CN103620016A - Microfluidics device and use thereof - Google Patents
Microfluidics device and use thereof Download PDFInfo
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- CN103620016A CN103620016A CN201280016505.6A CN201280016505A CN103620016A CN 103620016 A CN103620016 A CN 103620016A CN 201280016505 A CN201280016505 A CN 201280016505A CN 103620016 A CN103620016 A CN 103620016A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L2200/0652—Sorting or classification of particles or molecules
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0883—Serpentine channels
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/08—Regulating or influencing the flow resistance
- B01L2400/084—Passive control of flow resistance
- B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502746—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the means for controlling flow resistance, e.g. flow controllers, baffles
Abstract
A microfluidics device and a use thereof are provided. Specifically, a microfluidics device, a method for determining whether a biological sample has a hereditary variation, and a system for determining whether a biological sample has a hereditary variation are provided. The microfluidics device has a microfluidics passage applicable for monoplast separation.
Description
Micro fluidic device and application thereof priority information
It is the priority and rights and interests of 201 1 10092704.2 patent application that the application, which asks submitted to China national Department of Intellectual Property 201 1 on April 13, number of patent application, and by referring to being incorporated by herein.Technical field
The present invention relates to micro fluidic device and application thereof, in particular to micro fluidic device, determine whether biological sample has the method for hereditary variation and determines whether biological sample has the system of hereditary variation.Background technology
Microfluidic chip technology originates from analytical chemistry field, it is by Micrometer-Nanometer Processing Technology, the basic operation units such as preparation, reaction, separation, the detection of sample involved in biological, medical science and chemical field are integrated on several square centimeter chips, network is formed by microchannel, whole system, the technology of the various functions to replace conventional analysis laboratory are run through with controlled fluid.Microflow control technique shows extremely strong superiority in terms of cell separation, nucleic acid extraction, purifying, PCR amplifications.
However, current micro-fluidic chip still has much room for improvement.The content of the invention
It is contemplated that at least solving one of technical problem present in prior art.
In the first aspect of the present invention, the present invention proposes a kind of micro fluidic device.Embodiments in accordance with the present invention, the micro fluidic device, which has, to be suitable to separate single celled microfluidic channel.Thus, effectively it can be separated using micro fluidic device according to embodiments of the present invention from biological specimen unicellular.
In the second aspect of the present invention, the present invention proposes whether a kind of determination biological sample has the method for hereditary variation, it is characterised in that comprise the following steps：Using foregoing micro fluidic device, cell sample is separated from biological sample；At least a portion to the inhereditary material included in separated cell sample is expanded, to obtain amplified production；The amplified production is sequenced, to obtain sequencing result；And based on the sequencing result, determine whether the biological sample has hereditary variation.Pass through method according to embodiments of the present invention, effectively it can be separated by micro fluidic device according to embodiments of the present invention unicellular, so as to based on single celled inhereditary material such as full-length genome is sequenced, effectively obtained by being analyzed sequencing result with the presence or absence of abnormal in single celled inhereditary material such as full-length genome, so that it is determined that whether biological sample has hereditary variation.
In the third aspect of the present invention, the present invention proposes whether a kind of determination biological sample has the system of hereditary variation, it is characterised in that including：Foregoing micro fluidic device, the micro fluidic device is used to separate cell sample from biological sample；Amplification device, the amplification device is connected with the micro fluidic device, and is expanded suitable at least a portion to the inhereditary material included in separated cell sample, to obtain amplified production；Sequencing device, the sequencing device is connected with the amplification device, and suitable for the amplified production is sequenced, to obtain sequencing result；And analytical equipment, the analytical equipment is connected with the sequencing device, and suitable for being based on the sequencing result, determines whether the biological sample has hereditary variation.Whether there is the system of hereditary variation using determination biological sample according to embodiments of the present invention, foregoing method can effectively be implemented, effectively it can be separated by micro fluidic device according to embodiments of the present invention unicellular, so as to based on single celled inhereditary material such as full-length genome is sequenced, effectively obtained by being analyzed sequencing result with the presence or absence of abnormal in single celled inhereditary material such as full-length genome, so that it is determined that whether biological sample has hereditary variation.
Thus, embodiments in accordance with the present invention, the present invention is directed to the acquisition problem of low abundance cell, establish the low abundance cell screening model based on high-flux sequence, by the parameter of the biological information analog regulation model, detecting domains, sensitivity, accuracy and repeatability of the model in terms of the low abundance cell of examination are demonstrated.Specifically, the present invention initially sets up an automation micro-current controlled cell sorting model, produces a number of particular sequence data followed by amplification technique and new-generation sequencing technology and carries out effective examination to low abundance cell Different Variation.Microflow control technique, whole genome amplification technology and high throughput sequencing technologies are integrated, realization builds unicellular on micro-fluidic chip（Few cells）The function such as separation, enrichment, whole genome amplification, with reference to high throughput sequencing technologies, automated, the single celled detection and analysis of high flux, to the examination in terms of the hereditary variation of sample, the technology model can be applied to the research in terms of new science of heredity, such as find new mechanism of causing a disease etc..
Present invention demonstrates that a kind of method model for automating the low abundance cytometaplasia information of examination, its experiment flow is：（A) sample is made to enter micro fluidic device, it is unicellular by the purpose in multiple microchannels or the separation of small room and enriched sample；（B) toward addition lytic reagent in the microchannel on micro-fluidic chip or small room or sudden and violent in generation heat cleavage step under light（A) purpose obtained by is unicellular, is expanded using pyrolysis product DNA or R A as template；（C) to step（B) amplified production obtained by carries out building storehouse, is sequenced on high-flux sequence platform, the high-flux sequence platform includes but is not limited to Illumina/Solexa, ABI Solid and Roche 454;(d) to step（C) sequencing data obtained is handled.
Another aspect of the present invention provides a kind of micro fluidic device for being applied to the automation low abundance cytometaplasia information of examination, the inlet and outlet that the micro fluidic device is interconnected comprising at least one by microchannel or small room, is made up of micro-fluidic chip, drive system and detecting system.
In a preferred embodiment of the invention, the micro-fluidic chip of the micro fluidic device includes cell separation and enrichment unit and cell manipulation unit.
In a preferred embodiment of the invention, the cell separation and enrichment unit is provided with ^：Structure or ^:Barrier, the cell manipulation unit is provided with the gentle wealthy control of liquid stream.
In a preferred embodiment of the invention, the micro-fluidic chip of the micro fluidic device is also extracted and amplification unit comprising DNA and/or R A.
In a preferred embodiment of the invention, according to method of the micro fluidic device to the separation and concentration of low abundance cell, including the microchannel or small room for sample is flowed through the micro-fluidic chip, utilize the magnetic capture target cell in microchannel or microchamber.
In a preferred embodiment of the invention, according to the method for the micro fluidic device cell lysis, including making sample flow through the microchannel or small room of the micro-fluidic chip, using the magnetic capture target cell in microchannel or microchamber, and add lytic reagent or magnetic bead is sudden and violent in producing heat under light and cell lysis.
In a preferred embodiment of the invention, target dna or R A method are expanded from cell according to the micro fluidic device, including making sample flow through the microchannel or small room of the micro-fluidic chip, utilize the magnetic capture target cell in microchannel or microchamber, add lytic reagent or magnetic bead is sudden and violent in producing heat under light and cell lysis, target dna or R A are extracted, and is expanded using target dna or R A as template；The amplification includes whole genome amplification.
Thus, embodiments in accordance with the present invention, the present invention establishes the technology model of the low abundance cell screening based on microflow control technique and high-flux sequence, and demonstrate detecting domains, sensitivity, accuracy and the repeatability of the model, it can be achieved to carry out high throughput automated examination to the hereditary information of the low abundance cell in complex sample, realize that laboratory is high throughput automated, for genetics in terms of medical diagnosis research basis is provided.Embodiments in accordance with the present invention, the present invention devises the micro-fluidic chip with microchannel or small room, collect separation, gathering trace cell and to DNA or R A are extracted and expanded in cell function in a chip, reduce experimental provision equipment cost, it is easy to promote.
The additional aspect and advantage of the present invention will be set forth in part in the description, and partly will become apparent from the description below, or be recognized by the practice of the present invention.Brief description of the drawings
The above-mentioned and/or additional aspect and advantage of the present invention will be apparent and be readily appreciated that from description of the accompanying drawings below to embodiment is combined, wherein：
Fig. 1-Fig. 6 shows the structural representation of micro fluidic device according to embodiments of the present invention.Detailed description of the Invention
Embodiments of the invention are described below in detail, the example of the embodiment is shown in the drawings, wherein phase from beginning to end
The embodiment of figure description is exemplary, is only used for explaining the present invention, and is not considered as limiting the invention.In the description of the invention, it will be appreciated that, term " center ", " upper ", " under ", " preceding ", " rear ", " left side ", " right side ", " vertical ", " level ", " top ", " bottom " " interior ", the orientation of the instruction such as " outer " or position relationship be based on orientation shown in the drawings or position relationship, it is for only for ease of the description present invention and simplifies description, rather than indicate or imply that the device or element of meaning must have specific orientation, with specific azimuth configuration and operation, therefore be not considered as limiting the invention.
It should be noted that term " first ", " second " are only used for describing purpose, and it is not intended that indicating or implying relative importance or the implicit quantity for indicating indicated technical characteristic.Thus, " first " is defined, one or more this feature can be expressed or be implicitly included to the feature of " second ".Further, in the description of the invention, unless otherwise indicated, " multiple " are meant that two or more.
Term " low abundance cell ", " list（It is individual）Cell " and " few cells " is used interchangeably in the text, all referring in a complex sample comprising the cell for wanting type, but its content very little, general only one of which is to several.
Micro fluidic device
In the first aspect of the present invention, the present invention proposes a kind of micro fluidic device.Embodiments in accordance with the present invention, the micro fluidic device, which has, to be suitable to separate single celled microfluidic channel.Thus, effectively it can be separated using micro fluidic device according to embodiments of the present invention from biological specimen unicellular.
The form for being suitable to separate single celled microfluidic channel in embodiments in accordance with the present invention, micro fluidic device is not particularly restricted.For for separating the complexity of single celled sample and the rareness of aim cell, it can design and make on chip according to difference of the different type cell on physics, chemistry and biological characteristics unicellular to cell progress one step separation or multistep separates single celled microchannel, it is unicellular to obtain final purpose.It should be noted that can be by the various combinations for being suitable to separate single celled method, and be sequentially not particularly limited, as long as can realize to single celled separation.For example, biased sample cell can be passed into the first microfluidic channel first, preliminary separation is carried out, most interference cell is removed；Then again by the cell Jing Guo initial gross separation, it is passed into the 2nd ^：In stream control passage, the further enrichment of aim cell is carried out.
Embodiments in accordance with the present invention, microfluidic channel can the physics based on cell, chemical property, such as size, shape could deformation, cell surface property（Such as cell surface receptor, antigen, the permeability of film）, cell interior property（Such as express certain albumen, enzyme-specific）To realize to single celled separation.
According to one embodiment of present invention, multiple microtrabeculaes are formed with the microfluidic channel.Thus, it is possible to by forming post on microfluidic channel（It is referred to as barrier）, the obstacle flowed in the direction of flow as cell, so as to the difference based on cell size, realization is to single celled separation.Embodiments in accordance with the present invention, can build regularly arranged in microfluidic channel, and be spaced the barrier of fixed dimension（The form of barrier is not particularly restricted, and can be cylinder,
Ellipse is lived, square column etc.）, the lateral displacement of fixed dimension often occurs between row barrier.With reference to Fig. 1-3, gathered regularly arranged barrier by micro-fluidic chip channel interior（Such as multiple ^:Post is in the ^:It is in array distribution on stream control passage；), cell to be separated collides as the particle in fluid in flow channel with barrier, when cell is more than a certain size（Spacing between barrier is related）When, cell is according to certain angle（Lateral displacement between every row barrier is related）Shift, less than the size cell then according to former fluid trajectory flow, with this realize various sizes of cell separation (；Such as Fig. 1).Obstacle spacing, such as the distance between microtrabeculae, including horizontal spacing and longitudinal pitch can be adjusted according to the size of cell to be separated.In the vertical, the distance between two neighboring microtrabeculae is 10-100 microns to embodiments in accordance with the present invention, also, in the horizontal, the distance between two neighboring microtrabeculae is 10-100 microns.Thus, it is possible to further improve the single celled efficiency of separation.According to one embodiment of present invention, often relative 6-7 microns of the skew of row microtrabeculae.Thus, it is possible to further improve the single celled efficiency of separation.Barrier array structure can be arranged to single-stage or multistage, according to the difference of cellular environment to be separated, and the cell of fixed size, general list Grade ^ are separated such as from body fluid:Post array it is achieved that and as the complex sample of blood etc, there be the cell of sizes the inside, can carry out fine separation with multistage micro-pillar array.According to one embodiment of present invention, along flow direction, the ^：The diameter increase of post.Thus, it is possible to further improve the single celled efficiency of separation.Further, with reference to Fig. 2, the barrier array of multistage different spacing dimensions can be set up, the separation of various sizes of cell is further refined.Various sizes of cell is collected in exit, next step separation is carried out or is directly used in follow-up test experience.According to one embodiment of present invention, along flow direction, the diameter of microtrabeculae is constant.
In addition, embodiments in accordance with the present invention, the difference for the inertia force that can also be produced using various sizes of cell in bending channel, are realized in ^：Cell is separated in stream control passage.In bending channel, parabolic shape distribution is presented in the fluid of Stationary flow its flow velocity, in the channel between flow velocity it is maximum, the centrifugal force being subject to is maximum, and the rate of flow of fluid close to conduit wall is minimum.In order to keep the conservation of mass of fluid, vortex can be produced.Particle so in fluid receives the interaction of buoyancy and Dien power.Under certain flow conditions, formed and focus on flowing.The focused flow of the bigger particle of size closer to passage madial wall, the focused flow of the smaller particle of size further away from passage madial wall according to one embodiment of present invention, the microfluidic channel is crooked pipeline.Thus, by using crooked pipeline, cell can be efficiently separated to realize by producing inertia force.Just blunt according to the preferred embodiments of the present invention, the crooked pipeline used is selected from Archimedes spiral passage（As shown in Fig. 4）With nautilus line passage（As shown in Fig. 5）At least one.Thus, it is possible to by making various sizes of cell be passed into passage, form focused flow in bending channel, and reserved in different outlets, realize the different separation of big cellule, so as to further improve the single celled efficiency of separation.
In addition, embodiments in accordance with the present invention, can also be magnetic according to cell band and be separated.Cell band is magnetic, and is broadly divided into two kinds, one kind is that itself band is magnetic, such as red blood cell, and because red blood cell contains hemoglobin, hemoglobin is ferritin, therefore red blood cell has paramagnetism, it is possible to handled by deoxygenated, improves its magnetic；It is another be by can specificity capture aim cell antibody, aptamers etc. and magnetic microsphere connection, by antigen-antibody reaction, make specificity
Cell there is magnetic.Have magnetic cell by externally-applied magnetic field, magnetic cell can be separated with non-magnetic cell, as shown in Fig. 6.According to one embodiment of present invention, the micro fluidic device is further provided with magnet.Thus, it is possible to be magnetic based on cell band, and realize the separation to cell.Embodiments in accordance with the present invention, the set location of magnet is not particularly restricted, as long as enabling to microfluidic channel to be in the range of the effective magnetic field of magnet, embodiments in accordance with the present invention, the lower section of microfluidic channel can be disposed a magnet within, thus, it is possible to further improve separative efficiency.According to one embodiment of present invention, the width of the microfluidic channel is 0.5 millimeter, and length is 50 millimeters.Thus, it is possible to further improve the single celled efficiency of separation.
Micro fluidic device（Because chip is the primary clustering of realizing apparatus function, thus referred to herein as micro-fluidic chip）, can be prepared from known compounds by those skilled in the known methods.The making material of micro-fluidic chip includes silicon, glass, quartz, poly- Yue Ji Bing Women acid Yue esters（Poly (methylmethacrylate), PMMA), polystyrene（Polystyrene), poly- carbonic acid tenth of the twelve Earthly Branches purport（Polycarbonate) ^ polyethylene（Polyethylene) ^ silicon rubber（Such as poly- two Yue radical siloxanes (poly (dimethylsiloxane), PDMS), epoxy resin etc..Seem silicon, glass, quartz etc., ^ is carried out using the method for wet etching：The processing of structure channel；High when making depth-width ratio, structure is compared with ^：, it is necessary to carry out the processing of micro-structural using the method for deep reaction ion etching during thin silicon；And the high polymer material chip of heat curing-type is made, such as PDMS, epoxy resin are typically necessary structure mould, can be made of silicon, glass, optical cement, PDMS etc..Make the method for mould and typically have profound light, it is LIGA (Lithographie galvanoformung and abformung), profound1It is worm, soft profound1The technology such as worm power mouthful work.' and then utilize the method for cast to make micro-structured channels；And the high polymer of thermoplastics type, such as makrolon, PMMA, it is general to be made using the method for hot pressing.Some high polymer materials, such as PMMA, polystyrene can use the method for laser ablation to be made.Determine whether biological sample has the method and system of hereditary variation
In the second aspect of the present invention, the present invention proposes whether a kind of determination biological sample has the method for hereditary variation.Embodiments in accordance with the present invention, this method can comprise the following steps：
First, using foregoing micro fluidic device, cell sample is separated from biological sample, micro fluidic device is previously with regard to and detailed description has been carried out, do not repeating.It should be noted that all feature and advantage being previously with regard to described by micro fluidic device are applicable the method for determining whether biological sample has hereditary variation.
Next, at least a portion to the inhereditary material included in separated cell sample is expanded, to obtain amplified production.Term " inhereditary material " used in herein should be interpreted broadly, and it refers to that included in cell sample any material of its hereditary information can be carried, and can be that DNA can also be R A, can be full-length genome or transcript profile.According to one embodiment of present invention, the biological sample is at least one selected from blood, body fluid, tissue sample and cell culture.Thus, it is possible to it is effectively unicellular from biological sample separation, so as to improve the efficiency of subsequent analysis.
According to one embodiment of present invention, the cell sample be selected from erythroblast, tumour cell, embryonic stem cell, immunocyte at least one.It is preferred that the cell sample is at least one selected from fetal nucleated red blood and circulating tumor cell.Thus, it is possible to effectively unicellular the detecting to the separation of the biological specimen of particular source.According to one embodiment of present invention, at least a portion progress amplification to the inhereditary material included in separated cell sample further comprises：Separated cell sample is cracked, to discharge the inhereditary material of the cell sample；And the inhereditary material discharged is expanded, to obtain the amplified production.Thus, it is possible to effectively expand inhereditary material from separated cell.According to one embodiment of present invention, separated cell sample is cracked using lytic reagent, wherein, the lytic reagent is at least one selected from potassium hydroxide, sodium hydroxide, SDS and Proteinase K.Thus, it is possible to further improve the efficiency of amplification inhereditary material.Furthermore it is also possible to be cracked by physical method to cell sample, for example, cell sample is placed in acoustic wavefield, crack cell；By being heated to cell, crack cell.In order that low-abundance nucleic acid meets the demand being subsequently sequenced, it is necessary to carry out the extraction and amplification of full-length genome and transcript profile to it in individual cells.The extracting method of individual cells nucleic acid is various, and the genome or mR A in individual cells can be extracted using commercial kit;The amplification of genome or transcript profile can also directly be carried out, the extraction without carrying out nucleic acid according to the strategy in the technology such as MDA or transcript amplification after cracking；Or micro-fluidic technology is used, such as liquid-solid extraction, liquid-liquid extraction method extract genome or transcript profile；And unicellular full-length genome or the main method of full transcript profile amplification are the technology such as MDA, DOP-PCR, cDNA exponential amplification and the linear amplification based on T7 R A polymerases.Thus, according to one embodiment of present invention, the inhereditary material is the unicellular full-length genome of the cell sample.Thus, it is possible to effectively by the way that unicellular full-length genome is sequenced, and determine the hereditary information of sample.According to one embodiment of present invention, at least one progress that amplification is amplified reaction by PCR-based and isothermal amplification reactions is carried out to the inhereditary material that is discharged.It is preferred that, it is by MDA, DOP-PCR, cDNA exponential amplification and at least one progress of the RNA linear amplifications based on T7 RNA polymerases to carry out amplification to the inhereditary material discharged.Thus, it is possible to further improve the efficiency expanded to full-length genome.
After amplified production is obtained, resulting amplified production is sequenced, to obtain sequencing result.According to one embodiment of present invention, sequencing is carried out to the amplified production to further comprise：For the amplified production, sequencing library is built；And the sequencing library is sequenced, to obtain the sequencing result.Thus, it is possible to which effectively amplified production is sequenced.According to one embodiment of present invention, the genome sequencing library is sequenced using at least one selected from second generation high-flux sequence platform and third generation high-flux sequence platform and the sequencing library is sequenced.Thus, it is possible to further improve sequencing efficiency.Sequencing mainly has two major classes at present, and a class is the high throughput sequencing technologies of the second generation, includes the Gemone Anayzer systems of Illumina companies（I.e. Solexa sequenators, rear to develop into the systems of HisSeq 2000 again）, ABI companies Solid systems and the GS-FLX systems of Roche 454 Corp..Second generation sequencing technologies have its normal process, are sequenced according to normal process；It is another kind of, then it is third generation sequencing technologies, i.e. single-molecule sequencing technology includes true single-molecule sequencing technology, the real-time sequencing technologies of unimolecule of Pacific Biosciences companies of Helicos companies, and Oxford
Nano-pore sequencing technology of Nanopore Technologies companies etc..Such technology can be directly to DNA, R A, microR A, albumen equimolecular direct Sequencing, the amplification without carrying out full-length genome or transcript profile to the nucleic acid in unicellular, has more intuitively reacted the change of DNA, R A quantity and sequence in individual cells.
Finally, based on the sequencing result, determine whether the biological sample has hereditary variation.Pass through the method according to the invention, effectively it can be separated by micro fluidic device according to embodiments of the present invention unicellular, so as to based on single celled inhereditary material such as full-length genome is sequenced, effectively obtained by being analyzed sequencing result with the presence or absence of abnormal in single celled inhereditary material such as full-length genome, so that it is determined that whether biological sample has hereditary variation.According to one embodiment of present invention, the hereditary variation is selected from SNP, copy number variation, genome structure variation, alternative splicing, differential expression and at least one of transcript variation.According to one embodiment of present invention, based on the sequencing result, determine whether the biological sample further comprises with hereditary variation：The sequencing result is compared with reference gene group；And based on comparison result, determine whether the biological sample has hereditary variation.Thus, it is possible to further improve the efficiency for determining whether biological sample has hereditary variation.The data obtained based on high throughput sequencing technologies, with short sequence alignment tools（Such as SOAP) sequencing data is compared onto reference gene group, and then the genome signature of aim cell can be obtained according to the qualitative and quantitative parameter of comparison result.The conventional means of numerical analysis of genomic DNA sequencing has：Single nucleotide polymorphism (SNP) is analyzed, copy number variation (CNV) analysis, genome structure variation (SV) analysis etc.；Transcript profile R A sequencings common analysis has：Gene differential expression（DGE) analyze, alternative splicing (AS) detection, Gene Fusion detection etc.；Cell development and evolutionary analysis can also be carried out for cell colony sequencing data.Thus, be compared by the data of the cell to separation, correlation analysis, not only it will be seen that the generation of cell, development, evolutionary process, and Research foundation can be provided for gene medical diagnosis, examination or identification such as are carried out to existing or potential cancer, complex disease, early detection, the purpose of early treatment of disease is realized.
In the third aspect of the present invention, the present invention proposes whether a kind of determination biological sample has the system of hereditary variation, it is characterised in that including：Micro fluidic device, amplification device, sequencing device and analytical equipment.Wherein, micro fluidic device is used to separate cell sample from biological sample；Amplification device is connected with micro fluidic device, and is expanded suitable at least a portion to the inhereditary material included in separated cell sample, to obtain amplified production；Sequencing device is connected with amplification device, and suitable for resulting amplified production is sequenced, to obtain sequencing result；And analytical equipment is connected with sequencing device, and suitable for based on resulting sequencing result, determining whether biological sample has hereditary variation.Whether there is the system of hereditary variation using determination biological sample according to embodiments of the present invention, foregoing method can effectively be implemented, effectively it can be separated by micro fluidic device according to embodiments of the present invention unicellular, so as to based on single celled inhereditary material such as full-length genome is sequenced, effectively obtained by being analyzed sequencing result with the presence or absence of abnormal in single celled inhereditary material such as full-length genome, so that it is determined that whether biological sample has hereditary variation.According to one embodiment of present invention, amplification device further comprises：Crack in unit, the cracking unit and be provided with lytic reagent or extra power device, with
Just separated cell sample is cracked using lytic reagent or extra power, discharges the inhereditary material of the cell sample；And amplification unit, the amplification unit is connected with the cracking unit, and is suitable to expand the inhereditary material discharged, to obtain the amplified production.Thus, it is possible to effectively be expanded to inhereditary material.According to one embodiment of present invention, determine whether biological sample there is lytic reagent described in the system of hereditary variation to be at least one selected from potassium hydroxide, sodium hydroxide, SDS and Proteinase K, the extra power is light, at least one electrically and thermally.Just blunt according to one embodiment of the present of invention, the amplification unit is adapted at least one of MDA, DOP-PCR, cDNA exponential amplification and the R A linear amplifications based on T7 R A polymerases.Thus, it is possible to effectively be expanded to unicellular full-length genome.According to one embodiment of present invention, the sequencing device further comprises：Library construction unit, the library construction unit is used to be directed to the amplified production, builds sequencing library；And sequencing unit, the sequencing unit is connected with the library construction unit, and is suitable to the sequencing library is sequenced, to obtain the sequencing result.Thus, it is possible to which effectively amplified production is sequenced.According to one embodiment of present invention, the sequencing unit is at least one selected from second generation high-flux sequence platform and third generation high-flux sequence platform.Thus, it is possible to further improve sequencing efficiency.According to one embodiment of present invention, analytical equipment further comprises：Comparing unit, the comparing unit is used to the sequencing result being compared with reference gene group；And variation determining unit, the variation determining unit is connected with the comparing unit, and suitable for being based on comparison result, determines whether the biological sample has hereditary variation.Thus, it is possible to further improve the efficiency for determining whether biological sample has hereditary variation.
It should be noted that the feature and advantage for whether having the method for hereditary variation above for micro fluidic device and for determining biological sample are equally applicable to the system, repeat no more.The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that the following examples are merely to illustrate the present invention, and it should not be taken as limiting the scope of the invention.Unreceipted particular technique or condition in embodiment, according to the technology or condition described by document in the art（Write such as with reference to J. Pehanorm Brookers, what Huang Peitang etc. was translated《Molecular Cloning:A Laboratory guide》, the third edition, Science Press）Or carried out according to product description.Agents useful for same or the unreceipted production firm person of instrument, are that be able to can for example be purchased from Illumina companies by the conventional products of acquisition purchased in market.
The foundation of low abundance cell screening model of the conventional method based on high-flux sequence
The design of 1.1 micro-fluidic chips
1.1.1 the unicellular separation of micro-fluidic chip and enrichment unit
Due to the complexity and the rareness of aim cell of sample, the present embodiment designs the microchannel that multistep separation is carried out to cell according to difference of the different type cell on physics, chemistry and biological characteristics on chip, unicellular to obtain final purpose.
According to the physicochemical property of aim cell, micro-fluidic chip of the design with different structure carries out Selective Separation and the enrichment of aim cell.If the size of cell to be separated and other cells have significant difference, can micro-fluidic chip passage
In design the various structures such as micro-structural such as column, meander channel, pectination, weir shape, sieve-like, only the cell of certain size size is allowed to pass through, or the cell of retention certain size size, to separate cell or the principle using certainty lateral displacement, the barrier of certain intervals size is designed in passage, different size of cell enters different liquid streams by being collided with barrier, reaches the purpose of separation different size cell；Using hydromechanical principle, according to the difference of different size cell particle, the separation of cell is realized.According to inertia, bending channel is designed, liquid stream is controlled, realizes the migration of different size cell and separate.If cell can be deformed, such as erythrocyte, fetal nucleated red blood, the barrier of different spacing can be designed, allows obstacle spacing to be less than the diameter of red blood cell either fetal nucleated red blood, by deformable cell and non-deformable cell（Leucocyte, tumour cell etc.）Separated.
According to the difference of cell physical property, various fields, such as electric field, magnetic field, the separation of sound wave progress cell can be set up on chip.Such as dielectrophoresis, the electric field of different frequency can be added outside chip channel, by adjusting the spatial distribution of electric field, different types of cell can be made to be produced different degrees of drift by different degrees of polarization, realize the separation of cell, either utilize dielectrophoresis formation conductivity gradient, cell is driven to be moved to the low direction of electrical conductivity, until balance, electrical conductivity is 0, recycle gravity that cell is dragged in purpose passage, realize cell separation.Separated using magnetic field, it is similar with MACS, the biomolecule of specific capture cell is connected with magnetic bead surfaces, is combined, using controllable magnetic field, the cell of capture is separated by immuno-chemical reaction；Or the magnetic itself having using some cells, the paramagnetism and diamagnetism of such as red blood cell and leucocyte realize the separation of cell.Using the difference of the node characteristic stress of sound wave, according to properties such as the sizes, density, proportion of cell, the separation of cell is realized.
Because cell can express many specific surface moleculars, according to its immunochemistry property, the molecule such as antibody, aptamers etc. that can be combined in passage in connection with cell surface marker molecule, carry out the capture of specific cell and/or the removal of non-specific cell.The corresponding biomolecule for being connected with specificity capture aim cell or removing non-specific cell on the sorting of immunomagnetic beads, magnetic bead can also be carried out in passage, the separation and enrichment of specific cell is realized.
1.1.2 the cell of micro-fluidic chip manipulates unit
The requirement separated according to the integration of overall chip and progenitor cells, can select different control modes.Liquid stream and micro- wealthy control can be utilized, micro- wealthy opening and closing, entrapped cell is manipulated.If early stage carries out the sorting of cell using magnetic bead, individual cells can optionally be manipulated by the control in magnetic field（Few cells）To the conversion zone specified.Electric field can be added on manipulation passage, using the mode of EOF or electrophoresis by individual cells（Few cells）Drive into reaction tank.The passage of special construction is designed, individual cells Jiao amount cells can be only retained）.Capillary can also be utilized, carries out cell regularly arranged, is moved by displaying, by individual cells（Few cells）It is driven into the conversion zone specified.These control passages can be integrated, and realize simultaneously multiple unicellular（Few cells）Manipulation.
1.1.3 the unicellular nucleic acid extraction and amplification unit of micro-fluidic chip
Nucleic acid extraction and amplification unit can in the micro reaction pool of certain regular texture, can also pass through droplet (liquid
Drop）By individual cells（Few cells）Parcel, forms cell independent one by one and is used as reaction tank.In order to extract intracellular DNA or R A, we can be put into surface-functionalized magnetic bead in extraction unit, pass through magnetic bead sorting, obtain the few cells DNA molecular or R A molecules needed, by controlling magnetic bead, the DNA extracted or RNA molecule outflow outlet are driven, into amplification unit.Such as the nucleic acid molecules being collected into are put into PCR pipe, the WGA or full transcript amplification procedure of standard is carried out.Droplet technologies, individual cells can also directly be utilized（Few cells）It is wrapped in droplet (drops）In, such as PCR reaction solutions are directly wrapped up in drop, after cracking, amplified reaction is carried out directly in droplet.
1.2 high-flux sequence
The amplified production of acquisition, outlet is reserved by drive system, collects reaction solution.According to the normal process of second generation sequencing technologies, nucleic acid molecules are interrupted, storehouse is built, is then sequenced.
Have for DNA and R A and different build storehouse mode.If DNA, the DNA fragmentation of acquisition can be broken into small fragment at random, add given joint, be prepared into DNA library, cluster amplification is carried out on cBot, the sequencing of single end or double ends is directly carried out to DNA fragmentation.If that obtain is R A, mR A are isolated, the cDNA library of fragmentation is prepared into, cluster amplification is carried out on cBot, high pass sequencer is used further to.
It is main at present to carry out high-flux sequence, the Gemone Anayzer systems of Illumina companies using three kinds of technologies（I.e. Solexa sequenators, rear to develop into the systems of HisSeq 2000 again）, ABI companies Solid systems and the big high throughput sequencing technologies of GS-FLX systems three of Roche 454 Corp..And with the development of science and technology, third generation sequencing technologies will be applied, that is single-molecule sequencing technology, the real-time sequencing technologies of unimolecule of true single-molecule sequencing technology, Pacific Biosciences companies including Helicos companies, and Oxford Nanopore Technologies companies nano-pore sequencing technology etc..
1.3 data analysis
Based on high-flux sequence result, with short sequence alignment tools（Such as SOAP) sequencing data is compared onto reference gene group, and then the genome signature of aim cell can be obtained according to the qualitative and quantitative parameter of comparison result.
The conventional means of numerical analysis of genomic DNA sequencing has：Single nucleotide polymorphism（SNP) analyze, copy number variation (CNV) analysis, genome structure variation (SV) analysis etc.；Transcript profile R A sequencings common analysis has：Gene differential expression（DGE) analyze, alternative splicing (AS) detection, Gene Fusion detection etc.；Cell development and evolutionary analysis can also be carried out for cell colony sequencing data.
By to separation sample be compared, correlation analysis, not only it will be seen that the generation of cell, development, evolutionary process, and Research foundation can be provided for gene medical diagnosis, examination or identification such as are carried out to existing or potential cancer, complex disease, early detection, the purpose of early treatment of disease is realized.The heredity identification of the fetal nucleated red blood of embodiment 1
The ulnar vein peripheral blood for the pregnant woman that 1.1 5mL are pregnant 8-14 weeks, EDTA-K2 anti-freezings, 4 °C of vibrations are preserved, in 24 h
Tested.
The separation of 1.2 fetal nucleated red bloods：Separated using two steps.Separated first according to the different progress of red white corpuscle size, the chip structure in such as 2.1 removes most red blood cell；The cell of recovery is passed into Magnetic Isolation chip, separated using the difference of the magnetic of red white corpuscle.
The structure of first chip：Spacing between microtrabeculae is 15 μ η ι, 6.75 μ η ι of the often relative skew of row microtrabeculae.Micro-structural is built on silicon chip using deep reaction ion etching technology, channel depth is 150 μ η ι, is sealed using sheet glass.
The structure of second chip：Using Miltenyi LS Column (Miltenyi BioTech), 1.4T magnet is put in sunset fore-telling.
Blood and Slow fliud flushings are passed into first chip with the η ι of 100 μ Ι 7 speed using external drive equipment, Slow fliud flushings are iDPBS (containing 1% BSA, 2mM EDTA).Cell is reclaimed from exit.The cell of recovery is by centrifugation 2000 rpm, 15min.Sample is with 50 mM NaN02Handle 10min.Then pass in Miltenyi LS Column.Rinsed slowly using iDPBS Slow, wash away unadsorbed leucocyte.Then magnet is removed, is rinsed using Slow fliud flushings, the magnetic cell of tool is reclaimed.Cell, the single erythroblast of picking under fluorescence microscope are marked using FITC-epsilon- globulin, and is transferred into ^:In hole array.
1.3 cells are cracked：Use NaOH cell lysis.
The extraction and amplification of 1.4 nucleic acid：Directly unicellular whole genome amplification is carried out using REPLI-g Mini/Midi kits (Qiagen).
1.5 sequencing：Whole genome amplification product is sequenced by building storehouse with Illumina technologies.
1.6 data analysis：Carry out single nucleotide polymorphism（SNP) analyze, carry out single-gene site or polygenic locus mutation analysis.
Embodiment 2:The chromosome aneuploid of fetal nucleated red blood is identified extremely
2.1 sampling：With 1.1
The separation of 2.2 fetal nucleated red bloods：
The structure of used first chip：Such as the nautilus chip in Fig. 5, there are an injection port and 6 outlets, inlet passageway width is 0.22 mm, and passage bending curvature gradually amplifies according to a certain percentage, the channel width in exit is 3.88 mm.Chip channel configuration su-8 bears optical cement and makes mask through photoetching process, then the mixing of PDMS prepolymers is cast on mould, is formed by overmolded.Sealed using sheet glass.
The structure of used second chip：Such as Fig. 6.There are two entrances and two outlets.Channel width is 0.5mm, and length is 50mm.Chip channel configuration su-8 bears optical cement and makes mask through photoetching process, then the mixing of PDMS prepolymers is cast on mould, is formed by overmolded.Sealed using sheet glass.External magnet is added in the side of passage, magnetic chip is built into.
Separated using two steps.The first step removes most red blood cell using the first chip；Erythroblast will be contained again
Cell mixing sample be passed into the second chip, using red white corpuscle magnetic difference separated.
Using external drive equipment micro-injection pump, the blood that driving diluted concentration is 10%（Diluted using the iDPBS Slow fliud flushings containing 1%BSA）It is passed into passage, flow velocity is the η ι of 300 μ Ι 7.Cell solution is collected in exit.The cell of recovery is by centrifugation 2000rpm, 15min.Sample is with 50 mM NaN02Handle 10min.By cell from sample inlet is passed into chip, while another entrance is passed through iDPBS Slow fliud flushings, the two flow velocity is the η ι of 10 μ Ι 7.Aim cell is reclaimed in exit.Cell passes through FITC-CD71 antibody stainings, and the single positive cell of picking carries out subsequent experimental.
2.3 cells are cracked：Use KOH/DTT cell lysis.
The extraction and amplification of 2.4 nucleic acid：The amplification of individual cells genome is directly carried out using DOP-PCR (GenomePlex Single Cell Whole Genome Amplification Kit, Sigma) method.
2.5 sequencing：Whole genome amplification product is sequenced by building storehouse with Illumina technologies.
2.6 data analysis：Carry out copy number variation (CNV) analysis, genome structure variation (SV) analysis etc..
Embodiment 3:Cancer patient's circulating tumor cell (CTCs) examination
3.1 sampling：Extract ulnar vein peripheral blood 10 mL, the EDTA-K2 anti-freezing of tumor patient.Detected in 4 °C of preservations of blood sample, 24h.
3.2 CTCs separation：Separated using two step chips.The first step is separated according to the size of cell, the chip structure in such as Fig. 1, removes the outer cell of most red blood cell and part；Second step uses EpCAM antibody capture CTCs cells.Specifically,
The structure of first chip：Spacing between microtrabeculae is 20 μ η ι, 6 μ η ι of the often relative skew of row microtrabeculae.^ is built on silicon chip using deep reaction ion etching technology:Structure, channel depth is 150 mm, is sealed using sheet glass.
The structure of second chip：Using Miltenyi LS/MS Column (Miltenyi BioTech), CTCs is captured.
Blood and Slow fliud flushings are passed into first chip with the η ι of 100 μ Ι 7 speed using external drive equipment, Slow fliud flushings are iDPBS (containing 1% BSA, 2mM EDTA).Cell is reclaimed from exit.The cell of recovery is by centrifugation 300g, 10 min.Then the magnetic bead and mixing with cells that CD326 (EpCAM) antibody will be connected with are incubated.Rinsed using iDPBS.Cell and magnetic bead mix are passed into Miltenyi LS/MS Column.Rinsed slowly using iDPBS Slow, wash away unadsorbed leucocyte.Then magnet is removed, is rinsed using Slow fliud flushings, the magnetic cell of tool is reclaimed.And CTCs cells are counted.
3.3 cells are cracked：Use lysate KOH or NaOH cell lysis.
The extraction and amplification of 3.4 nucleic acid：The few cells being collected into are subjected to whole genome amplification according to MDA method.3.5 sequencing：Whole genome amplification product is sequenced by building storehouse with Illumina technologies.
3.6 data analysis：Carry out single nucleotide polymorphism（SNP) analyze, carry out single-gene site or polygenic locus mutation analysis.By comparing the related single celled Genetic Variation Analysis of separating sample and other normal cells, cancer solid tumor tissue, cancer, evidence is provided for the early detection, early diagnosis and aftertreatment of later cancer.
The heredity identification of the fetal nucleated red blood of embodiment 4
4.1 with 1.1
4.2 with 1.2
4.3 with 1.3
The extraction of 4.4 nucleic acid：Use magnetic bead（Invitrogen, Dynabeads) method directly extract it is unicellular in genome.
4.5 sequencing：Directly single-molecule sequencing is carried out using Oxford Nanopore Technologies.
4.6 data analysis：Carry out single nucleotide polymorphism（SNP) analyze, copy number variation (CNV) is analyzed, circulating tumor cell in the sample of embodiment 5 such as genome structure variation (SV) analysis（CTCs automation examination)
Sampling：Peripheral blood lOmL, EDTA-K2Anti-freezing, 4 °C of vibrations are preserved, and are tested in 24h.
Blood and PBS Slow fliud flushings are passed through to the separation and concentration unit of micro-fluidic chip using micro-injection pump in the way of laminar flow, regularly arranged cylindrical microtrabeculae is built with passage, microtrabeculae spacing is 16 μ η ι, haemocyte with microtrabeculae by colliding, the less red blood cell of size is separated according to cell size, larger-size leucocyte enters next split tunnel.Passage is built-in with the immunomagnetic beads for being connected with anti-EpCAM antibody molecules, and immunomagnetic beads captures the CTCs of the specific expressed EpCAM molecules of cell surface by the collision of the leucocyte with being flowed into passage.PBS is rinsed, and removes non-specific binding cell.Enzymolysis liquid is passed through, CTCs is discharged from magnetic bead.
Driven by fluid, make the regularly arranged entrance passage of cell, by the wealthy folding of gas, by single CTC Jiao amount CTCs) it is trapped in different cells.
The red blood cell and leucocyte separated through control, a small amount of cell enter it is respective only allow individual cells to arrange the passage that passes through, by the way that gas is wealthy and liquid flow control, by individual cells（Few cells）Independent cell is separated into, it is standby as compareing.
Gentle wealthy folding is controlled by fluid, individual cells are promoted（Few cells) from respective outlet outflow, collect respectively in PCR pipe.The individual cells being collected into（Few cells）Standard amplification is carried out according to MDA method.
Amplified production is collected, DNA fragmentation is broken into small fragment at random, given joint is added and sets up DNA fragmentation library, cluster amplification is carried out on cBot, is sequenced for Illumina Hiseq 2000.
Data analysis, detects cellular genome single nucleotide polymorphism and chromosome structure change based on high-flux sequence result, finds potential cancer driving factors and candidate locus.Cancer cell development tree is set up, cancer cell evo-devo process is reviewed.By comparing the related single celled Genetic Variation Analysis of separating sample and other normal cells, cancer solid tumor tissue, cancer, evidence is provided for the early detection, early diagnosis and aftertreatment of later cancer.
The heredity identification of the fetal nucleated red blood of embodiment 6
Sampling：The peripheral blood for the pregnant woman that 5mL is pregnant 14 weeks, EDTA-K2Anti-freezing, 4 °C of vibrations are preserved, and are tested in 24h.
Blood is passed into micro- magnetic current passage, in the presence of externally-applied magnetic field, paramagnetism, the diamagnetic principle of leucocyte, by red blood cell are had according to red blood cell, WRBCs in haemocyte（Including ^RBCs) separated with leucocyte, respectively enter different passages.
Red blood cell (including ^RBCs), which is entered, to be connected with the post passage of anti-CD71 antibody, by antigen-antibody immune response, captures ^RBCs.FNRBCs after desorption, is manipulated it and entered in control passage from microtrabeculae.
Control ^ RBCs, which enter, only allows the unicellular passage passed through.Under aobvious mirror, by the gentle wealthy control of liquid stream, single RBC is promoted to sequentially enter in array collecting pipe.
The leucocyte and red cell fraction separated in control blood enter the passage for only allowing individual cells to pass through, and by control, single leucocyte, living cells are forced into array collecting pipe, used as control.
Extract the cell R A in array collecting pipe, transcript amplification is carried out according to the linear amplification method based on T7 R A polymerases, detailed process is with reference to Eberwine, J. et al. Analysis of gene expression in single live neurons. Proc. Natl. Acad. Sci.USA89, 3010-3014 (1992) and Van Gelder, R.N. et al. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proc. Natl. Acad. Sci. USA87, 1663-1667 (1990) is carried out.
Amplified production cDNA is collected, the library construction document provided according to Illumina prepares library, cluster amplification is carried out on cBot, is sequenced for Illumina Hiseq 2000.Data analysis.
Based on high-flux sequence data analysis fetus transcript profile feature, basis is provided for medical molecule diagnosis research.
The research of the circulating tumor cell expression of embodiment 7
7.1 with 3.1
7.2 with 3.2
Obtain after single loop tumour cell, follow-up R A extract amplification procedure and the primer reagent etc. with reference to Tang F, Barbacioru C, Nordman E, et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols. 2010;5(3):516-535. carry out.
7.3 cells are cracked：Use NP40 cell lysis.
7.4 reverse transcription：It is directly added into reverse transcriptase and primer carries out reverse transcription, synthetic dsdna.
7.5 cDNA exponential amplifications：The reaction of first round PCR is carried out, is purified using QIAquick PCR purification kit；The product purified after by first round PCR carries out the second wheel amplification, uses QIAquick PCR purification kit
Purified.Gel reclaims the fragment of the 0.5-3 kb in PCR primer.
7.6 sequencing：CDNA amplified productions are sequenced by building storehouse with Illumina technologies.
7.7 data analysis：Sequencing gained reads is compared to transcript is referred to, transcript structures research, transcript variation research, gene expression dose research, noncoding region functional study etc. are carried out according to gained information is compared.Industrial applicibility
According to an embodiment of the invention micro fluidic device, determine biological sample whether have hereditary variation method and determine biological sample whether have hereditary variation system, can efficiently separate it is unicellular, so that it is determined that biological sample whether have hereditary variation.
Although the embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all teachings, various modifications and replacement can be carried out to those details, these change within protection scope of the present invention.The four corner of the present invention is provided by appended claims and its any equivalent.
In the description of this specification, the description of reference term " one embodiment ", " some embodiments ", " illustrative examples ", " example ", " specific example " or " some examples " etc. means to combine specific features, structure, material or the feature that the embodiment or example describe and is contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referring to the schematic representation of above-mentioned term.Moreover, specific features, structure, material or the feature of description can in an appropriate manner be combined in any one or more embodiments or example.
Claims (1)
- Claims1st, a kind of micro fluidic device, it is characterised in that the micro fluidic device, which has, to be suitable to separate single celled microfluidic channel.2nd, device according to claim 1, it is characterised in that multiple microtrabeculaes are formed with the microfluidic channel.3rd, device according to claim 2, it is characterised in that the multiple microtrabeculae is in array distribution on the microfluidic channel.4th, device according to claim 3, it is characterised in that in the vertical, the distance between two neighboring microtrabeculae is 10-100 microns, also, in the horizontal, the distance between two neighboring microtrabeculae is 10-100 microns, often relative 6-7 microns of the skew of row microtrabeculae.5th, device according to claim 2, it is characterised in that along flow direction, the diameter of the microtrabeculae increases.6th, device according to claim 1, it is characterized in that, the microfluidic channel is crooked pipeline, the crooked pipeline is selected from least one of Archimedes spiral passage and nautilus line passage, the throat width of the nautilus line passage is 0.22 millimeter, and outlet channel width is 3.88 millimeters.7th, device according to claim 1, it is characterised in that the micro fluidic device is further provided with magnet, and the width of the microfluidic channel is 0.5 millimeter, and length is 50 millimeters.8th, it is a kind of to determine whether biological sample has the method for hereditary variation, it is characterised in that to comprise the following steps：Using the micro fluidic device described in claim any one of 1-7, cell sample is separated from biological sample；At least a portion to the inhereditary material included in separated cell sample is expanded, to obtain amplified production；The amplified production is sequenced, to obtain sequencing result；AndBased on the sequencing result, determine whether the biological sample has hereditary variation.9th, method according to claim 8, it is characterised in that the inhereditary material is the unicellular full-length genome or full transcript profile of the cell sample, based on the sequencing result, determines whether the biological sample further comprises with hereditary variation：The sequencing result is compared with reference gene group or with reference to transcript profile；AndBased on comparison result, determine whether the biological sample has hereditary variation.10th, it is a kind of to determine whether biological sample has the system of hereditary variation, it is characterised in that including：Micro fluidic device described in claim any one of 1-7, the micro fluidic device is used to separate cell sample from biological sample；Amplification device, the amplification device is connected with the micro fluidic device, and is expanded suitable at least a portion to the inhereditary material included in separated cell sample, to obtain amplified production；Sequencing device, the sequencing device is connected with the amplification device, and suitable for the amplified production is sequenced, To obtain sequencing result；AndAnalytical equipment, the analytical equipment is connected with the sequencing device, and suitable for being based on the sequencing result, determines whether the biological sample has hereditary variation.
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| CN101086504A (en) * | 2006-06-06 | 2007-12-12 | 北京大学 | Microfluid centrifugal chip and its processing method |
| CN101305087A (en) * | 2005-09-15 | 2008-11-12 | 阿尔特弥斯康复公司 | Devices and methods for magnetic enrichment of cells and other particles |
| CN101643701A (en) * | 2009-07-23 | 2010-02-10 | 清华大学 | Cell sorter micro-fluidic chip based on immunomagnetic separation technology and application thereof in aspect of enrichment of rare cells |
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| US7390388B2 (en) * | 2004-03-25 | 2008-06-24 | Hewlett-Packard Development Company, L.P. | Method of sorting cells on a biodevice |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2004037374A2 (en) * | 2002-10-23 | 2004-05-06 | The Trustees Of Princeton University | Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields |
| WO2004037374A3 (en) * | 2002-10-23 | 2004-10-14 | Univ Princeton | Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields |
| CN101305087A (en) * | 2005-09-15 | 2008-11-12 | 阿尔特弥斯康复公司 | Devices and methods for magnetic enrichment of cells and other particles |
| CN101086504A (en) * | 2006-06-06 | 2007-12-12 | 北京大学 | Microfluid centrifugal chip and its processing method |
| CN101643701A (en) * | 2009-07-23 | 2010-02-10 | 清华大学 | Cell sorter micro-fluidic chip based on immunomagnetic separation technology and application thereof in aspect of enrichment of rare cells |
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| CN104991055A (en) * | 2015-06-19 | 2015-10-21 | 大连理工大学 | Blood sample time-delay flowing bionic control unit in thrombus POCT product |
| CN107262171A (en) * | 2017-07-12 | 2017-10-20 | 华讯方舟科技有限公司 | A kind of blood separation and culture chip and blood separating mechanism |
| CN107702973A (en) * | 2017-09-08 | 2018-02-16 | 深圳市太赫兹科技创新研究院有限公司 | A kind of whole blood blood plasma piece-rate system and method |
| CN111363795A (en) * | 2018-12-26 | 2020-07-03 | 清华大学 | Single cell whole genome sequencing method |
| CN109746064A (en) * | 2019-01-28 | 2019-05-14 | 武汉纺织大学 | A kind of gradient magnetic micro-fluidic chip |
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