Biological sample preparation systemTechnical field
The present invention relates to life science, and system is prepared more particularly, to a kind of biological sampleSystem and method.
Background technology
The preparation of biological sample to life science, medicine research and development, the prevention of disease and control,Agricultural research, the clinical diagnosis based on precisely medical treatment are all most important, due to biological sampleComplexity, its sample preparation procedure often complex, rely on substantial amounts of manual operations,It is difficult to automate.
So that two generations were sequenced as an example, the Sample Preparation Procedure of its sequencing library structure is related to nucleic acidFragmentation, multistep enzyme reaction, multistep nucleic acid purification, or even the link such as PCR, take, takePower, it is difficult to automate, high is required to experimenter's experimental skill, manually operated reappearanceDifference, it is readily incorporated manually operated error even mistake, the difficulty of sample preparation and constrains twoFor the development and application of sequencing technologies.
At present, the automation prepared to biological sample is often using commercial automation liquid relief workMake station to realize.Liquor removing workstation possesses single or multiple pipetting channels, is grasped according to manual experimentMake flow liquid the programme-control of process computer such as added to, mixed, removed to realize automationOperation, its advantage are can to carry out automatic fluid processing so as to which Sample Preparation Procedure is automaticChange, but inferior position is due to simply to repeat manual experiment, can not save single sample preparationTime, and due to preparing, writing, debugging, Optimal Experimental program, often than manualExperiment takes more time, and reduces efficiency.
By taking the library construction of two generations sequencing as an example, the purifying of multistep nucleic acid is the most in its flowOne of time-consuming step, current commercial liquor removing workstation coordinate suction nozzle to inhale using pipetting channel moreTake magnetic-particle to add in the solution containing nucleic acid for needing to purify, and blown and beaten and mixed with suction nozzle,Magnetic-particle is promoted to be combined with nucleic acid, it is then that it is close with magnet, inhaled magnetic-particleOn the inwall of reaction cavity, then waste liquid is suctioned out with suction nozzle, and magnetic-particle is stayed in insteadAnswer in cavity；Then cleaning fluid is added, is blown and beaten and mixed with suction nozzle, to reach purged of impuritiesPurpose, after it is close with magnet, waste liquid is suctioned out with suction nozzle, and magnetic bead is stayed in insteadAnswer in cavity；Eluent is eventually adding, is blown and beaten and mixed with suction nozzle, promotes nucleic acid and magneticGrain dissociation, after it is close with magnet, with suction nozzle by containing nucleic acid eluent suction out,Obtain purified product.This process is related to many more manipulations, and single purifying is time-consuming often small at oneWhen more than., it is necessary to be mixed biology enzyme with nucleic acid after purification with suction nozzle after nucleic acid purification,To carry out enzyme reaction, many liquor removing workstations can not carry out automating enzyme reaction, it is necessary to handIt is dynamic to be transferred into temperature control equipment, such as shifting is again returned in PCR instrument, after reactionLiquid work station continues nucleic acid purification process.Further relate among these by every step experiment needed for reagent,The process of liquor removing workstation is inserted or removed to consumptive material, biological specimen, and whole process is difficult to avoid that handDynamic operation, in addition to time-consuming, still can not thoroughly solve the problems, such as all to automate.
The content of the invention
The shortcomings that it is an object of the invention to overcome above-mentioned prior art.
Therefore, the invention provides a kind of biological sample preparation system, the system includes：OneOr multiple bar magnets, there is the end for being used for attracting magnetic-particle；Be socketed in it is one or moreSleeve pipe outside individual bar magnet；Liquid transfer device, the liquid transfer device can be one or many turnsLiquid relief body is into specified reaction cavity；Motion control mechanism, the motion control mechanism can be realNow and control one or more of bar magnets and described sleeve pipe and the liquid transfer device itBetween relative motion.
It can control using computer program and coordinate liquid transfer device, based on bar magnet and magneticThe motion of the extraction from biological material purification devices, consumptive material carrying mechanism of property particle, to whole samplePreparation process optimizes control, so as to maximize the efficiency of sample preparation.According to the present invention'sSystem has the advantage not available for technology before following a few projects：
First, liquid addition/transfer, biological extraction/separation/purifying, biochemical reaction can be parallelProcessing, causes speed faster, more efficient, the unit interval can handle more biological specimens；
Second, coordinate sleeve pipe to complete the transfer of magnetic-particle with bar magnet, and then realize and be incorporated into magneticThe separation and purifying of biomolecule on property particle, compared to existing liquor removing workstation technology, moreAccelerate speed, convenient, efficiency high, whole process is automatically finished, and be related to during itReagent (include but are not limited to magnetic-particle suspension, magnetic-particle and biomolecule combination liquid,Cleaning fluid, eluent) filling process automatically completes；
3rd, without manual operations, avoid mistake or liquid operation mistake that manual operation is triggeredDifference, reliability is improved, and saved labour turnover.
In the present invention, term " biological sample " refers to the material and material for coming from life entity,The nucleic acid that includes but is not limited to come from human body, animal body, plant, microbial body, albumen,Polypeptide, metabolite and its derivative.
In the present invention, term " bar magnet " refers to have magnetic slender type object, including butBe not limited to it is elongated bar-shaped, such as cylinder-shaped magnet or electromagnet.
In the present invention, term " consumptive material " refers to the running stores for biological or chemical experiment,Include but is not limited to hold the EP pipes of reagent and biological sample, eight platoon pipes, PCR pipe, 96Orifice plate, suction nozzle and its suction nozzle box.
In the present invention, term " reagent " refers to the liquid for biological or chemical experiment, bagInclude but be not limited to biology enzyme, enzyme reaction system, magnetic-particle suspension, magnetic-particle and biologyIt is molecule combination liquid, cleaning fluid, eluent, PCR primer, sequence measuring joints, water, ethanol, differentThe mixed liquor of propyl alcohol and aforesaid liquid.
In one embodiment, the lateral dimension of the end is less than or equal to 10mm, preferablyGround, it is 1 to 6mm.Such bar magnet go for small size (such as 200 microlitres withUnder) in system, more preferable effect can be obtained in many applications (such as the sequencing of two generations),And reach the purpose saved reagent and save precious biological sample.
Preferably, the biological sample preparation system also includes the control of one or more fluid temperaturesDevice, the liquid temperature control device can include conductor temperature controller and/or water-bathTemperature controller and/or thermocirculator and/or PCR instrument.
The liquid transfer device can include piston driving pin pump, the pin pump containing liquid filling body,The pin pump or peristaltic pump of no liquid filling.
Preferably, described sleeve pipe is made of plastics.
In one embodiment, the bar magnet is in array arrangement (such as 1 × 8 array, 12 × 8Array, 24 × 16 arrays), and described sleeve pipe also arranges in identical array.
Advantageously, the bar magnet is integrated can be synchronized with the movement on the same substrate, and describedEntirety is interconnected to form between sleeve pipe.
Advantageously, the biological sample preparation system also includes reagent storage rack and/or consumptive material is depositedStore up frame.
Present invention also offers a kind of method prepared by biological sample, this method comprises the following steps：
Step A：Solution comprising biological sample and magnetic-particle is provided；
Step B：The magnetic-particle of biological sample will be combined with from institute using the bar magnet with sleeve pipeState in solution and suction out；
Step C：The magnetic-particle for being combined with biological sample is transferred in another solution；
Above step A to C is repeated, to realize the purifying of biological sample.
Preferably, methods described also includes step D：To the biological sample included after purificationThe reagent needed for reacting in next step is added in solution, it is followed at a certain temperature or through overheatRing carries out biochemical reaction.
Preferably, methods described also includes step E：Biological sample after biochemical reaction is repeatedStep A to C, is purified again.
According to practical application, step A to D can be circulated arbitrarily repeatedly, finally to realize sampleThe purpose of product preparation.
Brief description of the drawings
The present invention is described in further detail below with reference to accompanying drawings.People in the artMember is it is easily understood that these accompanying drawings are intended solely for illustrative purposes, and are not intended to thisThe protection domain of invention is construed as limiting.Identical reference represents same or analogous in figurePart.For illustrative purposes, these figures and non-fully drawn to scale.
Fig. 1 is schematically bowing for biological sample preparation system according to a first embodiment of the present inventionView；
Fig. 2 is schematically bowing for biological sample preparation system according to a second embodiment of the present inventionView；
Fig. 3 is schematically bowing for biological sample preparation system according to a third embodiment of the present inventionView；
Fig. 4 is schematically bowing for biological sample preparation system according to a fourth embodiment of the present inventionView；
Fig. 5 is the schematic side elevation of Fig. 1 liquid transfer/adding sets into Fig. 4.
Embodiment
Fig. 1 schematically shows biological sample according to a first embodiment of the present invention with top viewProduct preparation system 1.The system include liquid filling/transition range 110, biochemical reaction zone 120 withAnd biological sample separated/extracted/zone purification 130.
One or more (Fig. 1 of consumptive material receiving position 111 are provided with liquid filling/transition range 110Schematically illustrate 3).In the present embodiment, each consumptive material receiving position 111 includes8 pipe positions 112, pipe position 112 are the devices that can receive Reagent Tube, such as size and reagentThe hole that the diameter of pipe matches.Can be accommodated in Reagent Tube such as suspension containing magnetic-particle,Magnetic-particle and biomolecule combination liquid, cleaning fluid, eluent, enzyme for biochemical reaction etc..Various reagents can be stored in different Reagent Tubes respectively, to avoid cross pollution.According toThe biological sample preparation system 1 of this embodiment can be used together with eight platoon pipes, be realized flexibleBut prepared by the biological sample of small throughput.Those skilled in the art can be set according to the actual requirementsPipe position included by the quantity of consumptive material receiving position 111 and each consumptive material receiving position 111112 quantity.Consumptive material can be placed at a part of consumptive material receiving position 111 (in this exampleFor eight platoon pipes) liquid filling/transfer operation is carried out, and in idle consumptive material receiving position 111Place's storage consumptive material.
One or more consumptive material receiving positions 121 also are provided with biochemical reaction zone 120, and (Fig. 1 showsShow 1 to example property).Equally, each consumptive material receiving position 121 includes 8 pipe positions,It can be used for receiving such as eight platoon pipes.Temperature control equipment 122 is equipped with each pipe position.Liquid temperature control device 122 can include conductor temperature controller, bath temperature controller,Thermocirculator, PCR instrument or temperature control equipment or combinations thereof based on other principles.Temperature control equipment 122 at each pipe position works independently from each other or unitedOne control.Preferably, temperature controlling range is between 0 degree Celsius to 100 degrees Celsius.BorrowHelp the temperature control equipment, the biochemical reaction in Reagent Tube can be set in advance a certainAt a temperature of or temperature range in carry out.
One or more (Fig. 1 of bar magnet 131 are provided with bio-separation/extraction/zone purification 130Schematically illustrate 8) and the sleeve pipe 132 that is socketed in outside each bar magnet.These sleeve pipe energyEnough isolate bar magnet and reagent, reach the purpose for protecting bar magnet not extended its service life by burn into.In addition, up and down concussion of the sleeve pipe in reagent can also play mictomagnetism particle and biomolecule,Cleaning or the effect of elution biomolecule.Also, it is arranged right below one or more in bar magnetConsumptive material receiving position (Fig. 1 schematically illustrates 1), equally, each consumptive material received bitPut including 8 pipe positions, can be used for receiving such as eight platoon pipes.One end of each bar magnet 131Motion control mechanism is connected to, the other end is used to stretch into adsorb magnetic-particle in reagent.These magneticRod 131 may be coupled to respective motion control mechanism, so as to independently of each other along verticalDirectly moved in the direction of paper, realize that stretching into reagent neutralizes the action withdrawn from from reagent.Also may be usedWith this 8 bar magnets 131 link together (for example, integrating on the same substrate), by sameThe control of one motion control mechanism, is synchronized with the movement so as to realize.Motion control can so be simplifiedMechanism, improve the reliability of control.Sleeve pipe 132, sleeve pipe 132 are equipped with outside each bar magnet 121Gap be present between bar magnet 121, the gap is sized so that sleeve pipe 132 and bar magnetRelative motion can occur between 121, also magnetic force caused by bar magnet 121 is enough reagentIn whole magnetic-particles be attracted on the outer wall of sleeve pipe 132.Preferably, the gap is less than2mm.Identically with bar magnet, these sleeve pipes 132 may be coupled to respective motion control mechanism,So as to be moved independently of each other along the direction perpendicular to paper, reagent neutralization is stretched into realizationThe action withdrawn from from reagent.This 8 sleeve pipes 132 can also be interconnected to an entirety,Controlled by same motion control mechanism, be synchronized with the movement so as to realize.
Bar magnet 121 can be made up of materials such as permanent magnet or electromagnets, and those skilled in the art canIt is actually needed with basis and designs its shape and size.For example, in the sequencing application of two generations, magneticThe end for being used to adsorb magnetic-particle of rod 121 preferably has the horizontal stroke less than or equal to 10mmTo size (i.e. perpendicular to the size of its length direction), it is highly preferred that the size of 1 to 6mm.Sleeve pipe 132 can be made up of any corrosion-resistant material, preferably be made of plastics.
Above three region 110,120,130, which can be respectively equipped with, extra is used for storing reagentAnd/or one or two in the position of consumptive material and/or sample, or these three regions is provided with thisThe position of sample.Certainly, it will be understood by those skilled in the art that this position can also individually be setPut in the 4th region outside above three region.
As shown in figure 1, biological sample preparation system 1 also includes one or more liquid transfer dressPut 140 (Fig. 1 schematically illustrates 1) and one or more for carrying experiment consumptive materialThe manipulator 150 (Fig. 1 schematically illustrates 1) of (such as eight platoon pipes), manipulatorThere is the handgrip 151 for carrying consumptive material on 150.Manipulator 150 is slidably connected to support 160,Can be along arrow B bidirectional-movements.Liquid transfer device 140 is slidably attached at support 161On, can be along arrow C bidirectional-movements, and support 161 is then slidably attached at support 160On, can be along arrow A bidirectional-movements.Therefore, liquid transfer device 140 can reach lifeAny position in thing sample preparation system 1, to any required adding liquid in any regionAny liquid is added on position in the cavity of any consumptive material.Pipetting volume is generally 0.3 microlitre extremelyBetween 1000 microlitres.Manipulator 150 can also catch experiment consumptive material (such as eight platoon pipes)On any position in any region of the system, to complete in the same area or between different zonesThe transfer of sample.
Liquid transfer device 140 can be any suitable liquid transfer device, for example, can be withIncluding suction nozzle (as shown in Figure 5) and drive device, the pin pump that is driven such as piston, containing filling liquidPin pump, the pin pump or peristaltic pump of no liquid filling of body.
Biological sample preparation system as described above can have a variety of applications.It is sequenced below with two generationsExemplified by the experiment flow of library construction, illustrate how the system works.
DNA or RNA separated/extracted/purifying such as nucleic acid interrupt after purifying, end repairPurifying afterwards, end add the purifying after adenosine, end add sequence measuring joints after purifying,Purifying after PCR, carried out in region 130.First, liquid filling/transition range 110 byLiquid transfer device 140 fills magnetic-particle in consumptive material (such as eight platoon pipes) and contains DNAOr RNA solution, then manipulator 150 consumptive material is transported to bio-separation/extraction/purifyingArea 130, it is placed into immediately below bar magnet 131 and sleeve pipe 132；Then, sleeve pipe 132 is movingUnder the control of mechanism with certain speed, vibration frequency and amplitude include magnetic-particle andVibration is (i.e. along perpendicular to the direction of paper in DNA or RNA consumptive material (such as eight platoon pipes)Move back and forth), to allow magnetic-particle and DNA or RNA to combine；Then bar magnet 131Stretch into sleeve pipe 132, the solution entered together with sleeve pipe 132 in consumptive material, magnetic-particle is connectedIt is attracted to the biomolecule that is incorporated on magnetic-particle on the outer wall of sleeve pipe 132, and from reagentIn withdraw from；Then, the consumptive material for including waste liquid is transferred to any idle consumption by manipulator 150Material receiving position, and the consumptive material of cleaning fluid will be included (in advance by liquid transfer device 140It is populated in liquid filling/transition range 110) it is placed into immediately below bar magnet 131.Then, weightTo carry out follow-up impurity cleaning process, (what magnetic rod sleeve stretched into is cleaning fluid to multiple above stepIn), DNA or RNA elution processes (magnetic rod sleeve stretch into be in eluent).This areaTechnical staff is appreciated that impurity cleaning process may be carried out for several times.
After DNA/RNA purifying is completed, it will be contained with manipulator 150 purifiedDNA/RNA eight platoon pipes are transferred to liquid transfer/fill area 110, by liquid transfer device140 enzymes and/or other reagents added required for reacting in next step, and can enter as neededRow mixing.
Then, manipulator 150 removes the eight platoon pipes containing the DNA/RNA solution for having added enzymeBiochemical reaction zone 120 is transported to, carries out biochemical reaction at a certain temperature or by thermal cycle.
Finally, manipulator 150 shifts the eight platoons pipe to bio-separation/extraction/zone purification 130,Purified next time.
Above-mentioned steps move in circles, and finally realize the biological sample for two generation sequencing libraries structureIt is prepared by product.It can be seen that whole process realizes full automation without any manual operations.Further, since liquid addition, three biochemical reaction, nucleic acid purification steps can be carried out parallel,Sample preparation is carried out simultaneously at least three piece of eight platoon pipe, sample preparation can be saved with high degreeTime, improve efficiency, compared to traditional liquor removing workstation library construction protocols save up to about2/3 operating time.
Fig. 2 schematically shows biological sample according to a second embodiment of the present invention with top viewProduct preparation system 2.Compared to the first embodiment shown in Fig. 1, difference is：The system 2There is no obvious region division, any consumptive material receiving position 230,240 may be used to liquidFilling, biochemical reaction, biological sample separated/extracted/purifying；There is no manipulator in the system,Without carrying and shifting consumptive material (such as eight platoon pipes)；Bar magnet 211 and sleeve pipe 221 are except canBeyond being moved back and forth on the direction perpendicular to paper, additionally it is possible in its respective motion control machineRespectively along arrow B, C bidirectional-movement under the control of structure 210 and 220, therefore, pass through byThe magnetic-particle for being combined with biological sample is attracted on the outer wall of sleeve pipe 221 and moves the sleeve pipeMove specified location, it is possible to achieve the position transfer of biological sample；, can be with according to practical applicationTemperature control equipment 231 is set at any desired pipe position 241, and (Fig. 2 is merely illustrativelyShow to be provided with temperature control equipment at the pipe position of two consumptive material receiving positions 230 on the right).ByAny position can be reached in bar magnet and sleeve pipe, liquid addition and biochemical reaction can be in any positionsProgress is put, therefore the program is more flexible and reliable.
Biological sample preparation system as described above can have a variety of applications.It is sequenced below with two generationsExemplified by the experiment flow of library construction, illustrate how the system works.
First, in some or multiple consumptive material receiving positions 230,240 by liquid transfer device250 in the consumptive material (such as eight platoon pipes) filling magnetic-particle and containing DNA or RNA solution,Then bar magnet 211 and sleeve pipe 221 are moved to above the consumptive material receiving position；Then, sleeve pipe221 are including magnetic under the control of motion with certain speed, vibration frequency and amplitudeProperty particle and DNA or RNA consumptive material (such as eight platoon pipes) in vibration (i.e. edge perpendicular toThe direction of paper moves back and forth), to allow magnetic-particle and DNA or RNA to combine；SoBar magnet 211 is stretched into sleeve pipe 221 afterwards, the reagent entered together with sleeve pipe 221 in consumptive material, willMagnetic-particle is attracted to the outer wall of sleeve pipe 221 together with the biomolecule being incorporated on magnetic-particleOn, and withdrawn from from reagent；Then, bar magnet 211 and sleeve pipe 221 divide with biology is combined withThe magnetic-particle of son is moved to the consumptive material for including cleaning fluid (in advance by liquid transfer device 250It is populated) top.Then, above step is repeated to carry out follow-up impurity cleaning process (magneticWhat rod sleeve pipe stretched into is in cleaning fluid), (magnetic rod sleeve stretches into DNA or RNA elution processesBe in eluent), the position of elution is needed with function of temperature control, can carry out biochemical reaction.It will be understood by those skilled in the art that impurity cleaning process may be carried out for several times.
After DNA/RNA purifying is completed, liquid transfer device 250 is moved to relevant position,The enzyme and/or other reagents added required for reacting in next step, and can be mixed as neededClose.Then start temperature control mechanism, carry out at a certain temperature or by thermal cycle biochemical anti-Should.
Finally, liquid transfer device 250 is moved to relevant position and adds magnetic bead and nucleic acid purificationRequired liquid, bar magnet 211 and sleeve pipe 221 are moved to above the consumptive material receiving position, are enteredRow purifies next time.Above-mentioned steps move in circles, and finally realize for two generation sequencing library structuresIt is prepared by the biological sample built.Whole process realizes full automation without any manual operations.Because three liquid addition, biochemical reaction, nucleic acid purification steps can be carried out parallel, at leastThree piece of eight platoon pipe carries out sample preparation simultaneously, and the time of sample preparation can be saved with high degree,Efficiency is improved, the library construction protocols compared to traditional liquor removing workstation save about 2/3 operationTime.
Fig. 3 schematically shows biological sample according to a third embodiment of the present invention with top viewProduct preparation system 3.The embodiment is similar to the first embodiment shown in Fig. 1, and difference existsIn：Each consumptive material receiving position includes 96 pipe positions in this embodiment, can be with the market96 orifice plates are used cooperatively；There is reagent/consumptive material deposit position in liquid filling/transition range 310, such asSupplies racks and/or reagent trough 311, depositing for storing reagent pipe (such as EP pipes, magnetic bead pipe)Store up frame 313 (including multiple storage holes 312 for being used to receive Reagent Tube), for depositing 96 holesThe plate position 315 of the microwell plates such as plate.
It can be used together according to the biological sample preparation system 3 of this embodiment with 96 orifice plates,Realize prepared by high flux, a large amount of biological samples rapidly and efficiently.
Fig. 4 schematically shows biological sample according to a fourth embodiment of the present invention with top viewProduct preparation system 4.The embodiment is similar to the second embodiment shown in Fig. 2, and difference existsIn：Each consumptive material receiving position includes 96 pipe positions in this embodiment, can be with the market96 orifice plates are used cooperatively；The system also has reagent/consumptive material deposit position, such as supplies racks and/or examinationAgent groove 411, for storing reagent pipe (such as EP pipe, magnetic bead pipe) storage rack 413 (includingIt is multiple to be used to receive the storage holes 412 of Reagent Tube), the plate for depositing the microwell plates such as 96 orifice platesPosition 415.
It can be used together according to the biological sample preparation system 4 of this embodiment with 96 orifice plates,Realize prepared by high flux, a large amount of biological samples rapidly and efficiently.
It is understood that those skilled in the art can according to the consumptive material of suitable practical application comeThe quantity and size of consumptive material receiving position are set, are allowed to for example, any of the above-described system can be transformedIt can be used together with 384 orifice plates.The quantity of liquid transfer device and position, manipulator (Can be the carrying mechanism of other forms) quantity and position can also set according to the actual requirementsMeter.
Accompanying drawing and the non-limiting specific embodiment for foregoing description describing the present invention.In order to teachInventive principle is led, simplifies or eliminates some conventional aspects.Those skilled in the art shouldUnderstand that the modification from these embodiments is fallen within the scope of the present invention.Those skilled in the artIt should be appreciated that features described above can combine to form multiple modifications of the present invention in a variety of ways.Thus, the invention is not limited in above-mentioned specific embodiment, and only by claim and theyEquivalent limit.