, first time Magnetic Isolation go supernatant, for the first time add 80% ethanol cleaning DNA sample make 80% ethanol and magnetic
Pearl mix 1-2 minutes, second of Magnetic Isolation go supernatant, add for second 80% ethanol cleaning DNA sample make 80% ethanol with
Magnetic bead, which mixes 1-2 minutes, third time Magnetic Isolation removes supernatant, magnetic bead is dried 5 minutes plus elution buffer makes elution buffer
With magnetic bead blending incubation 5 minutes, Magnetic Isolation and keeping 2-3 minutes and transfer supernatant into fresh sample pipe, wherein magnetic bead with
DNA sample blending incubation 5 minutes, 80% ethanol and magnetic bead mix and elution buffer to be mixed with magnetic bead be by sample-adding dress
Put 5 ceaselessly pressure-vaccum DNA sample make its mix realize, it is that magnetic separation device is moved on into sample well bottom that Magnetic Isolation, which removes supernatant,
Portion adsorbs magnetic bead and removes waste liquid with sample adding device 5 again, and waste liquid is moved on into reagent by the mobile test tube shelf 4 of Y-direction transmission mechanism 15
In the waste liquid hole of bar, pipette tips are dropped into waste liquid box 6, the use of new pipette tips are to the mobile test tube shelf 4 of transmission mechanism 14 and by X
Three transmission mechanisms 11 drive sample-adding needle assemblies to move up and down realization, and adding magnetic bead and 80% ethanol and elution buffer is
Sample-adding needle assemblies are driven to move up and down realization by the mobile test tube shelf 4 of Y-direction transmission mechanism 15 and the 3rd transmission mechanism 11, magnetic bead
Drying is to realize heat drying by drying at room temperature or heater 7.The namely reading reagent box information of barcode reader 12
When starting purifying after confirmation, X drives rack for test tube 4 to move to transmission mechanism 14 so that sample adding device 5 is located at reagent strip and magnetic bead is housed
Hole in, draw magnetic bead, then drive rack for test tube 4 magnetic bead is put into sample well, sample-adding pump 51 drive sample needle ceaselessly
Pressure-vaccum sample well 5 minutes, makes DNA sample fully be mixed with magnetic bead, and magnetic separating device 8 moves on to sample well bottom, waits 1-2 points
Clock, using the Aspirate supernatant of sample adding device 5, X drives rack for test tube 4 to move to transmission mechanism 14, and supernatant is discharged into reagent strip
In waste product hole, pipette tips are discharged into waste liquid box 6, Y-direction transmission mechanism 15 drives rack for test tube 4 to move, and sample adding device 5 is reused newly
Pipette tips, X drives rack for test tube 4 to move to transmission mechanism 14, and sample adding device 5 draws a certain amount of 80% ethanol, adds sample well
In, the second drive device removes magnetic separating device 8, and using sample adding device 5, ceaselessly pressure-vaccum sample well 1-2 minutes, Magneto separate fill
Put 8 and move on to sample well bottom, wait 1-2 minutes, using the Aspirate supernatant of sample adding device 5, supernatant is drawn using sample adding device 5
Liquid, X drive rack for test tube 4 to move, supernatant are discharged into the waste product hole of reagent strip, pipette tips are discharged into waste liquid box to transmission mechanism 14
In 6, Y-direction transmission mechanism 15 drives rack for test tube 4 to move, and sample adding device 5 reuses new pipette tips, then takes the repetition of 80% ethanol clear
Wash once, after reject supernatant, heater 7 is moved on at sample well, insert the test tube of sample well and incubate in groove 71, heating 5
Minute drying magnetic bead, Y-direction transmission mechanism 15 drive rack for test tube 4 to move, and sample adding device 5 reuses new pipette tips, and X is to driver
Structure 14 drives rack for test tube 4 to move, and takes a certain amount of eluent to add in sample well using sample adding device 5, is not stopped using sample adding device 5
Pressure-vaccum sample well 5 minutes, eluent is fully mixed with magnetic bead, DNA is dissolved into from magnetic bead in eluent, by Magneto separate
Device 8 moves on to well bottom, waits 1-2 minutes, and siphoning away purifying DNA sample using sample adding device 5 is transferred in new test tube.
Second step：The DNA after purification of acquisition is processed into DNA small fragments, digestion or ultrasonic wave are used outside instrument
The mode for interrupting DNA is realized；
3rd step：Library is built, the heating of heater 7 is reached 30 degree, taken the photograph successively by End repairing reagents 30
Family name's degree incubates 30 minutes, A-tailing reagents incubate 30 degrees Celsius of 30 minutes and 30 degrees Celsius of Ligation reagents and incubate 15 points
Clock, incubate be required for the mixed liquor purifying of acquisition after terminating every time, purification process is identical with the purification process of the first step, finally
Sequenator of the DNA direct Sequencings purified as being adapted to Illumina；Or taken the photograph successively by End repairing reagents 30
Family name's degree incubates 30 minutes and 30 degrees Celsius of Ligation reagents incubate 15 minutes, incubates be required for the mixed of acquisition after terminating every time
Liquid purifying is closed, purification process is identical with the purification process of the first step, finally obtains the DNA direct Sequencings of purifying as being adapted to Ion
Torrent sequenator.
The advantages of the present embodiment：
1st, thoroughly solve the problems, such as that the manual sample for preparing is time-consuming low with flux, the system whole flow process is by one-stop work
Station is automatically performed, and is avoided malfunctioning and is more suitable for the standardization in laboratory；
2nd, the DNA sample after dispensing is directly used in the survey of two generations after the small multi-channel library construction instrument processing of the present invention
Sequence, it is not necessary to extra process.8 reagent strip positions, 8 sample preparations can be carried out simultaneously, greatly improve sample preparation efficiency.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, although with reference to foregoing reality
Apply example the present invention is described in detail, for those skilled in the art, it still can be to foregoing each implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic.All essences in the present invention
God any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection with principle.
Claims (8)
- A kind of 1. small multi-channel library construction instrument, it is characterised in that including frame, the frame be provided with the first supporting plate and Controller, first supporting plate, which is provided with, to be used to deposit reagent strip and the rack for test tube and sample adding device of pipette tips, the rack for test tube Side is provided with waste liquid box, and the rack for test tube bottom is movably equipped with heater and magnetic separating device respectively, the heater and The magnetic separating device is connected to the first transmission mechanism and the second transmission mechanism, and the top of the rack for test tube is provided with described add Sampling device, the sample adding device connect the 3rd transmission mechanism, first transmission mechanism, second transmission mechanism and described the Three transmission mechanisms are electrically connected the controller.
- 2. small multi-channel library construction instrument according to claim 1, it is characterised in that be movably equipped with the rack for test tube For scanning the barcode reader of kit information, the barcode reader connects the 4th transmission mechanism, the 4th driver Structure is electrically connected the controller.
- 3. small multi-channel library construction instrument according to claim 1 or 2, it is characterised in that the sample adding device includes Sample-adding pump, sample-adding needle assemblies and elevating mechanism fixed plate, the elevating mechanism fixed plate is in first supporting plate, institute State and connected between sample-adding pump and the sample-adding needle assemblies by flexible pipe, the 3rd transmission mechanism is and the sample-adding needle assemblies phase The 3rd linear electric motors for connecting and driving the sample-adding needle assemblies to be moved up and down in the elevating mechanism fixed plate, the test tube Frame bottom is provided with Y-direction transmission mechanism and the driving examination for driving the rack for test tube to make to move forward and backward along the frame width Pipe support makees the X of side-to-side movement to transmission mechanism along the frame length direction.
- 4. small multi-channel library construction instrument according to claim 3, it is characterised in that the rack for test tube and described first The second supporting plate is provided between supporting plate, Y-direction transmission mechanism is respectively equipped with first supporting plate and second supporting plate Supported respectively with the rack for test tube and described second to transmission mechanism, the Y-direction transmission mechanism and the X to transmission mechanism with X Plate is connected, and the Y-direction transmission mechanism and the X are electrically connected the controller to transmission mechanism.
- 5. small multi-channel library construction instrument according to claim 4, it is characterised in that the Y-direction transmission mechanism includes First timing belt, the first motor gear for being located at the first timing belt both ends and the first driven gear, the first motor with And two the first guide rails being parallel to each other, first guide rail is parallel with first timing belt, and the bottom surface of the rack for test tube is set There are a first straight line bearing for crossing first guide rail and the timing belt tabletting being connected with first timing belt, described Two supporting plates are provided with first motor gear, first driven gear and first guide rail, second supporting plate Bottom is provided with second straight line bearing, and the X includes the 4th linear electric motors in first supporting plate to transmission mechanism With two the second guide rails being parallel to each other, second guide rail crosses the second straight line bearing, the 4th linear electric motors with Second supporting plate is connected, and first supporting plate is provided with long strip through hole, and first motor passes through the strip Through hole is connected with first motor gear.
- 6. the small multi-channel library construction instrument according to claim 4 or 5, it is characterised in that the heater includes Several are used to insert the incubation groove of test tube, the incubation groove fixed plate for fixing the incubation groove and for incubating groove heating Adding thermal resistance or heating cushion, first transmission mechanism include several first straight line guide rails, with the first straight line guide rail The first straight line motor that several intersecting first sliding blocks of activity and the driving heater move up and down, described first slides Block and the first straight line motor are in the incubation groove fixed plate, the first straight line motor and the controller phase Even, short transverse of the first straight line guide rail along the frame is located at the side of the rack for test tube.
- 7. the small multi-channel library construction instrument according to claim 1 or 2 or 4 or 5 any one, it is characterised in that institute Stating magnetic separating device includes magnet fixed plate and is respectively arranged on the magnetic field fixed plate and strong magnet at the magnet fixed plate both ends, Second transmission mechanism include 2 second straight line guide rails, with movable 2 the second sliding blocks intersected of the second straight line guide rail with And second straight line motor, second sliding block are located at the both ends of the magnetic field fixed plate, the second straight line motor is located at described In the fixed plate of magnetic field and the controller that is electrically connected, width of the second straight line guide rail along the frame are located at the test tube The both sides of frame.
- 8. the method in small multi-channel library construction instrument structure library is as follows：Use DNA purification kits and library construction reagent Two kinds of kits of box, and two kinds of kits use all in the form of reagent strip.The first step：DNA is purified, and is comprised the following steps that：DNA sample to be purified is put into the sample well of purification kit, opens small multi-channel library construction instrument, instrument is opened Inspection is started from, after self-test is qualified, the reagent strip equipped with DNA sample and pipette tips are put into the rack for test tube, the barcode reader Start to purify after reading reagent box validation of information, make magnetic bead and DNA sample blending incubation 5 minutes by adding magnetic bead successively, the Magnetic Isolation go supernatant, add for the first time 80% ethanol cleaning DNA sample make 80% ethanol and magnetic bead mixing 1-2 minutes, Second of Magnetic Isolation goes supernatant, second of addition 80% ethanol cleaning DNA sample 80% ethanol and magnetic bead is mixed 1-2 points Clock, third time Magnetic Isolation go supernatant, magnetic bead to dry 5 minutes, add elution buffer to make elution buffer and magnetic bead blending incubation 5 Minute, the 4th Magnetic Isolation simultaneously keep 2-3 minutes and transfer supernatant into fresh sample pipe, wherein being mixed in magnetic bead and DNA sample It is even be incubated 5 minutes, 80% ethanol and magnetic bead mix and elution buffer and magnetic bead mixing be by the sample adding device not Stopping ground pressure-vaccum DNA sample makes it mix what is realized, and it is that the magnetic separation device is moved on into the sample that Magnetic Isolation, which removes supernatant, Bottom hole portion adsorbs magnetic bead and removes waste liquid with the sample adding device again, and moving the rack for test tube by the Y-direction transmission mechanism will Waste liquid is moved on in the waste liquid hole of reagent strip, and pipette tips are dropped into the waste liquid box, the use of new pipette tips is to driver by the X Structure moves the rack for test tube and the 3rd transmission mechanism drives the sample needle to move up and down realization, adds magnetic bead and 80% Ethanol and elution buffer are to move the rack for test tube and the 3rd transmission mechanism drive by the Y-direction transmission mechanism The sample needle moves up and down realization, and it is to realize heat drying by drying at room temperature or the heater that magnetic bead, which is dried,；Second step：The DNA after purification of acquisition is processed into DNA small fragments, interrupted outside instrument using digestion or ultrasonic wave DNA mode is realized；3rd step：Library is built, the heating devices heat is reached 30 degree, taken the photograph successively by End repairing reagents 30 Family name's degree incubates 30 minutes, A-tailing reagents incubate 30 degrees Celsius of 30 minutes and 30 degrees Celsius of Ligation reagents and incubate 15 points Clock, incubating be required for the mixed liquor purifying of acquisition after terminating every time, purification process is identical with the purification process of the first step, Finally obtain the DNA direct Sequencings of purifying；Or incubate 30 minutes by 30 degrees Celsius of End repairing reagents successively and 30 degrees Celsius of Ligation reagents incubate 15 minutes, incubate be required for the mixed liquor purifying of acquisition, purifying side after terminating every time Method is identical with the purification process of the first step, finally obtains the DNA direct Sequencings of purifying.
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