WO2016038670A1 - Cell analysis system and cell analysis method using same - Google Patents

Abstract

The purpose of the present invention is to provide a cell analysis system whereby a comprehensive gene analysis of a cell population and a specific gene analysis of a single cell contained in the population can be carried out at the same time. The cell analysis system according to the present invention is provided with an array device, said array device comprising a plurality of cell traps each capable of capturing a single cell and nucleic acid traps each capturing a nucleic acid extracted from a cell trapped by a cell trap, and a reagent addition unit for adding a reagent to the array device, wherein a nucleic acid trap contains an immobilized first probe for capturing a nucleic acid and the reagent addition unit supplies at least a second probe and a third probe to the array device.

Description

Cell analysis device and cell analysis method using the same

The present invention relates to a cell analysis apparatus and a cell analysis method using the same, which can simultaneously realize gene analysis of a single cell and comprehensive gene analysis of a cell population including the cell.

In recent years, when performing genome analysis, gene expression analysis, or protein analysis of living tissue composed of a large number of cells, it is important to analyze single cells by paying attention to differences in the genome, gene expression, and protein of each cell. Sex is beginning to be recognized. Particularly in cancer and iPSC (induced pluripotent stem cells) research, it is known that a small number of cells may behave differently from the average behavior of biological tissues, The importance of single cell analysis in such fields has been pointed out.

In the conventional general analysis method (bulk analysis), DNA and RNA are extracted and analyzed using a large number of cells (10 ³ to 10 ⁶ or more cells) sampled from a living tissue as one type of sample. For example, by using a next-generation sequencer that has recently become widespread, as used in Patent Document 1, it is possible to determine the entire genome sequence of one person in one measurement, or more than tens of thousands of types It became possible to perform expression analysis on all existing genes. However, in bulk analysis, only average data of all cells can be obtained, and evaluation cannot be made even if the abundance of DNA or RNA in individual cells deviates from the average value. In addition, since many of the bulk analysis methods require a minimum sample amount of 1000 to 1 million times that of a single cell, it is difficult to apply it directly to the analysis of a single cell from the viewpoint of necessary sensitivity.

On the other hand, the present inventors have previously used a method for performing gene analysis of a single cell, using a pore sheet to which a nucleic acid probe including a tag sequence that differs depending on the position and a sequence for capturing a test nucleic acid is fixed. In addition, a method has been proposed in which a cDNA library is prepared while maintaining positional information in a cell tissue, and the library is amplified and analyzed (Patent Document 2). In order to realize such single cell analysis, various other methods for extracting a very small amount of genes from cells with high efficiency and performing analysis with high accuracy have been proposed.

US Patent Publication No. 2013/0184999 International Publication No. 2014/020657

In such a background, there is a demand for comparing and analyzing the gene analysis results of the cell population and the single cell gene analysis results. However, when cell population analysis and single cell analysis are performed separately, even if cells are sampled from the same tissue, random changes in gene expression in individual cells and the processing conditions for analysis Due to the differences, it was difficult to make a strict comparison.

In order to reduce the effects of fluctuations in gene expression in the cells and reaction efficiency bias at each reaction opportunity when processing nucleic acids for measurement, multiple analyzes to be compared must be performed on the same sample. Performing simultaneously is an effective means for solving the above problems. Such multiple analyzes are performed by editing the analysis of all genes (ie, exhaustive gene analysis) and editing the data for every cell (ie, single cell analysis) for each cell. It is feasible. However, since the analysis cost is proportional to the product of the number of measured genes and the number of measured cells, there is a problem that the cost for analysis becomes high.

The present inventors have completed a device for cell analysis that enables simultaneous analysis of a gene population of a cell population and a gene analysis specific to a single cell included in the population, The cell analysis method used was found. The gist of the present invention is as follows.

(1) An array device having a plurality of cell traps that can capture cells one by one, a nucleic acid trap that captures nucleic acid extracted from the cells trapped in the cell trap, and a reagent for adding a reagent to the array device With an addition device,
A nucleic acid trap is present for each cell trap, and the nucleic acid trap includes a fixed first probe for capturing the nucleic acid;
The reagent addition apparatus provides at least a second probe and a third probe to the array device,
The first probe has a different cell recognition sequence for each individual nucleic acid trap, and a nucleic acid capture sequence that hybridizes with a nucleic acid extracted from the cell, and optionally a first nucleic acid amplification primer sequence. You can,
The second probe has a second nucleic acid amplification primer sequence, and a sequence that hybridizes to the end of the nucleic acid including a sequence synthesized using the nucleic acid captured by the first probe as a template,
The third probe has a second nucleic acid amplification primer sequence, and a sequence that hybridizes to a known sequence contained in the nucleic acid captured by the first probe,
The reagent addition apparatus supplies an enzyme and a substrate for synthesizing a complementary strand using the captured nucleic acid as a template after the nucleic acid extracted from the cell is captured by the first probe of the nucleic acid trap, and then the second A cell analysis apparatus characterized by supplying a probe and a third probe simultaneously or successively.

(2) the first probe has a first nucleic acid amplification primer sequence,
The cell analysis device according to (1), wherein the reagent addition device supplies a first nucleic acid amplification primer and a second nucleic acid amplification primer after supplying the second probe and the third probe.

(3) the first probe does not have the first nucleic acid amplification primer sequence,
The reagent addition device supplies the fourth probe having the first nucleic acid amplification primer sequence and the cell recognition sequence after the supply of the second probe and the third probe, and then for the first nucleic acid amplification The cell analysis device according to (1), wherein a primer and a second nucleic acid amplification primer are supplied.

(4) The known sequence contained in the nucleic acid captured by the first probe is present at a position of 150 to 250 bases from the 5 ′ end of the captured nucleic acid, in any of (1) to (3) The cell analysis apparatus described.

(5) The cell analyzer according to any one of (1) to (3), wherein the first probe further has a molecular recognition sequence that does not depend on the nucleic acid trap and is different for each probe.

(6) The cell according to any one of (1) to (3), wherein the nucleic acid capture sequence of the first probe is a poly-T sequence, and the nucleic acid extracted from the cell and captured by the first probe is mRNA. Analysis device.

(7) The cell analysis device according to any one of (1) to (3), wherein the end of the nucleic acid to which the second probe hybridizes comprises a poly A sequence.

(8) A method for simultaneously performing a comprehensive analysis of a cell population and a single cell analysis for individual cells included in the cell population,
Multiple cell traps capable of capturing cells one by one, and a fixed first probe for capturing nucleic acid extracted from the cells captured in the cell trap, and corresponding to each cell trap Preparing an array device having a nucleic acid trap;
A step of extracting nucleic acids by destroying cells captured by the nucleic acid trap;
A first cell may have a cell recognition sequence different for each individual nucleic acid trap, and a nucleic acid capture sequence that hybridizes with a nucleic acid extracted from the cell, and optionally a first nucleic acid amplification primer sequence. A step of allowing the probe to capture the extracted nucleic acid, then supplying an enzyme and a substrate for complementary strand synthesis using the captured nucleic acid as a template to the array device, a second nucleic acid amplification primer sequence, and a first A second probe having a sequence that hybridizes to the end of the nucleic acid containing the sequence synthesized using the nucleic acid captured by the probe as a template;
Supplying a second nucleic acid amplification primer sequence and a third probe having a sequence that hybridizes to a known sequence contained in the nucleic acid captured by the first probe to the array device simultaneously or sequentially. , Said method.

(9) the first probe has a first nucleic acid amplification primer sequence,
The method according to (8), further comprising supplying a first nucleic acid amplification primer and a second nucleic acid amplification primer after supplying the second probe and the third probe.

(10) the first probe does not have the first nucleic acid amplification primer sequence,
After supplying the second probe and the third probe, a fourth probe having a first nucleic acid amplification primer sequence and a cell recognition sequence is supplied, and then the first nucleic acid amplification primer and the second nucleic acid The method according to (8), further comprising a step of supplying an amplification primer.

(11) The known sequence contained in the nucleic acid captured by the first probe is present at a position of 150 to 250 bases from the 5 ′ end of the captured nucleic acid, any of (8) to (10) The method described.

(12) The method according to any one of (8) to (10), wherein the first probe further has a molecular recognition sequence that does not depend on the nucleic acid trap and is different for each probe.

(13) The method according to any one of (8) to (10), wherein the nucleic acid capture sequence of the first probe is a poly-T sequence, and the nucleic acid extracted from the cell and captured by the first probe is mRNA. .

(14) The method according to any one of (8) to (10), wherein the end of the nucleic acid to which the second probe hybridizes comprises a poly A sequence.

(15) The cell analysis device according to any one of (1) to (3), comprising an array device and a reagent addition device,
A monitoring device for observing cells captured in the cell trap of the array device,
A memory device storing the sequence information of the first probe, which is different for each nucleic acid trap of the array device, and the sequence information of other probes supplied by the reagent addition device,
A control device for controlling the reagent addition device with reference to the memory device;
A cell analysis system comprising a sequencer for analyzing a nucleic acid amplification product obtained from an array device, and an analysis device for analyzing data obtained from the sequencer with reference to a memory device as necessary.

According to the cell analysis apparatus and the cell analysis system of the present invention, comprehensive gene analysis of a cell population and gene analysis specific to a single cell included in the population are simultaneously performed with high accuracy and low cost. It becomes possible.

The schematic diagram which shows an example of a structure of a two-dimensional array device. FIG. 2 is an enlarged view of a partial structure around one cell trap 3 of the two-dimensional array device 1 shown in FIG. The enlarged view of the surface of the bead 7 (when using the first DNA probe 31 of the basic example). The enlarged view of the surface of the bead 7 (when using the first DNA probe 31 of the basic example). The enlarged view of the surface of the bead 7 (when using the first DNA probe 31a of the application example). The enlarged view of the surface of the bead 7 (when using the first DNA probe 31a of the application example). The enlarged view of the partial structure around one cell trap 3 in the two-dimensional array device 1 of the application example. 1 is a schematic diagram of the structure of a nucleic acid preparation device 100. FIG. The figure explaining the cell analysis apparatus and cell analysis system using a two-dimensional array device.

In the present specification, “gene expression analysis” refers to quantitative analysis of the expression of a gene in a sample (cell, tissue section, etc.), that is, the target test nucleic acid, and expression of the gene (test nucleic acid) in the sample. Analyzing the distribution means obtaining correlation data between specific cells in the sample and the expression level of the gene (test nucleic acid). The sample is not particularly limited as long as it is a biological sample to be analyzed for gene expression, and any sample such as a cell sample, a tissue sample, or a liquid sample can be used. The organism from which the sample is derived is not particularly limited, and includes vertebrates (eg, mammals, birds, reptiles, fishes, amphibians), invertebrates (eg, insects, nematodes, crustaceans), protists, Samples derived from any living body such as plants, fungi, bacteria, and viruses can be used. In the present invention, messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, and DNA, and fragments thereof can be used as the target test nucleic acid.

(Array device)
FIG. 1 is a schematic diagram showing an example of the configuration of a two-dimensional array device according to the cell analysis apparatus of the present invention. 1A is a top view, and FIG. 1B is a cross-sectional view taken along the alternate long and short dash line between AA ′ shown in FIG. 1A. The two-dimensional array device 1 includes a cell trap 3 for fixing cells 2 introduced into the device one by one, a nucleic acid trap 4 for capturing nucleic acids contained in the cell 2 extract, and a cell trap 3 A flow path 5 communicating with the nucleic acid trap 4 is provided. The flow path 5 can be provided, for example, by installing a substrate having a micropore or a porous resin sheet below the nucleic acid trap 4. The arrow in FIG. 1 (b) indicates the flow direction of the cell 2 extract. Such a device can be manufactured, for example, by applying a semiconductor manufacturing process.

FIG. 2 is an enlarged view for explaining in detail a partial structure around one cell trap 3 of the two-dimensional array device 1 shown in FIG. When a solution in which cells are suspended flows from the upper region of the two-dimensional array device 1, the two-dimensional array device 1 is connected so that liquid can flow from the upper region to the lower region. Move on the flow and reach cell trap 3. Since the opening diameter (for example, 3 to 30 μm) of the cell trap 3 is smaller than the diameter of the cell 2, the reached cell is fixed to the cell trap 3. Since the fixed cells 2 serve as plugs for the cell trap 3, the flow of the solution goes to the cell trap 3 where the cells are not yet fixed, and another cell is also fixed there.

When the required number of cells 2 has been reached, the solution is then replaced with a solution in which the cells are suspended, and a Lysis buffer (eg, Tween 20) is used to break down the cells and proteolysis The enzyme mixture) is flowed from the upper region of the two-dimensional array device 1. At the same time, if necessary, an electric field is applied in the direction indicated by E in FIG. 2, and the nucleic acid 6 (mRNA) obtained by destroying the cells 3 is moved to the nucleic acid trap 4 by electrophoresis. The nucleic acid 6 may be moved by the flow of the solution regardless of electrophoresis. The nucleic acid trap 4 is filled with beads 7 on the surface of which probes for capturing the nucleic acid 6 are fixed. The diameter of the bead 7 is larger than that of the flow path 5 so that the bead 7 does not flow out.

3 and 4 are enlarged views of the surface of the bead 7. A first DNA probe 31 is fixed on the surface of the bead 7. The immobilization can be performed, for example, by modifying the 5 ′ end of the DNA probe with biotin and binding it with streptavidin immobilized on the surface of the beads 7 in advance. The first DNA probe 31 has a poly T sequence 32 at the 3 ′ end, and captures the mRNA 41 by hybridizing with the poly A sequence at the 3 ′ end of the mRNA 41. In addition, when it is desired to analyze microRNA or the like instead of mRNA, a known sequence that hybridizes with the analysis target may be used instead of the poly T sequence.

The first DNA probe 31 has a first PCR amplification primer sequence 33 at the 5 ′ end fixed to the bead 7, and further the individual cells 2 fixed to the cell trap 3 of the two-dimensional array device 1. Includes a cell recognition sequence 34 for identification. The primer sequence for PCR amplification is not particularly limited as long as it is a known sequence having an appropriate length for nucleic acid amplification, and those skilled in the art can appropriately design such a sequence. For example, the primer sequence for PCR amplification can be 10-50 bases, 15-50 bases, 15-40 bases, 15-30 bases, 15-20 bases in length. By including a common primer sequence in the probe, an amplification reaction in the subsequent nucleic acid amplification step can be easily performed.

The first DNA probe 31 immobilized on the surface of the bead 7 has a different cell recognition sequence 34 for each of the nucleic acid traps 4 existing in the two-dimensional array device 1, so that the sequence analyzed later can be It is possible to determine whether it originates. For example, when the cell recognition sequence 34 is a random sequence of 5 bases, it becomes possible to identify 4 5, that is, 1024 cells. Therefore, the cell recognition sequence 34 can be arbitrarily set according to the number of nucleic acid traps 4 to be identified, specifically, in the range of 5 to 30 bases, 5 to 20 bases, 5 to 15 bases, or 5 to 10 bases. can do.

FIG. 3 (a) shows a state in which the mRNA 41 extracted from the cell 2 is captured by the first DNA probe 31. FIG. mRNA 41 contains a known gene-specific sequence 42. Next, the enzyme and substrate necessary for the reverse transcription reaction are supplied to the nucleic acid trap 4, and the mRNA 41 captured by the first DNA probe 31 is used as a template for the 1st cDNA strand 51 (one containing a sequence complementary to the mRNA 41). Strand DNA) is synthesized, and then unnecessary mRNA 41 is removed by enzymatic degradation or the like. In this way, a cDNA library is constructed on the beads 7. In the present invention, a plurality of DNA amplification processes are simultaneously performed on the cDNA library to prepare samples for a plurality of types of analysis.

FIG. 3 (b) is a diagram showing the configuration of the 1st cDNA strand 51. The 1st cDNA strand 51 includes a gene-specific sequence 52 that is complementary to the gene-specific sequence 42 contained in the mRNA. Next, as shown in FIG. 3 (c), a poly A continuous sequence (poly A tail 53) is further added to the 3 ′ end of the 1st cDNA strand 51.

The present invention is characterized in that at least two types of DNA probes are used for PCR amplification of the 1st cDNA strand 51.

FIG. 4 (a) is a diagram for explaining the procedure for obtaining a PCR amplification product containing the full length of the first cDNA strand 51 using the second DNA probe 61. FIG. The second DNA probe 61 is composed of a second PCR amplification primer sequence 62 and a poly T sequence 63, and functions as a primer by hybridizing to the poly A tail 53 added to the 1st cDNA strand 51. Using 2st cDNA strand 51 as a template, 2nd cDNA strand 64 (single-stranded DNA containing a sequence complementary to 1st cDNA strand 51) was synthesized (Figure 4 (a) (i)), and the first PCR amplification was performed there. PCR amplification product containing double-stranded cDNA 65 having a sequence corresponding to the entire length of mRNA 41 is obtained by adding PCR primer 33 and second PCR amplification primer 62 (FIG. 4 (a)) ii) and (iii)). The product obtained here is used for exhaustive analysis of the cell population.

FIG. 4 (b) is a diagram for explaining a procedure for obtaining a PCR amplification product containing the gene-specific sequence 52 contained in the 1st cDNA strand 51 using the third DNA probe 71. The third DNA probe 71 is composed of a second PCR amplification primer sequence 62 and a sequence 73 complementary to the gene-specific sequence 52, and hybridizes to the gene-specific sequence 52 of the 1st cDNA strand 51 and functions as a primer. To do. 2nd cDNA strand 74 is synthesized using 1st DNA strand 51 as a template (Figure 4 (b) (i)), and PCR amplification is performed by adding first PCR amplification primer 33 and second PCR amplification primer 62 As a result, a PCR amplification product containing a double-stranded cDNA 75 having the gene-specific sequence 52 is obtained (FIGS. 4 (b) (ii) and (iii)). The product obtained here is used for specific analysis of a single cell.

When at least two types of DNA probes described above are supplied simultaneously or sequentially, the resulting PCR amplification product has a relatively short double-stranded cDNA 65 having a sequence corresponding to the entire length of mRNA 41 and a gene-specific sequence 52. Based on samples obtained under the same PCR amplification reaction conditions, a comprehensive analysis for the entire cell population using the former and a specific analysis for a single cell using the latter are included. Can be done.

The nucleic acid amplification was described on the assumption that a PCR amplification reaction was used, but the Nucleic Acid Sequence-Based Amplification (NASBA) method, Loop-Mediated Isothermal Amplification (LAMP) method, rolling circle amplification (RCA) reaction, etc. Other amplification methods may be used.

(Application example of the first DNA probe)
FIG. 5 and FIG. 6 are diagrams for explaining a case where the first DNA probe 31a according to the application example is used. The first DNA probe 31a does not have the first PCR amplification primer sequence at the 5 ′ end fixed to the bead 7, and the 5 ′ end is the cell recognition sequence 34. It is different from the one. FIG. 5 is a diagram showing a state in which the 1st cDNA strand 51a is synthesized and a poly A tail 53 is added to the 3 ′ end in the same manner as in the basic example described above.

FIG. 6 (a) is a diagram for explaining a procedure for obtaining a PCR amplification product containing the full length of the 1st cDNA strand 51, and FIG. 6 (b) is a PCR amplification product containing the gene-specific sequence 52 contained in the 1st cDNA strand 51. It is a figure explaining the procedure which obtains. In this application example, the second DNA probe 61 and the third DNA probe 71 are used to synthesize a 2nd cDNA strand 64a or 74a, and then a fourth PCR amplification primer sequence 33 and a cell recognition sequence 34 are provided. This is also different from the basic example described above in that a complementary strand synthesis reaction is performed using the DNA probe. By using the fourth DNA probe, it is possible to amplify only a sequence derived from a specific cell. After the complementary strand synthesis reaction, the first PCR amplification primer 33 and the second PCR amplification primer 62 are added and the PCR amplification reaction is performed, so that the sequence corresponding to the full length of mRNA 41 is obtained as in the basic example described above. A PCR amplification product is obtained which comprises a double stranded cDNA 65 having a relatively short double stranded cDNA 75 having a gene specific sequence 52.

(Molecular recognition sequence)
The first DNA probe 31 described with reference to FIG. 3 (a) and the first DNA probe 31a described with reference to FIG. 5 further have different molecular recognition sequences (not shown) for each probe. You may do it. The molecular recognition sequence is preferably arranged on the 3 ′ end side of the cell recognition sequence 34. For example, when a molecular recognition sequence of 7 bases is introduced, it becomes possible to identify 4 to the 7th power, that is, 1.6 × 10 ⁴ molecules. If the molecular recognition sequence has a length of, for example, 5 to 30 bases, 5 to 25 bases, or 5 to 20 bases, a sufficient number of molecules can be identified. By introducing a molecular recognition sequence, it is possible to identify from which molecule (cDNA) amplification products having the same gene sequence derived from the same cell are derived. According to this, it becomes possible to correct | amend the amplification bias between the genes which arose in amplification reaction, and it becomes possible to quantify mRNA with very high precision.

(Known sequence)
The known sequence 52 contained in the 1st cDNA strand 51 and the mRNA 41 and the third DNA probe 71 containing sequences complementary thereto is 150 bases or more, particularly 180 bases or more, especially 190 from the 5 ′ end of the 1st cDNA strand 51. It is preferably present at a position of at least 400 bases, in particular 220 bases or less, particularly 210 bases or less, specifically 150 to 250 bases, particularly 180 to 220 bases, particularly 190 to 210 bases. If the known sequence 52 is present at such a position, the length of the double-stranded cDNA 75 contained in the PCR amplification product obtained using the third DNA probe 71 may be about several hundred bases, particularly around 200 bases. In addition, it is preferable because procedures such as fraction purification (electrophoresis, gel cutting and PCR product extraction and purification) and fragmentation can be omitted in sequencer analysis.

(Application examples of array devices)
In the two-dimensional array device 1 described with reference to FIGS. 1 and 2, the nucleic acid trap 4 is filled with the beads 7 on which the first DNA probe 31 is fixed, but the beads 7 are not necessarily used. FIG. 7 is an enlarged view for explaining in detail a partial structure around one cell trap 3 of the two-dimensional array device 1 of the application example corresponding to the structure described with reference to FIG. In this application example, the cell trap 3 is formed by providing a through hole in a substrate having a thickness about the diameter of the cell. The thickness of the substrate may be in the range of several μm to several mm as long as cells can be captured by the through-hole. A substrate having a pore or a porous resin sheet is provided under the substrate constituting the cell trap 3, and the first DNA probe 31 is fixed in the pore instead of the bead. In the two-dimensional array device 1 of such an application example, the use of a transparent material as a substrate or the like has an advantage that cell observation with a microscope becomes easy.

In order to fix the first DNA probe 31 to the pores of the substrate made of a silicon oxide film material, for example, the following is performed. First, an aqueous solution containing 0.3 mg / mL silane coupling agent GTMSi (3- Glycidoxypropyltrimethoxysilane) and acid catalyst 0.02% acetic acid was introduced into the substrate for silane treatment of the inner wall of the pores, and then mineral oil was immediately added. Introduce solution to separate and react for 2 hours. After washing with ethanol, all the solution is drained and heat-reacted at 110 ° C. for 2 hours. Next, 1 μM streptavidin solution is introduced into the substrate and reacted at room temperature for 6 hours to fix streptavidin in the pores. After the washing, a 10 mM TrisHCl solution containing the first DNA probe 31 whose biotin is modified at the 5 ′ end and 1 M NaCl and 0.1% Tween 20 is introduced into the substrate, reacted for 30 minutes, and then washed.

According to the above treatment, one DNA probe 31 can be immobilized at a ratio of 1 to 30 to 100 nm ² and it is estimated that 10 ⁷ order DNA probes can be immobilized per one nucleic acid trap 4. . Since it is known that the number of mRNA copies in one cell is 10 ⁶ or less, it is considered that a sufficient number of first DNA probes 31 can be immobilized to capture all mRNA contained in the cell. It is done.

In the above example, it is possible to capture all mRNAs in one cell and perform gene analysis, but this requires a relatively large area for the cell trap 3 and nucleic acid trap 4, so 1 cm ² The number of cell traps 3 that can be placed per unit is about 1000. On the other hand, by limiting the number of genes to be analyzed to tens to hundreds of specific ones, the required area can be narrowed, and the number of cell traps 3 that can be installed is increased from 100,000 to 1 million. Is also possible.

(Cell analysis device and cell analysis system)
FIG. 9 is a diagram for explaining a cell analysis apparatus and a cell analysis system using a two-dimensional array device having a configuration as described above. A two-dimensional array device 1 is incorporated in the nucleic acid preparation device 100, and a reagent addition apparatus 201 is connected to the nucleic acid preparation device 100. The cell analysis apparatus of the present invention includes a nucleic acid preparation device 100 in which a two-dimensional array device 1 is incorporated and a reagent addition device 201. The reagent addition device 201 driven by the control device 202 supplies the nucleic acid preparation device 100 with substrates, enzymes, and various probes and primers necessary for nucleic acid amplification.

The nucleic acid amplification product obtained by the nucleic acid preparation device 100 is sent to the sequencer 203 for sequence analysis, and the result is sent to the data analysis device 204. The sequencer 203 may have preprocessing necessary for sequence analysis (for example, individual amplification processing such as emulsion PCR and bridge amplification). The memory device 205 stores the sequence information of various probes and primers supplied by the reagent adding device 201 and the sequence information of the first DNA probe linked to the position information in the two-dimensional array device 1, and the data The analysis device 204 performs analysis while referring to the data in the memory device 205 as necessary. The control device 202 also controls the reagent adding device 201 while referring to the data in the memory device 205 as necessary. By adopting such a configuration, the cell analysis system 200 associates the analysis result of the nucleic acid amplification product obtained from the nucleic acid preparation device 100 with the sequence data of various probes, and performs comprehensive analysis on the cell population and single cell. It is possible to simultaneously perform specific analysis for.

In the nucleic acid preparation device 100, a microscope (not shown) may be installed to enable observation of cells captured by the two-dimensional array device 1. The image obtained by a microscope is preferably an optical image that can be obtained without significantly destroying the shape of the cell, such as a fluorescent image, a bright field image, or a non-linear microscope image. If the position of the cell being observed with the microscope in the two-dimensional array device 1 is known, the cell recognition sequence at that position can be specified, so the observation result by the microscope image can be linked to the result of the sequence analysis. is there. It is also possible to destroy only specific cells with a laser or the like through microscopic observation.

(Application examples)
The cell analysis device of the present invention can be applied not only to nucleic acid analysis as described above but also to analysis of biomolecules. For example, an arbitrary biomolecule contained in a cell is recognized as a sequence for capturing the biomolecule (for example, a ligand that can specifically bind to the biomolecule, specifically, an antibody or an aptamer). It is also possible to use the first probe including the sequence. Examples of biomolecules to be analyzed include arbitrary molecules such as proteins, peptides, other macromolecules, small molecules, and the like, in which a ligand that specifically binds exists.

Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited to these examples.

[Example 1]
Genetic analysis was performed according to the method of the present invention using the nucleic acid preparation device 100 shown in FIG. FIG. 8 (a) is a top view of the nucleic acid preparation device 100, and FIG. 8 (b) is a cross-sectional view. The upper and lower sides of the nucleic acid preparation device 100 have a structure in which a two-dimensional array device 109 is sandwiched between an upper electrode 107 and a lower electrode 108 in which a transparent electrode (ITO) is provided by sputtering on a transparent substrate. An electric field can be applied to the solution filled in the inner region including 106. The upper electrode 107 and the lower electrode 108 have a transmission characteristic of 40% or more in the wavelength range of 400 to 900 nm, and the cells can be observed from above with an optical microscope. The distance between the upper electrode 107 and the lower electrode 108 was 2 mm. In order to realize PCR temperature cycle, a heat block with heater (aluminum alloy or copper alloy) and a temperature controller were provided separately.

On the surface of the two-dimensional array device 109, holes having a diameter of 10 μm were arranged in an array at 125 μm intervals as cell traps 112. The two-dimensional array device 109 was a square having a side of 1.3 mm, and 11 × 11 cell traps 112 were arranged. A nucleic acid trap 113 having a diameter of 50 μm was provided immediately below the cell trap 112, and magnetic beads having a diameter of 1 μm were packed in the nucleic acid trap 113. The packing of the magnetic beads was performed by discharging 2 nL of the magnetic bead solution (7 × 10 ⁹ pieces / mL) to the nucleic acid trap 113 by an ink jet printer.

The structure of the cell trap 112 and the nucleic acid trap 113 described above was fabricated using a semiconductor process using a PDMS (polydimethylsiloxane) substrate, but instead of the resin (polycarbonate, cyclic polyolefin) by nanoimprint technology or injection molding. , Polypropylene), or a commercially available nylon mesh or track-etched membrane can be used.

Under the nucleic acid trap 113, there was provided a porous membrane 114 having pores with a diameter of 0.2 μm, the inner walls of the pores being subjected to a hydrophilic treatment, and absorbing water but retaining magnetic beads. As the porous membrane 114, a pore array sheet obtained by anodizing alumina was used, but instead, a monolith sheet made of porous glass, a capillary plate obtained by bundling capillaries and sliced, a nylon membrane or A gel thin film or the like can also be used. The pores of the porous membrane 114 function as a flow path that connects the nucleic acid trap 113 and the lower region 106. The structure comprising the PDMS substrate and the porous film 114 are bonded by plasma bonding, but thermal bonding can also be used depending on the material. Note that the structure of the above-described two-dimensional array device 109 can also be fabricated by integral processing by a semiconductor process.

(First DNA probe)
Streptavidin is immobilized on the magnetic beads in advance, and a first DNA probe (SEQ ID NO: 1) whose 5 ′ end is modified with biotin is immobilized thereon. The first DNA probe is a molecular recognition sequence consisting of a 30-base PCR amplification consensus sequence (Forward) (SEQ ID NO: 2), a 5-base cell recognition sequence (1024 types), and a 7-base random sequence from the 5 'end. And an 18-base oligo (dT) sequence and a 2-base VN sequence.

(Cell introduction and mRNA extraction)
After washing about 1000 cells or less (here, suspension culture cell THP1) with 500 μL of 1 × PBS buffer so as not to damage the cells, remove the washing solution so that PBS does not remain as much as possible. 50 μL of 1 × PBS buffer cooled to 4 ° C. was added. The cells 111 were introduced from the cell inlet 101, and the buffer was discharged from the lower outlet 102, whereby the cells 111 were adsorbed to the cell trap 112 and arranged in an array. Excess cells were drained from the upper outlet 103.

Next, 250 μL of 4% SeaPrep agarose solution (Cambrex Bio Science Rockland) solution, 495 μL of Lysis solution (TaqMan MicroRNA Cell-to-CT Kit, Applied Biosystems), and 5 μL of DNase I are introduced from the top inlet 104 Then, the PBS buffer was discharged from the lower outlet 102 to replace the upper region 105 with the Lysis buffer. After confirming that the solution near the cell trap 112 has gelled, raise the temperature to 20 ° C. and react for 8 minutes, add 50 μL of Stopping solution that deactivates DNase on the gel, react for 5 minutes, Cooled to 4 ° C. The agarose gel used here may be replaced with a polyacrylamide gel, or a PBS buffer having no gel component may be used.

Add 0.5 mL of 10 mM Tris buffer (pH 8.0) containing 0.03% polyethylene oxide (PEO) (molecular weight 600,000), 0.03% polyvinyl pyrrolidone (PVP), and 0.1% Tween 20, upper region 105 and lower region 106 was filled with Tris buffer. While maintaining the temperature of the solution and the device at 4 ° C., +5 V was applied for 2 minutes using the upper electrode 107 as the cathode (GND) and the lower electrode 108 as the anode, and the negatively charged mRNA was added to the nucleic acid trap 113 below the cell trap 112. Electrophoresis was performed in the direction of. (Instead of the DC voltage, a pulse voltage with an on level of 10 V, an off level of 0 V, a frequency of 100 kHz, and a duty ratio of 50% may be applied.) By the above operation, most mRNA was immobilized in the nucleic acid trap 113. Although it was thought that it was captured by the first DNA probe, some mRNA was not captured and flowed to the lower region 106, and the temperature of the solution and device was raised to 70 ° C and waited for 5 minutes. While reversing the polarity of the voltage applied to the electrode 108 (firstly, −5V was repeated for 1 minute, then + 5V and −5V were repeated 10 times each for 1 minute), the electrode 108 was cooled to 4 ° C. at a cooling rate of −0.1 ° C./second. In this case, the mRNA is guided to the nucleic acid trap 113 by electrophoresis. However, the mRNA may be guided to the nucleic acid trap 113 by continuously flowing the PBS buffer from the upper inlet 104 toward the lower outlet 102.

While introducing the Tris buffer from the upper inlet 104 and draining it from the upper outlet 103, changing the solution in the upper region 105, the temperature of the solution and device was raised to 35 ° C to dissolve the agarose gel, and unnecessary tissue and agarose Was removed.

(1st cDNA strand synthesis)
585 μL of 10 mM Tris buffer (pH = 8.0) containing 0.1% Tween20, 40 μL of 10 mM dNTP mix, 225 μL of 5 × RT buffer (SuperScript III, Invitrogen), 40 μL of 0.1 M DTT, 40 μL of RNaseOUT (Invitrogen), and reverse transcriptase ( 40 μL of Superscript III (Invitrogen) was mixed. The solution filling the device was discharged from the lower outlet 102 and the upper outlet 103, and immediately the solution containing the reverse transcriptase mixed above was injected from the upper inlet 104. The reverse transcription reaction was completed by raising the temperature of the solution and the device to 50 ° C. and maintaining it for 50 minutes, and a 1st cDNA strand having a structure having a complementary sequence to mRNA at the 3 ′ end of the first probe was synthesized.

The temperature was raised to 85 ° C. and held for 1.5 minutes to inactivate the reverse transcriptase. After cooling to 4 ° C., 10 mL of 10 mM Tris buffer (pH = 8.0) containing RNase and 0.1% Tween 20 was injected from the upper inlet 104 and discharged from the lower outlet 102 and the upper outlet 103 to decompose the mRNA. Further, the same amount of alkali modifier was flowed in the same manner to remove the residue and decomposition products on the device.

(Addition of poly A tail to 1st cDNA strand)
Prepare a reagent containing MgCl ₂ (1.5 mM), dATP (3 mM), RNaseH enzyme (0.1 U / μL) and TdT enzyme (0.75 U / μL) in 1 × PCR buffer, inject from the top inlet 104, 37 The reaction was carried out at 30 ° C. for 30 minutes. The temperature was then raised to 70 ° C. and held for 5 minutes to inactivate the enzyme and washed with a sufficient amount of buffer.

(Second DNA probe)
The second DNA probe (SEQ ID NO: 3) for obtaining the full-length cDNA used for the comprehensive analysis is a 23 base PCR consensus sequence (Reverse) from the 5 ′ end (SEQ ID NO: 4: Forward of SEQ ID NO: 2) Different sequences), an 18 base oligo (dT) sequence and a 2 base VN sequence.

(Third DNA probe)
The third DNA probe (SEQ ID NOs: 5 to 24) for obtaining a fragment cDNA containing a gene-specific sequence for single cell analysis is a 23 base PCR amplification consensus sequence (Reverse) (sequence) from the 5 'end. Number 4) and 20 genes (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27, and OAZ1) Each having a specific sequence. The gene-specific sequence uses 20 ± 5 bases 109 ± 8 bases upstream from the polyA tail of the target gene so that the size of the fragment cDNA obtained as a PCR product is unified to about 200 bases, which is a complicated size Fraction purification (electrophoresis, gel cutting and PCR product extraction and purification) can be omitted.

(Synthesis of 2nd cDNA strand using 2nd and 3rd DNA probes)
690 μL of sterilized water, 100 μL of 10 × Ex Taq buffer (Takara Bio Inc.), 100 μL of 2.5 mM dNTP mix, 100 μL of 10 μM second DNA probe solution or 20 types of third DNA probe solutions (each 10 μM), and 100 μL each 10 μL of Ex Taq Hot start version (Takara Bio Inc.) was mixed. The solution filling the device was discharged from the lower outlet 102 and the upper outlet 103, and the reaction solution mixed above was immediately injected from the upper inlet 104. The reaction was performed at 95 ° C. for 3 minutes → 44 ° C. for 2 minutes → 72 ° C. for 6 minutes to synthesize a 2nd cDNA strand (full length or fragment) using the 1st cDNA strand as a template.

(Synthesis and amplification of double-stranded cDNA)
Sterile water 495 μL, 10 × High Fidelity PCR buffer (Invitrogen) 100 μL, 2.5 mM dNTP mix 100 μL, 50 mM MgSO ₄ 40 μL, 10 μM common sequence primer for PCR amplification (Forward, SEQ ID NO: 25) 100 μL, 10 μM PCR amplification 100 μL of common sequence primer (Reverse, SEQ ID NO: 4) solution and 15 μL of Platinum Taq Polymerase High Fidelity (Invitrogen) were mixed. The solution filling the device was discharged from the lower outlet 102 and the upper outlet 103, and the reaction solution mixed above was immediately injected from the upper inlet 104. The three-step process of 94 ° C. for 30 seconds → 55 ° C. for 30 seconds → 68 ° C. for 30 seconds was repeated 25 cycles. Finally, the mixture was held at 68 ° C. for 3 minutes and then cooled to 4 ° C. for PCR amplification. The PCR amplification product solution was recovered and purified using a PCR purification kit (Qiagen), and the remaining free common primer for PCR amplification and residual reagents were removed.

When the second DNA probe was used in the synthesis process of the 2nd cDNA strand, a full-length cDNA was obtained. Samples that can be sequenced by fragmentation and adapter ligation, introduced into emPCR amplification or bridge amplification devices, and applied to next-generation sequencers (Life Technologies (Solid / Ion Torrent), Illumina (High Seq), Roche 454) Sequence analysis was performed.

When a third DNA probe is used in the synthesis process of the 2nd cDNA strand, each target gene has a common sequence for PCR amplification (Forward and Reverse) at both ends, and also gene-specific sequence, cell recognition sequence and molecular recognition Fragment cDNA containing single sequence for single cell analysis was obtained and applied to the sequencer for gene analysis specific to single cells. In addition, PCR amplification bias was corrected using molecular recognition sequences, and highly accurate quantitative data of gene expression levels were obtained.

[Example 2]
Using the cell analysis system 200 shown in FIG. 9, gene analysis was performed according to the method of the present invention. The nucleic acid preparation device 100 is the same as that described with reference to FIG. 8, and a microscope (not shown) is installed so that the cells 111 adsorbed on the cell trap 112 of the nucleic acid preparation device 100 can be observed.

In this example, the first DNA probe (SEQ ID NO: 25) does not have a 30-base PCR amplification consensus sequence (Forward), and is derived from a cell recognition sequence, a molecular recognition sequence, an oligo (dT) sequence, and a VN sequence. The difference from Example 1 was used. According to the same procedure as in Example 1, 1st cDNA strand was synthesized and poly A tail was added. Next, using the second DNA probe (SEQ ID NO: 3), a 2nd cDNA chain (full length) was synthesized using the 1st cDNA chain as a template in the same manner as in Example 1.

Next, four cells 111 to be subjected to detailed single cell analysis were determined based on the results of observation with a microscope in advance. Information on individually different cell recognition sequences corresponding to the place where the cell 111 is adsorbed is taken out from the memory device 205 via the control device 202, and transmitted to the reagent adding device 201, and the fourth corresponding to the cell recognition sequence. The reagent adding apparatus 201 sequentially supplies the solution containing the DNA probe to the nucleic acid preparation device 100. The fourth DNA probe (SEQ ID NOs: 26 to 29) was composed of a 30-base PCR amplification consensus sequence (Forward) and a cell recognition sequence. To the solution containing the fourth DNA probe, a PCR enzyme and substrate and a PCR amplification common sequence primer (Forward, SEQ ID NO: 25 / Reverse, SEQ ID NO: 4) were added, and the PCR cycle was repeated, Each full-length cDNA of the identified cells 111 was obtained. Conditions such as PCR reaction were all the same as in Example 1. The obtained PCR product is made into a sample that can be sequenced by fragmentation and adapter ligation, and then introduced into the sequencer 203. From the result, the data analyzer 204 performs comprehensive analysis across multiple cells and is specific to a single cell. Genetic analysis.

1 ... 2D array device, 2 ... cell, 3 ... cell trap, 4 ... nucleic acid trap, 5 ... channel, 6 ... nucleic acid, 7 ... bead, 31 ... first DNA probe, 32 ... poly T array, 33 ... First PCR amplification primer (sequence), 34 ... cell recognition sequence, 41 ... mRNA, 42 ... gene specific sequence, 51 ... 1st cDNA strand, 52 ... gene specific sequence, 53 ... poly A tail, 61 ... first 2 DNA probes, 62 ... second PCR amplification primer (sequence), 63 ... poly T sequence, 64 ... 2nd cDNA strand, 65 ... double stranded cDNA, 71 ... third DNA probe, 73 ... gene specific Sequence complementary to sequence 52, 74 ... 2nd cDNA strand, 75 ... double stranded cDNA, 81 ... fourth DNA probe, 100 ... nucleic acid preparation device, 101 ... cell inlet, 102 ... lower outlet, 103 ... upper outlet, 104 ... upper entrance, 105 ... upper region, 106 ... lower region, 107 ... upper electrode, 108 ... lower electrode, 109 ... two-dimensional array device, 111 ... cell, 112 ... cell trap, 113 ... nucleic acid trap, 114 ... many Shitsumaku, 200 ... cell analysis system, 201 ... reagent addition device 202 ... controller, 203 ... nucleic acid sequence analyzer 204 ... data analyzer, 205 ... memory device

Claims (15)

1. A cell trap that can capture cells one by one, an array device that has a nucleic acid trap that captures nucleic acids extracted from the cells trapped in the cell trap, and a reagent addition device for adding reagents to the array device Prepared,
   A nucleic acid trap is present for each cell trap, and the nucleic acid trap includes a fixed first probe for capturing the nucleic acid;
   The reagent addition apparatus provides at least a second probe and a third probe to the array device,
   The first probe has a different cell recognition sequence for each individual nucleic acid trap, and a nucleic acid capture sequence that hybridizes with a nucleic acid extracted from the cell, and optionally a first nucleic acid amplification primer sequence. You can,
   The second probe has a second nucleic acid amplification primer sequence, and a sequence that hybridizes to the end of the nucleic acid including a sequence synthesized using the nucleic acid captured by the first probe as a template,
   The third probe has a second nucleic acid amplification primer sequence, and a sequence that hybridizes to a known sequence contained in the nucleic acid captured by the first probe,
   The reagent addition apparatus supplies an enzyme and a substrate for synthesizing a complementary strand using the captured nucleic acid as a template after the nucleic acid extracted from the cell is captured by the first probe of the nucleic acid trap, and then the second A cell analysis apparatus characterized by supplying a probe and a third probe simultaneously or successively.
2. The first probe has a first nucleic acid amplification primer sequence,
   2. The cell analysis device according to claim 1, wherein the reagent adding device supplies the first nucleic acid amplification primer and the second nucleic acid amplification primer after supplying the second probe and the third probe.
3. The first probe does not have the first nucleic acid amplification primer sequence,
   The reagent addition device supplies the fourth probe having the first nucleic acid amplification primer sequence and the cell recognition sequence after the supply of the second probe and the third probe, and then for the first nucleic acid amplification 2. The cell analysis device according to claim 1, wherein the primer and the second primer for nucleic acid amplification are supplied.
4. The cell according to any one of claims 1 to 3, wherein the known sequence contained in the nucleic acid captured by the first probe is present at a position of 150 to 250 bases from the 5 'end of the captured nucleic acid. Analysis device.
5. The cell analyzer according to any one of claims 1 to 3, wherein the first probe further has a molecular recognition sequence that does not depend on the nucleic acid trap and is different for each probe.
6. The cell analyzer according to any one of claims 1 to 3, wherein the nucleic acid capture sequence of the first probe is a poly-T sequence, and the nucleic acid extracted from the cell and captured by the first probe is mRNA.
7. The cell analysis device according to any one of claims 1 to 3, wherein the terminal of the nucleic acid to which the second probe hybridizes comprises a poly A sequence.
8. A method of simultaneously performing a comprehensive analysis of a cell population and a single cell analysis of individual cells included in the cell population,
   Multiple cell traps capable of capturing cells one by one, and a fixed first probe for capturing nucleic acid extracted from the cells captured in the cell trap, and corresponding to each cell trap Preparing an array device having a nucleic acid trap;
   A step of extracting nucleic acids by destroying cells captured by the nucleic acid trap;
   A first cell may have a cell recognition sequence different for each individual nucleic acid trap, and a nucleic acid capture sequence that hybridizes with a nucleic acid extracted from the cell, and optionally a first nucleic acid amplification primer sequence. A step of allowing the probe to capture the extracted nucleic acid, then supplying an enzyme and a substrate for complementary strand synthesis using the captured nucleic acid as a template to the array device, a second nucleic acid amplification primer sequence, and a first A second probe having a sequence that hybridizes to the end of the nucleic acid containing the sequence synthesized using the nucleic acid captured by the probe as a template;
   Supplying a second nucleic acid amplification primer sequence and a third probe having a sequence that hybridizes to a known sequence contained in the nucleic acid captured by the first probe to the array device simultaneously or sequentially. , Said method.
9. The first probe has a first nucleic acid amplification primer sequence,
   9. The method according to claim 8, further comprising supplying a first nucleic acid amplification primer and a second nucleic acid amplification primer after supplying the second probe and the third probe.
10. The first probe does not have the first nucleic acid amplification primer sequence,
    After supplying the second probe and the third probe, a fourth probe having a first nucleic acid amplification primer sequence and a cell recognition sequence is supplied, and then the first nucleic acid amplification primer and the second nucleic acid 9. The method according to claim 8, further comprising supplying an amplification primer.
11. The method according to any one of claims 8 to 10, wherein the known sequence contained in the nucleic acid captured by the first probe is present at a position of 150 to 250 bases from the 5 'end of the captured nucleic acid. .
12. The method according to any one of claims 8 to 10, wherein the first probe further has a molecular recognition sequence that does not depend on the nucleic acid trap and is different for each probe.
13. The method according to any one of claims 8 to 10, wherein the nucleic acid capture sequence of the first probe is a poly-T sequence, and the nucleic acid extracted from the cell and captured by the first probe is mRNA.
14. The method according to any one of claims 8 to 10, wherein the end of the nucleic acid to which the second probe hybridizes comprises a poly A sequence.
15. The cell analysis device according to any one of claims 1 to 3, comprising an array device and a reagent addition device,
    A monitoring device for observing cells captured in the cell trap of the array device,
    A memory device storing the sequence information of the first probe, which is different for each nucleic acid trap of the array device, and the sequence information of other probes supplied by the reagent addition device,
    A control device for controlling the reagent addition device with reference to the memory device;
    A cell analysis system comprising a sequencer for analyzing a nucleic acid amplification product obtained from an array device, and an analysis device for analyzing data obtained from the sequencer with reference to a memory device as necessary.

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