JP6244439B2 - Two-dimensional cell array device and apparatus for gene quantification and sequence analysis - Google Patents

Description

  The present invention relates to gene expression analysis, cell function analysis, biological tissue analysis method, disease diagnosis, drug discovery, and the like. Specifically, it relates to an mRNA analysis method at the level of one cell.

  In gene expression analysis, mRNA is extracted from a group of cells, a complementary strand cDNA is prepared, its copy number is amplified by PCR, etc., and the target is placed at the corresponding probe position using a DNA probe array (DNA chip). A method of capturing and detecting fluorescence is used. However, methods using PCR amplification and DNA chips have low quantitative analysis accuracy, and a highly accurate gene expression profile analysis method has been desired. With the completion of human genome analysis, there is an increasing demand for quantitatively examining gene expression. Recently, a method of extracting mRNA from one cell and performing quantitative analysis is desired. Quantitative PCR is an analytical method with good quantitativeness. This involves preparing a standard sample with the same DNA sequence as the target, performing PCR amplification under the same conditions, and monitoring the progress of amplification with a fluorescent probe for comparison. This is a method for quantitative analysis. When the target is a single cell, the number of mRNA originally present is small and difficult to quantitatively analyze. In addition, for quantitative analysis of multiple gene expressions, the sample is divided and quantitative analysis is performed independently, so if there are many genes of interest and genes with low expression levels are included, In some cases, it cannot be measured.

  Under such circumstances, the inventors have devised a method of converting all mRNAs into cDNAs, preparing a cDNA library (cDNA aggregate containing all cDNAs) retained on beads, and using it for quantitative analysis. There, it was shown that the expression level of a plurality of genes contained in one cell can be accurately measured by eliminating a measurement error of a small amount of expressed gene by dividing a sample by repeatedly using a cDNA library (Non-patent Document 1).

  In the above method, all processes such as mixing and purification of reaction reagents into a solution containing a sample by hand are performed manually. Therefore, the volume of the reaction solution is limited to the order of several hundred nanoliters to microliters or more due to the problems of dispensing accuracy and solvent evaporation. Therefore, even when analyzing a single cell, since an appropriate concentration of reagent must be used, the amount of the reaction reagent (number of moles) increases in proportion to the reaction volume, and the reagent cost increases proportionally. To increase. If statistically significant data cannot be obtained unless a large number of cells are measured, the reagent cost is very large. Therefore, there is a need for a method for reducing the reaction volume with a structure that does not have dispensing accuracy and evaporation problems.

  In order to solve this problem, conventionally, a method using a device called a microfluidic combined with a small flow path has been adopted. An example of applying microfluidics for single cell analysis is described in Non-Patent Document 2. FIG. 1 in Non-Patent Document 2 describes a device structure diagram. The chip described here has 300 unit structures arranged so that 300 cells can be processed simultaneously. 3 × 50 unit structures are arranged on the chip. The unit structure is horizontally long, and the sample solution flows in the longitudinal direction and sequentially reacts while flowing. The reaction volume is 10 nL during the reverse transcription reaction and 50 nL during the PCR reaction, and the volume is reduced.

  Furthermore, in order to process a large number of cells simultaneously, it is necessary to perform gene expression analysis at a low cost on many cells at once. In order to realize this, Patent Document 1 discloses a method of constructing a cDNA library using a porous membrane or the like instead of beads. This method uses a device that can obtain a two-dimensional distribution of gene expression and realize gene expression analysis in a large number of cells. When analyzing the expression of a gene in a single cell using this device, it is not necessary to isolate the cell, and mRNA can be directly extracted from a cell in a section of a living tissue and the gene expression can be analyzed. However, in order to increase the number of genes that can be analyzed, it was necessary to repeat fluorescence measurement and chemiluminescence measurement in proportion to the number of measured genes.

JP 2009-276883

Nature Method Vol.6 No.7 2009 p. 503-506 (Nature Method vol. 6, no. 7, 2009, p.503-506) Proceeding of the National Academy of Sciences 108, 34, 2011 p. 13999-14004 (Proceeding of the National Academy of Sciences vol. 108, no. 34, 2011 p. 13999-14004)

  For basic understanding of regenerative medicine, diagnosis using genes, or life phenomena, not only the expression level of genes as an average of tissues, but also quantitative analysis of the contents of each cell that constitutes tissues Is beginning to be emphasized. For this purpose, it is desired to quantify the biological material in a large number of cells in order to obtain statistically significant data in combination with taking out the cells one by one and analyzing them in detail. In particular, it is desired to quantitatively monitor the expression levels of various genes. At this time, the biological substance to be quantified becomes mRNA in the cell. Here, for simplicity, it is assumed that the gene and the mRNA have a one-to-one correspondence, and quantifying gene expression and quantifying mRNA have the same meaning. In general, “measuring gene expression” has a broader meaning. In this case, the measurement takes into account the presence of multiple mRNA variants (variants) read from a single locus, and the various processes that occur during mRNA maturation and protein translation. Although the number of substances to be measured increases as necessary, here, in order to simplify the discussion, it refers to quantifying mature mRNA.

  There is a possibility that mRNA corresponding to a gene expressed in one cell is distributed from about several molecules to tens of thousands of molecules. In order to accurately quantify mRNA corresponding to a gene with a small number of molecules, it is necessary to efficiently convert it into a form that can be quantified. For this purpose, a single-cell-derived cDNA library is efficiently constructed (80% or more) on the surface of the beads described in Non-Patent Document 1, and the gene expression of multiple genes is accurately quantified by repeatedly measuring this. However, since the number of repetitions is limited to about 10 to 20, the number of measurable genes is limited to 10 to 20. The number of cells that can be measured simultaneously is about 100 cells or less, and the cost of the necessary reagents is also very expensive. Therefore, it is considered that it is an industrially important technique to measure only the number of genes that need to be measured for a large number of cells.

  In order to perform gene expression analysis in single cells, the cells are isolated and introduced into individual reaction wells, and reagents for cell disruption, reverse transcription, and PCR amplification are added to these reaction wells. Must be dispensed into. Therefore, in order to automate the analysis, a robot for dispensing is required, and the analysis apparatus becomes large and expensive.

  Furthermore, in order to eliminate dispensing by robots, when extracting mRNA from cells using microfluidics and amplifying nucleic acids, the reaction channel must be arranged in a row, so the number of parallel lines is increased. Since the chip size increases in proportion, there is a problem that the size of the microfluidic device increases and becomes expensive.

  Actually, as shown in the lower left of FIG. 1 of Non-Patent Document 2, the cell to be analyzed is introduced into the leftmost reaction tank, and the cell is crushed. Next, move the sample solution to the reaction tank on the right side, perform the reverse transcription reaction, and further move the sample to the right to perform the PCR reaction. Run. Finally, collect the processed sample from the far right. In the case of using microfluidics, the solution needs to move in the substrate surface, which increases the footprint of the unit structure. In addition, if the arrangement of the flow path system for introducing the sample or reagent into the unit structure or the flow path system necessary for discharging the sample after processing consumes the chip area, the number of arrangements of the unit structure is increased. There is a problem that the chip area increases and the cost of the chip increases.

  In this non-patent document 2, it is unclear as to what percentage of mRNA in a cell is processed as a sample so that it can be measured and quantified. In order to quantify chemical substances, it is an essential task to enable efficient measurement of target molecules (especially mRNA). For example, mRNA that has less than 10 molecules in one cell must be measured, but if the number of molecules in the sample processed so that it can be measured decreases, an inherent measurement error (sampling error according to the Poisson distribution) occurs. End up.

  Patent Document 1 discloses a method using a cDNA library sheet in order to realize gene expression analysis of a large number of cells at a low cost at a time. Although it is possible to measure many cells at once, it was necessary to repeatedly measure fluorescence using a cDNA library in order to increase the number of genes that can be analyzed. Therefore, there was a limit to the number of genes analyzed.

  In order to provide the above-mentioned problem, that is, to increase the number of cells that can be measured at one time and to efficiently sample molecules (here, mRNA) in the cells and to provide a measurement device or apparatus that is inexpensive, The device and apparatus configuration are as follows.

  Unit structures are arranged in a two-dimensional plane so that sample processing can be performed for each cell, such as fixing cells in a predetermined position, extracting mRNA as a molecule to be quantified from nucleic acid, and reverse transcription to construct a cDNA library. The cell-derived sample flows in a direction perpendicular to the planar device surface, and the unit structure is arranged in a chip surface, thereby reducing the area where the unit structure is tightened on the chip. In addition, the sample solution can be collected in a tag array that can be found by analyzing the sample in which position the unit structure is processed even if the sample processed by the unit structure arranged on the plane is mixed and recovered. (Tag molecule) is introduced in the course of sample processing. This eliminates the need for a sample collection mechanism for each unit structure.

  That is, the present invention includes the following inventions.

(1) a cell trapping unit for fixing one cell each;
A flow path through which the nucleic acid extract for extracting nucleic acid from the cells flows from the top to the bottom through the cell trapping section;
A nucleic acid trapping section that is connected to the cell trapping section via the flow path and is arranged below the cell trapping section and fixes the extracted nucleic acid;
A flow path for discharging the solution after nucleic acid extraction from the nucleic acid trapping part to the opposite side of the cell trapping part,
A nucleic acid extraction device, wherein the cell trapping section, the two flow paths, and the nucleic acid trapping section form a pair in the vertical direction, and a plurality of the pairs are arranged in a planar direction.

(2) The nucleic acid extraction device according to (1), wherein the nucleic acid trapping section includes beads to which DNA for nucleic acid trap is fixed.

(3) The nucleic acid extraction device according to (1), wherein the nucleic acid trapping portion includes a porous membrane in which DNA for nucleic acid trap is fixed in a pore.

(4) The nucleic acid extraction device according to any one of (1) to (3), wherein a substance that chemically binds to a cell surface substance is fixed to the cell trapping portion.

(5) The nucleic acid extraction device according to (2) or (3), wherein a part of the DNA for the nucleic acid trap includes a sequence for specifying a position on the chip.

(6) The nucleic acid extraction device according to (2) or (3), wherein a part of DNA for nucleic acid trap includes a different sequence for each trapped nucleic acid molecule.

(7) The nucleic acid extraction device according to (6), further comprising means for introducing an enzyme for reverse transcription of RNA trapped in the nucleic acid trapping section.

(8) The nucleic acid extraction device according to any one of (1) to (7), wherein the cell trapping portion is made of an optically transparent material.

(9) The nucleic acid extraction device according to any one of (1) to (8), wherein a nucleic acid trapping portion is provided immediately below the cell trapping portion.

(10) The nucleic acid extraction device according to any one of (1) to (8), wherein a nucleic acid trapping portion is provided in a region other than immediately below the cell trapping portion.

(11) A nucleic acid processing apparatus comprising the nucleic acid extraction device according to any one of (1) to (10) and a means for introducing a reagent for constructing a cDNA library.

(12) A nucleic acid treatment comprising the nucleic acid extraction device according to any one of (1) to (10), a reagent for constructing a cDNA library, and a means for introducing a reagent for nucleic acid amplification apparatus.

(13) The nucleic acid extraction device according to any one of (1) to (10), and a microscope unit for observing the cells trapped in the cell trapping unit with a differential interference microscope, a phase contrast microscope, a Raman microscope, or a coherent Raman microscope A nucleic acid processing apparatus comprising:

(14) A method for extracting nucleic acid from a cell using a nucleic acid extraction device comprising a cell trapping unit and a nucleic acid trapping unit disposed below the cell trapping unit,
Contacting a cell with the cell trapping portion and trapping each one cell in the cell trapping portion;
Flowing a nucleic acid extract for extracting nucleic acid from cells through a flow path passing through the cell trapping section from top to bottom;
Immobilizing the extracted nucleic acid in the nucleic acid trapping portion;
Discharging the solution after nucleic acid extraction from the nucleic acid trapping section to the opposite side of the cell trapping section through a flow path,
In the nucleic acid extraction device, the cell trapping section, the two flow paths, and the nucleic acid trapping section form a pair in the vertical direction, and a plurality of the pairs are arranged in a planar direction. Method.

  In devices that quantify and sequence biomolecules such as nucleic acids in a single cell, and identify the type of biomolecule, increase the number of cells that can be measured at one time and increase the efficiency of molecules in the cell (eg, mRNA) It is possible to provide an inexpensive measuring device or apparatus that can perform sample processing well.

It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the usage method of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid used with the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the usage method of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the usage method of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid processing apparatus of this invention. It is the schematic which shows one Embodiment of the nucleic acid processing apparatus of this invention. It is the schematic which shows one Embodiment of the nucleic acid processing apparatus of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the nucleic acid extraction device of this invention. It is the schematic which shows one Embodiment of the processing method of the nucleic acid extraction device of this invention.

  A specific structure of the nucleic acid extraction device of the present invention can be illustrated in, for example, FIG. FIG. 1 (a) is a cross-sectional view perpendicular to the surface of the planar device, and FIG. 1 (b) is a cross-sectional view taken along the alternate long and short dash line in FIG. A cell trapping unit 2 for fixing cells 1 introduced into the device one by one (in FIG. 1, the cell trapping unit has holes for fixing cells one by one), and a nucleic acid for extracting nucleic acid from the cells The extraction liquid passes through the cell trapping section and flows from top to bottom, and is connected to the cell trapping section via the flow path and is arranged below the cell trapping section to fix the extracted nucleic acid. A nucleic acid trapping section; and a flow path for discharging the solution after nucleic acid extraction from the nucleic acid trapping section to the opposite side of the cell trapping section. The cell trapping section, the two flow paths, and the nucleic acid A nucleic acid extraction device characterized in that trapping portions form a pair in the vertical direction and a plurality of pairs are arranged in the plane direction is a basic structure of the present invention. This structure is configured on and in the flat substrate 6, and an upper reaction region 7 for introducing cells and a lower reaction region 8 for discharging processed nucleic acids are added as necessary. The dotted arrow 9 is an example of the movement trajectory of the cell, and the arrow 10 indicates the movement direction of the nucleic acid extracted from the cell and the processed sample.

  In the device described in Non-Patent Document 2, a cell trap chamber (cell capture chamber), a reverse transcription portion (RT chamber), and a PCR portion (qPCR chamber) are arranged in the plane. A larger area than that required for the cell trap must be provided for the two types of reaction vessels, and a valve and a flow path for controlling the liquid flow must also be provided in the plane. Therefore, a device for arranging a unit structure for processing one cell (in the structure of Non-Patent Document 2, a structure related to one cell processing including a cell trap part, a reverse transcription part, a PCR part, and a flow path system). The upper area will become larger. Since the device cost is roughly proportional to the area of the device, increasing the number of cells that can be processed at the same time increases the device cost.

  In the embodiment of FIG. 1, in order to solve this problem, a nucleic acid trapping unit is provided immediately below the cell trapping unit, a cDNA library is constructed from the extracted nucleic acid in the nucleic acid trapping unit, and subsequent processing is performed on this library. Thus, a plurality of reactions can be executed in one reaction region.

  In order to increase the efficiency of the nucleic acid trap, the nucleic acid trapping portion preferably has a structure in which a large number of beads are packed or a porous structure. By increasing the surface area as a reaction field in such a unit volume, the efficiency is high even in a small reaction tank, and the reaction can be performed in a short time.

  Furthermore, when the constructed cDNA is used as a template and nucleic acid amplification by PCR or transcription reaction is performed on the device, the sample is collected and quantified by sequencing using a next-generation sequencer. By making it differ depending on the trapped position, it becomes possible to identify which cell-derived gene expression level is the sequencing for the collected sample solution.

  This eliminates the need for a valve mechanism or flow path for isolating the unit structure and controlling the movement of the sample.

  In the configuration of FIG. 1, a reagent necessary for the nucleic acid trapping unit 4 is supplied from the upper reaction region 7 to cause a PCR reaction in the nucleic acid trapping unit, and a PCR amplification product is recovered from the lower reaction region 8. At this time, it is preferable to provide a mechanism on the outside of the device so that the temperature of the entire device becomes a temperature cycle suitable for the PCR reaction.

  Furthermore, as a desirable structure, in order to measure the state of cells using a non-invasive microscope in advance and to quantify biological substances in the cells by gene expression analysis etc. for the same cells, directly under the cells fixed An example is a structure in which the nucleic acid trapping portion is not disposed but is disposed in the periphery.

  Specifically, the structure is as shown in FIG. FIG. 2 (a) is a cross-sectional view perpendicular to the surface of the planar device, and FIG. 2 (b) is a cross-sectional view taken along the alternate long and short dash line in FIG. In order to perform microscopic observation before extracting the nucleic acid from the cell 1, the parts 21, 22, and 23 in the device use an optically transparent material. For the microscopic observation, a transmission microscope, differential interference microscope, phase contrast microscope, coherent standing Stokes Raman microscope (CARS microscope), or the like can be used.

  After completion of microscopic observation, the sample and reagent are flowed in the direction of arrow 10 in order to perform nucleic acid extraction and reverse transcription. The process of sample processing is the same as the case without microscopic observation.

(Example 1)
The present embodiment relates to a nucleic acid extraction device and a sample processing apparatus in which a nucleic acid trapping unit is configured by packing a large number of DNAs (DNA probes) for nucleic acid traps immobilized on beads.

  The basic structure of the unit structure of the nucleic acid extraction device of this example is the same as that shown in FIG. However, in this example, not only nucleic acid is extracted from cells and mRNA is captured, but a cDNA library is constructed, and this is used as a template, with a known sequence at the end that can be sequenced, and a sufficient amount of nucleic acid amplification product. The device configuration is such that can be obtained.

  A sectional view of a unit structure for processing one cell in the nucleic acid extraction device corresponding to this example is shown in FIG. 3 (a). In addition, in (b) to (f) of FIG. 3 and FIG. 4, nucleic acid extraction and nucleic acid (mRNA) capture (b), cDNA synthesis (c), nucleic acid from each cell after cell capture possible with this device A conceptual diagram of the steps of synthesis (d), (e) and PCR amplification (f) of the 2nd strand into which known terminal sequences necessary for amplification (PCR) and sequencing were introduced is shown. Further, FIG. 5 shows an overall configuration diagram of the nucleic acid extraction device. FIG. 5 (a) is an overall view of a cross section of the nucleic acid extraction device corresponding to FIG. 1 (a) and FIG. 3 (a), and FIG. 5 (b) is a cross-sectional view taken along the line AA ′ of FIG. There is a drawing corresponding to FIG. FIG. 5C is a cross-sectional view corresponding to the B-B ′ cross section of FIG.

  Next, the operation of the nucleic acid extraction device will be described in the order of processing. In FIG. 3 (a), in order to fix the cell 1 suspended in the solution to a specific position (cell trapping portion (opening)) 2 on the device, the upper reaction region 7 to the lower reaction region Allow the solution to flow through the cell trapping section towards 8. The cells move along the flow of the solution and reach the cell trapping portion. Since the opening diameter of the cell trapping portion is smaller than the cell diameter, the cells are fixed here. Since the trapped cells act as plugs for the solution flow, the flow moves to a cell trapping section that has not yet captured the cells. Therefore, the remaining cells move to the part where the cells are not yet captured and are captured. When the desired number of cells 1 has been captured, the solution flowing in the upper reaction region 7 is replaced with a nucleic acid extract such as Lysis buffer (for example, a mixture of a surface active agent such as Tween 20 and a protease) to destroy the cells. Is done. At the same time, an electric field is applied in the direction of 11, and the nucleic acid (mRNA) in the cell is moved to the nucleic acid trapping unit 4 by electrophoresis. The nucleic acid trapping unit 4 is a region between the channel 3 between the cell trapping unit 2 and the channel 5 between the lower reaction region 8, and a bead 12 on which a DNA probe 31 for trapping nucleic acid is fixed. Is the packed region.

  FIG. 3 (b) shows an enlarged view of the surface of the bead 12 on which the DNA probe 31 is fixed. An electric field 11 is applied so that the mRNA 32 extracted from the cells is captured on the beads in the nucleic acid trapping portion immediately below the captured cells. In order to store the information on the position of the cell where the captured mRNA originally existed as sequence information, the DNA probe 31 fixed to the beads includes a sequence that differs depending on the position of the nucleic acid trapping portion, that is, a cell recognition sequence. The 3 'end of the DNA probe 31 has a poly T sequence, and the mRNA is captured by hybridizing with the poly A sequence at the 3' end of the mRNA. Nucleic acid trapping sections arranged on the two-dimensional array shown in 4 of FIGS. 1 (b) and 5 (c) are packed with beads on which DNA probes 31 having different cell recognition sequences are immobilized. Yes.

In this example, the DNA probe 31 for capturing mRNA has a slightly more complicated sequence structure. As shown in FIG. 3 (b), a 30-base PCR amplification is performed from the 5 ′ end as the DNA probe 31. Common sequence (Forward direction), tag sequence for cell recognition of 5 bases, tag sequence for molecular recognition consisting of a random sequence of 15 bases, and an oligo (dT) sequence of 18 bases + a VN sequence of 2 bases. By introducing the common sequence for PCR amplification into the DNA probe 31, it can be used as a common primer in the subsequent PCR amplification step. As for the cell recognition tag, for example, in the case of a random sequence of 5 bases, 4 ⁵ = 1024 single cells can be recognized. That is, a cDNA library can be prepared from 1024 single cells at a time, and as described above, in the sequence data of the next-generation sequencer finally obtained, which cell is derived can be recognized. Is possible. Furthermore, 4 ⁷ = 1.6x10 ⁴ molecules can be recognized by introducing a molecular recognition tag sequence (for example, 7 bases) into the DNA probe 31, so that DNA sequence data for amplification products obtained with the next-generation sequencer Therefore, it is possible to recognize which molecule is derived from the amplification product having the same gene sequence derived from the same cell. That is, since the amplification bias between genes generated in the amplification process can be corrected, the amount of mRNA initially present in the sample can be quantified with high accuracy. However, when the expression level of the same gene in a single cell is larger than the variation of the molecular recognition tag sequence, the accuracy of amplification bias correction is reduced. The oligo (dT) sequence located at the most 3 ′ side hybridizes with the poly A tail added to the 3 ′ side of mRNA32 and is used to capture mRNA32 (FIG. 3 (b)).

  In this example, a poly-T sequence was used as a part of the supplemental DNA probe 31 to analyze mRNA, but in order to perform microRNA or genomic analysis, a random sequence is complementary to the sequence to be analyzed instead of the poly-T sequence. Needless to say, a part of such a sequence may be used.

  Next, 1st cDNA strand 33 is synthesized using mRNA 32 captured by DNA probe 31 on the bead as a template. In this step, the voids of the beads packed with a solution containing reverse transcriptase and a synthetic substrate are filled, and the temperature is slowly raised to 50 ° C. and a complementary strand synthesis reaction is performed for about 50 minutes (FIG. 3 (c)). After completion of the reaction, RNase is flowed through the region packed with beads to decompose and remove mRNA32. Next, a solution containing an alkali modifier and a washing solution are passed through the voids of the beads to remove the residue and decomposition products. A cDNA library array as shown in FIG. 5 (c) is constructed on the beads packed in the nucleic acid trapping portion by the process so far, reflecting the positions of individual cells captured by the cell trapping portion.

  Next, Lysis buffer is flowed from the lower reaction region 8 toward the upper reaction region 7 in order to remove cell debris remaining in the cell trapping portion. Subsequently, multiple (~ 100) target gene-specific sequence primers 41 with a common sequence (Reverse) for PCR amplification were annealed to the 1st cDNA strand (Fig. 4 (d)), and the 2nd cDNA strand was obtained by complementary strand extension reaction. 42 is synthesized (FIG. 4 (e)). In other words, 2nd cDNA strand synthesis is performed under multiplex conditions. As a result, for a plurality of target genes, a double-stranded cDNA chain having a common amplification sequence (Forward / Reverse) at both ends and containing a cell recognition tag, a molecular recognition tag, and a gene-specific sequence is synthesized. The In this example, as an example, 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27 and For the gene-specific sequence of OAZ1), 20 ± 5 bases upstream of 109 ± 8 bases from the poly A tail of the target gene were used, but this unified the PCR product size to about 200 bases in the subsequent PCR amplification process. It is to do. By unifying the PCR product size, complicated size fraction purification steps (electrophoresis-> gel excision-> PCR product extraction / purification) can be eliminated, and parallel amplification from one molecule (emulsion PCR, etc.) Has an effect that can be used directly. Subsequently, PCR amplification was performed using a common sequence for amplification (Forward / Reverse) to prepare a PCR product 43 derived from a plurality of types of genes (FIG. 4 (f)). Even if an amplification bias occurs between genes or molecules in this step, high-accuracy quantitative data can be obtained because the amplification bias can be corrected using the molecular recognition tag after acquiring next-generation sequencer data. I can do it.

The number of mRNA per cell is approximately 10 ⁶ , and 1.1 × 10 ⁵ magnetic beads 12 are packed in the nucleic acid trap portion for capturing the mRNA. Streptavidin is immobilized on the surface of the magnetic beads, and is modified to the surface of the magnetic beads via streptavidin by modifying biotin at the 5 ′ end of the DNA probe 31.

  So far, 2nd cDNA has been synthesized using dozens of gene-specific sequence primers in which a common sequence for PCR amplification is added to the cDNA generated on the bead surface (1st cDNA). Disclosed. Of course, other amplification methods such as rolling circle amplification (RCA), NASBA, and LAMP may be used.

Next, a method for producing a nucleic acid extraction device will be described in detail. The nucleic acid trapping part, the cell trapping part, and the flow path connecting them packed with magnetic beads were produced as a substrate 6 made of PDMS (polydimethylsiloxane) using a semiconductor process. In the cell trapping portion, through holes having a diameter of 10 μm are arranged in an array at intervals of 125 μm. The size of the substrate side is a square 13 mm, the cell trapping portion is disposed 10 ⁴ therein. Directly under the cell trapping part, the diameter of the through hole is as wide as 50 μm, and magnetic beads are packed in this part. A pore array sheet (porous membrane) 35 was placed under the substrate 6 in which the through holes were arranged in an array. The pore diameter of the pore array sheet is smaller than 1 μm which is the diameter of the magnetic beads.

  Thereafter, the ink jet printer head is filled individually, and beads having different sequences fixed thereon are individually filled into the nucleic acid trapping unit 4 by 2 nL.

  The inner wall of the pore is processed into a hydrophilic surface and absorbs water but can hold the beads in the nucleic acid trapping portion. There are various types of pore array sheets, such as monolithic sheets made of porous glass, capillary plates obtained by bundling capillaries and slicing, nylon membranes, and gel thin films. A fine pore array sheet was used. Such a sheet can be made by anodic oxidation, but a sheet having a pore diameter of 20 nm to 200 nm and a diameter of 25 mm is commercially available. From this, it cut out into a square of 13 mm on a side and used it. The pores formed in the sheet are the flow paths 5 that connect the nucleic acid trapping portion and the lower reaction region.

  The PDMS substrate and the pore array sheet were bonded by plasma bonding.

  Here, instead of the PDMS substrate, a substrate made of a resin (polycarbonate, cyclic polyolefin, polypropylene) produced by nanoimprint technology or injection molding, a commercially available nylon mesh, or a track etch membrane may be used. Adhesion with the pore array sheet can be performed by thermal adhesion.

  Needless to say, this reaction layer may be integrally processed using a semiconductor process.

Next, a magnetic bead solution (7 × 10 ⁹ pieces / mL) with a diameter of 1 μm to which a DNA probe modified with a 5 ′ biotin group is immobilized is injected into the nucleic acid trapping unit 4 by 2 nL for each region by the same technique as an inkjet printer. To do. At this time, DNA probes having different cell recognition tag sequences (1024 types) for each region are discharged. The magnetic bead solution is discharged through the channel 5, leaving only the beads. The method for immobilizing mRNA capture DNA probes with different cell recognition tag sequences is to mix magnetic beads and DNA probe solution in separate reaction tubes and use Tris buffer (pH 7.4) containing 1.5M NaCl. Mix and incubate with rotation for 10 minutes.

  Hereinafter, an apparatus system for creating a nucleic acid extraction device described so far and obtaining a gene expression profile with a next-generation (large-scale) sequencer will be described with reference to FIG. After washing about 1000 cells or less with 500 μL of 1 × PBS so as not to damage the cells, the solution was removed so as not to leave PBS as much as possible, and 50 μL of 1 × PBS buffer cooled to 4 ° C. was added. Cells are introduced from the cell inlet 308, and the buffer is discharged from the lower outlet 307, whereby the cells are arranged in an array in the cell trap portion. Excess cells drain from the upper outlet 306. Next, the Lysis buffer is introduced from the upper inlet 305, the PBS buffer is discharged from the channels 306 and 307, and the upper reaction region 7 is replaced with the Lysis buffer. The upper and lower sides of the nucleic acid extraction device are sandwiched between a transparent upper substrate 301 and a lower substrate 302. Transparent (ITO) electrodes are formed inside these substrates by sputtering, an electric field is applied, and nucleic acids are moved to a nucleic acid trapping portion directly under the cells by electrophoresis. The reason for making the electrode transparent is that the cells can be observed with an optical microscope. The ITO transparent electrode used this time has a transmission characteristic of 40% or more in the wavelength range of 400 to 900 nm.

  Lysis Solution 495uL (TaqMan MicroRNA Cell-to-CT Kit; Applied Biosystems Inc.) and DNase I 5uL are injected from inlet 305. After confirming that the solution has gelled, raise the temperature to 20 ° C and react for 8 minutes, then add 50uL of Stopping Solution (solution that deactivates DNase) on the gel and react for 5 minutes. Cooled to 4 ° C. Next, 0.5 mL of 10 mM Tris Buffer (pH 8.0) containing 600,000, 0.03% PEO (polyethylene oxide), 1 million, 0.03% PVP (polyvinylpyrrolidone) and 0.1% Tween 20 was added. . At this time, the distance between the upper electrode 301 and the lower electrode 302 is 2 mm, and the upper reaction region 7 and the lower reaction region 8 are completely filled with the Tris buffer. While maintaining the solution temperature at 4 ° C, the upper electrode 301 is used as the cathode (GND), the lower electrode 302 is used as the anode, and + 5V is applied for 2 minutes using the power supply 311. Electrophoresis in the direction of region 8. As the electrophoresis conditions, pulse electrophoresis with an on level of 10 V, an off level of 0 V, a frequency of 100 kHz, and a duty of 50% may be used instead of applying a DC voltage. During this process, mRNA is almost trapped in the oligo (dT) part of the DNA probe fixed to the beads. However, some mRNAs are not trapped by the secondary structure and move to the lower reaction region 8 below the beads. To trap the mRNA completely with the DNA probe, raise the temperature of the solution to 70 ° C, wait 5 minutes, and then reverse the polarity of the voltage applied to the lower electrode 302 every minute at -0.1 ° C / sec. It was cooled to 4 ° C. (at first, −5 V was applied for 1 minute, and then repeatedly applied from +5 V to −5 V 10 times per minute). Next, by introducing the above tris buffer from the inlet 305 and discharging from the outlet 306, while exchanging the solution in the upper reaction region 7, the temperature of the solution is raised to 35 ° C. to dissolve the agarose gel, No cell tissue and agarose were washed and removed. Furthermore, 10 mM Tris Buffer (pH = 8.0) containing 0.1% Tween20 585 μL and 10 mM dNTP 40 μL and 5xRT Buffer (SuperScript III, Invitrogen) 225 μL, 0.1 M DTT 40 μL and RNaseOUT (Invitrogen) 40 μL and Superscript III (reverse transcriptase) Invitrogen) 40 μL was mixed, the solution filling the upper reaction region 7 and the lower reaction region 8 was discharged from the outlets 306 and 307, and the above solution containing reverse transcriptase was immediately injected from the inlet 305. Thereafter, the temperature of the solution was raised to 50 ° C. and kept for 50 minutes to complete the reverse transcription reaction, and 1st cDNA having a sequence complementary to mRNA was synthesized.

  A cDNA immobilized on the surface of many beads for each cell was obtained as a library. This is a so-called 1-cell cDNA library array, which is fundamentally different from the averaged cDNA library obtained from many cells so far.

  The expression level for each gene is quantitatively measured for various genes from the cDNA library array thus obtained. Since there are 10,000 pores per cell, the average number of cDNA per pore is 100. For one type of cDNA, when the number of cDNA copies per cell is 10,000 or less, the average is 1 or less per bead.

  After synthesizing the first cDNA strand, reverse transcriptase is inactivated by maintaining at 85 ° C for 1.5 minutes, cooled to 4 ° C, and 10 mL of 10 mM Tris Buffer (pH = 8.0) containing RNase and 0.1% Tween20 is injected from inlet 305. Then, the RNA was decomposed by discharging from the outlets 306 and 307, and the same amount of alkali denaturant was flowed in the same manner to remove and wash the residue and degradation products. Subsequently, 690 μL of sterilized water, 100 μL of 10 x Ex Taq Buffer (TaKaRa Bio), 100 μL of 2.5 mM dNTPMix and 20 gene-specific sequence primers mixed with 10 μM of each PCR amplification consensus sequence (Reverse) Mix 100 μL and Ex 10 μL of Taq Hot start version (TaKaRa Bio) was mixed, the solution filling the device was discharged from outlets 306 and 307, and the above solution containing reverse transcriptase was immediately injected from inlet 305. Then, 95 ° C for 3 minutes → 44 ° C for 2 minutes → 72 ° C for 6 minutes, annealing the primer-specific sequence using the 1st cDNA strand as a template (Figure 4 (d)), and then complementary strand extension reaction To synthesize a 2nd cDNA strand (FIG. 4 (e)).

Next, 495 μL of sterilized water, 100 μL of 10 x High Fidelity PCR Buffer (Invitrogen), 2.5 mM dNTP mix 100 μL, 50 mM MgSO ₄ 40 μL, and 10 μM common primer for PCR amplification (Forward) 100 μL and 10 μM common sequence primer for PCR amplification (Reverse) Mix 100 μL and Platinum Taq Polymerase High Fidelity (Invitrogen) 15 μL, drain the solution filling upper reaction zone 7 and lower reaction zone 8 from outlets 306 and 307, and immediately inject the above solution from inlet 305 did. After that, keep the solution at 94 ° C for 30 seconds, repeat 40 cycles of 3 steps of 94 ° C 30 seconds → 55 ° C 30 seconds → 68 ° C 30 seconds, finally hold 68 ° C for 3 minutes, then cool to 4 ° C A PCR amplification step was performed (FIG. 4 (f)). In order to realize such a temperature cycle, a heat block with heater (aluminum alloy or copper alloy) 309 and a temperature controller 310 may be added. This amplifies the target portion of 20 target genes, but the PCR product size is almost uniform at 200 ± 8 bases. Collect the PCR amplification product solution accumulated in the solution. For the purpose of removing residual reagents such as free PCR amplification common sequence primers (Forward / Reverse) and enzymes contained in this solution, purification is performed using PCR Purification Kit (QIAGEN). After applying this solution to emPCR amplification or bridge amplification, the solution is applied to a next-generation sequencer of each company (Life Technologies (Solid / Ion Torrent), Illumina (High Seq), Roche 454) and analyzed.

  Next, a method for reducing amplification bias using a molecular recognition tag will be described. FIG. 6 schematically shows a state where data sequenced in the same sequence except for the molecular recognition region is obtained (relevant portions of the obtained sequencing data are schematically shown). In FIG. 6, 601, 602, 603, 604, and 605 are the same sequence including the molecular recognition tag sequence that is a random sequence, and show the cases where 1, 7, 4, 2, and 2 reads are obtained, respectively. Yes. These sequences are all one molecule at the time when the 2nd strand is synthesized in FIG. 4 (e), and the number of molecules increases at the same time as the number of molecules increases by subsequent PCR amplification. Therefore, the same lead of the molecular recognition tag may be regarded as the same molecule, and all are regarded as one molecule. As a result, the unevenness of the number of molecules for each sequence due to PCR amplification in the process after the synthesis of the second strand and adsorption to the inside of the pore array sheet when the solution is taken out is eliminated by the above-mentioned identification.

  Here, as the obtained data, 1, 7, 4, 2, and 2 are apparent counts (the same sequence except for the molecular recognition tag). The number of molecules in each cell is recounted as 1 count, for a total of 5 counts (1, 7, 4, 2, and 2 each corresponding to 1 count). That is, it is estimated that there were 5 molecules corresponding to sequences other than the molecular tag before amplification. Actually, in the data, reads having different sequences other than the molecular recognition tag are also obtained as a sequencing result. At this time, by counting the number of reads in which the molecular recognition tags are different and the other sequences are the same, it is possible to execute the count for the sequence to be known. It can be presumed that the original sample contains mRNA with the number of molecules proportional to this count.

  The prepared sheet can be used repeatedly, and for gene groups that need to know the expression level, a gene-specific sequence primer Mix solution with a common sequence primer (Reverse) for PCR amplification added is prepared. Similarly, synthesis of the 2nd cDNA strand, PCR amplification, and emPCR may be performed and analyzed by a next-generation sequencer. That is, by repeatedly using a cDNA library, it is possible to perform highly accurate expression distribution measurement for a necessary type of gene.

(Example 2)
In this example, beads were used to construct a cDNA library from a group of cells arranged in an array while retaining information on which cells derived mRNA contained in individual cells. Instead of a nucleic acid extraction device having a nucleic acid trapping portion, a pore array sheet having a DNA probe immobilized thereon is used as the nucleic acid trapping portion. Moreover, T7 promoter is used instead of PCR amplification for nucleic acid amplification after constructing the cDNA library.

  The structure of the nucleic acid extraction device and the extraction / treatment method using it in this example are shown in FIGS.

  FIG. 7 (a) shows a cross-sectional view of the unit structure of the nucleic acid extraction device. The cells 1 are captured by the cell trapping unit 2 by flowing a buffer solution containing cells from the upper reaction region 7 to the lower reaction region 8 so as to penetrate the device. The cell trapping unit 2 is in a cell array device 6 made of PDMS. The diameter of the cell trapping part is 16 μm, which is slightly larger than the cell diameter. By setting the diameter, two or more cells are prevented from being trapped in the cell trapping portion. The pore array sheet 71 that is a nucleic acid trapping portion has a large number of pores 72 penetrating the sheet, and a DNA probe is fixed to the inner wall of the pore 72. A cell array device 6 made of PDMS for cell trap and a pore array sheet (porous membrane) 71 for nucleic acid trap are basic elements of the nucleic acid extraction device. These two elements are directly overlapped, and these elements play a role in the flow path connecting them. As in Example 1, after cell trapping, the cell-containing buffer is replaced with Lysis buffer, and the cells are disrupted while an electric field is applied in a direction perpendicular to the device. Thus, mRNA in the disrupted cell is captured by hybridization to the DNA probe 73 on the inner wall of the pore 72 immediately below the cell trap position.

  The DNA probe 73 fixed inside the cell array sheet consists of a T7 promoter sequence from the 5 'end direction, a common sequence for emPCR amplification (Forward direction), a cell recognition tag sequence, a molecular recognition tag sequence, and an oligo (dT) sequence. Consists of. By introducing the T7 promoter sequence into the DNA probe, the target sequence can be amplified by the subsequent cRNA83 amplification step (FIG. 8 (e)) by IVT (In Vitro Transcription).

  As will be described later, when a transcription reaction from cDNA to cRNA by a transcription factor is performed in the nucleic acid amplification step, the DNA probe preferably further contains a promoter sequence of the transcription factor. As such a promoter sequence, T7 is used, but SP6, T3 and the like are also included. The activity of T7 RNA polymerase is used for nucleic acid amplification.

  In this example, a T7 promoter sequence is used. This sequence is recognized by T7 RNA polymerase, and a transcription (cRNA83 amplification) reaction is initiated from the downstream sequence.

  Nucleic acid amplification using the promoter sequence of transcription factor is isothermal amplification, which not only eliminates the need for a temperature controller to add a temperature cycle, but also reduces the possibility of detachment of probe DNA immobilized on the device surface at high temperatures. can do.

Similarly, by introducing a common sequence for PCR amplification, it can be used as a common primer in the subsequent emPCR amplification step. Further, as in Example 1, it is possible to recognize 4 ⁵ = 1024 single cells by introducing a cell recognition tag (for example, 5 bases) into a DNA probe. Furthermore, 4 ⁷ = 1.6x10 ⁴ molecules can be recognized by introducing a molecular recognition tag sequence (for example, 7 bases) into a DNA probe. Similar to the first embodiment, it is possible to recognize the origin. In other words, since the amplification bias between genes generated in the amplification process such as IVT / emPCR can be corrected, the amount of mRNA present in the sample can be quantified with high accuracy. The oligo (dT) sequence located at the most 3 ′ side hybridizes with the poly A tail added to the 3 ′ side of the mRNA and is used to capture the mRNA (FIG. 7 (a)).

  Next, a method for producing a pore array sheet constituting the nucleic acid trapping portion will be described.

As the pore array sheet, a commercially available product prepared by an anodic oxidation method is available. Here, a pore array sheet 71 having a pore diameter of 200 nm, a thickness of 60 μm, and a 13 mm square (cut from a 25 mm diameter sheet) is used. An example using this will be described. The pores 72 formed in the pore array sheet 71 penetrate in the thickness direction of the pore array sheet 71, and the pores are completely independent. The pore 72 also functions as the flow path 5. The surface is hydrophilic, the protein adsorption to the surface is extremely small, and the enzymatic reaction proceeds efficiently. First, the surface of the pore array sheet 71 is treated such as silane coupling to fix the DNA probe 73 to the pore surface. Since the DNA probes 73 are fixed to the surface at an average ratio of 30 to 100 nm ^{2, 4} to 10 × 10 ⁶ DNA probes are fixed to one hole. Next, the surface is coated with a surface coating agent in order to prevent surface adsorption. This surface coating may be performed simultaneously with the probe fixation. The DNA probe density is such that mRNA passing through this space can be captured by the DNA probe with an efficiency of almost 100%.

Next, a method for immobilizing a DNA probe inside the pore will be described in detail. The surface of the pores inside the pore array sheet is a surface that does not adsorb nucleic acids such as mRNA and PCR amplification primers, and proteins such as reverse transcriptase and polymerase at the same time that DNA probes are immobilized at high density. There is a need. In this example, a silane coupling agent for immobilizing a DNA probe and a silanized MPC polymer for preventing adsorption were simultaneously covalently immobilized on the pore surface at an appropriate ratio to increase the DNA We realized density fixation and stable adsorption inhibition of nucleic acids and proteins. Actually, the alumina pore array sheet is first immersed in an ethanol solution for 3 minutes, then treated with UVO3 for 5 minutes, and washed with ultrapure water three times. Next, MPC _0.8 -MPTMSi _0.2 (MPC: 2-Methacryloyloxyethyl phosphorylcholine / MPTMSi: 3-Methacryloxypropyl trimethoxysilane) (for example, Biomaterials 2009, 30: 4930-4938, and Lab Chip 2007, 7: 199-206) 2 in 80% ethanol solution containing 3 mg / ml and 0.3 mg / ml silane coupling agent GTMSi (GTMSi: 3-Glycidoxypropyltrimethoxysilane Shin-Etsu Chemical) and 0.02% acetic acid as acid catalyst. Soaked for hours. After washing with ethanol, it was dried in a nitrogen atmosphere and heat-treated at 120 ° C. for 30 minutes in an oven. Next, in order to immobilize the DNA, 1 μM 5 ′ amino group-modified DNA probe and 0.05 M borate buffer (pH 8.5) containing 7.5% glycerol and 0.15 M NaCl are the same as the inkjet printer on the pore array sheet. Depending on the technique, DNA probes containing different cell recognition tag sequences (1024 types) for each 25 μm × 25 μm region of 100 pL were discharged. Then, it was made to react at 25 degreeC in a humidification chamber for 2 hours. Finally, to wash unreacted glycidic groups and remove excess DNA probe, wash with borate buffer (pH 8.5) containing sufficient 10 mM Lys, 0.01% SDS and 0.15 M NaCl for 5 minutes. Then, the DNA was washed with 60 mM sodium citrate buffer (2 × SSC, pH 7.0) containing 0.01% SDS and 0.3 M NaCl to remove excess DNA. This completed the DNA probe immobilization and surface treatment.

  Next, each step of the reaction will be described in order. As shown in FIG. 7 (a), mRNA 74 is captured by an 18-base poly-T sequence that is complementary to the poly-A sequence at the 3 ′ end of mRNA, as in the previous example. Next, the 1st cDNA strand 79 is synthesized to construct a cDNA library (FIG. 7 (b)). Next, anneal multiple target gene-specific sequence primers 80 corresponding to the gene you want to quantify to the 1st cDNA strand 79 (Figure 8 (c)), and synthesize 2nd cDNA strand 81 by complementary strand extension reaction. (FIG. 8 (d)). In other words, 2nd cDNA strand synthesis is performed under multiplex conditions. As a result, for a plurality of target genes, a double-stranded cDNA having a common amplification sequence (Forward / Reverse) at both ends and containing a cell recognition tag, a molecular recognition tag, and a gene-specific sequence is synthesized. . In this example, as an example, 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27, And 20 ± 5 bases 109 ± 8 bases upstream from the poly A tail of the target gene were used for the gene-specific sequence of OAZ1), and this resulted in an amplification product size of approximately 200 bases in the subsequent IVT amplification step. It is for unifying. By unifying the PCR product size, complicated size fraction purification steps (electrophoresis-> gel excision-> PCR product extraction / purification) can be eliminated, and parallel amplification from one molecule (emulsion PCR, etc.) Has an effect that can be used directly. Subsequently, T7 RNA polymerase is introduced into the pore to synthesize cRNA83 (FIG. 8 (e)). Through this process, about 1000 copies of cRNA are synthesized. Furthermore, in order to synthesize double-stranded DNA for emPCR, using the amplified cRNA as a template, multiple (~ 100 species) target gene-specific sequence primers with a common PCR amplification sequence (Reverse) added. Are hybridized (FIG. 9 (f)) to synthesize cDNA (FIG. 9 (g)). Furthermore, after cRNA is degraded using an enzyme in the same manner as in the previous example, the second strand 92 for emPCR is synthesized by synthesizing the 2nd strand 92 using the forward common primer (FIG. 9 (h)). . This amplification product has the same length and can be directly applied to emPCR and next-generation sequencers. Even if an amplification bias occurs between genes or molecules in this step, high-accuracy quantitative data can be obtained because the amplification bias can be corrected using the molecular recognition tag after acquiring next-generation sequencer data. The thing that can be done is the same as in the previous embodiment.

  Next, a specific series of steps will be described with the apparatus configuration shown in FIG. 10 having the unit structure of the nucleic acid extraction device shown in FIG. 10B is a cross-sectional view taken along the line A-A ′ in FIG. 10A, and FIG. 10C is a cross-sectional view corresponding to the cross-sectional view taken along the line B-B ′ in FIG. The procedure is the same as in Example 1 until cell 1 is introduced from cell inlet 308, the cell is trapped in cell trapping portion 2, and the 1st cDNA strand is synthesized. After synthesizing the first cDNA strand, reverse transcriptase is inactivated by maintaining at 85 ° C for 1.5 minutes, cooled to 4 ° C, and injected with 10 mL of 10 mM Tris Buffer (pH = 8.0) containing RNase and 0.1% Tween20 from inlet 305 Then, the RNA was decomposed by discharging from the outlets 306 and 307, and the same amount of alkali denaturing agent was flowed in the same manner to remove and wash the residue and decomposition products in the pores. Next, 690 μL of sterilized water, 100 μL of 10 x Ex TaqBuffer (TaKaRa Bio), 100 μL of 2.5 mM dNTP Mix, and 20 gene-specific sequence primers to be added with 10 μM of each PCR amplification consensus sequence (Reverse) Mix 100 μL and Ex Taq Hot start version (TaKaRa Bio) 10 μL is mixed, the solution filling upper reaction region 7 and lower reaction region 8 is discharged from outlets 306 and 307, and the above solution containing reverse transcriptase is immediately injected from inlet 305. did. Then, 95 ° C for 3 minutes → 44 ° C for 2 minutes → 72 ° C for 6 minutes. After annealing the primer-specific sequence using the 1st cDNA strand as a template, the complementary strand extension reaction is performed to obtain the 2nd cDNA strand. Synthesized.

  Subsequently, 10 mL of 10 mM Tris Buffer (pH = 8.0) containing 0.1% Tween 20 was injected from the inlet 305 and discharged from the outlets 306 and 307 to remove and wash the residue and decomposition products in the pores. Furthermore, 340μL of sterilized water, AmpliScribe 10 X Reaction Buffer (EPICENTRE) 100μL, 100mM dATP 90μL, 100mM dCTP 90μL, 100mM dGTP 90μL, 100mM dUTP 90μL, 100mM DTT, and AmpliScribe T7 Enzyme Solution (EPICENTRE) 100L The solution filling the upper reaction region 7 and the lower reaction region 8 was discharged from the outlets 306 and 307, and the solution containing reverse transcriptase was immediately injected from the inlet 305. Thereafter, the temperature of the solution was raised to 37 ° C. and maintained for 180 minutes to complete the reverse transcription reaction, and cRNA amplification was performed. As a result, target portions of 20 target genes are amplified, and the size of the cRNA amplification product is almost uniform at 200 ± 8 bases. Collect the cRNA amplification product solution accumulated in the solution inside and outside the pores of the membrane. In order to remove residual reagents such as enzymes contained in this solution, it is purified using PCR Purification Kit (QIAGEN) and suspended in 50 μL of sterile water. To this solution, mix 10 μL of 10 mM dNTP mix and 30 μL of 50 ng / μL random primer, heat at 94 ° C. for 10 seconds, lower the temperature to 30 ° C. at 0.2 ° C./second, heat at 30 ° C. for 5 minutes, and further increase to 4 ° C. To lower. Then, 20 μL of 5 × RT Buffer (Invitrogen), 5 μL of 0.1M DTT, 5 μL of RNase OUT, and 5 μL of SuperScript III are mixed, heated at 30 ° C. for 5 minutes, and the temperature is increased to 40 ° C. at 0.2 ° C./second. In order to remove residual reagents such as enzymes contained in this solution, it is purified using PCR Purification Kit (QIAGEN), applied to emPCR amplification, and then applied to next-generation sequencers of each company (Life Technologies, Illumina, Roche) And analyze.

(Example 3)
It is possible to identify the individuality / state of a cell by a nucleic acid extraction device that realizes gene analysis for each cell. On the other hand, in non-invasive microscope observation, it is possible to measure cell morphology and chemical composition while keeping cells alive. However, it is extremely difficult to distinguish the state of a cell from information only from a microscopic image because the individuality / state of the cell is various and unstable. In this example, a device and an apparatus configuration for combining identification of cell individuality by gene analysis for each cell and noninvasive imaging are shown. When microscopic observation is performed with cells captured using the device structure shown in FIG. 3 (a) or FIG. 7 (a), even if beads and pore sheets are made of a transparent material, Since the refractive index of the material is generally different from the refractive index of the solution, excitation light and illumination light are scattered, causing problems such as a decrease in resolution and an increase in background light level. In the present embodiment, an example in which a nucleic acid extraction device and an optical microscope are combined with the configuration of FIG. 2 as a typical example is shown.

  FIG. 11 shows the structure and apparatus of the nucleic acid extraction device in this example. FIG. 11B is a cross-sectional view taken along the line A-A ′ of FIG. 11A, and FIG. 11C is a cross-sectional view corresponding to the cross-sectional view taken along the line B-B ′ of FIG. In this example, the shape of the substrate 6 made of PDMS was devised so that a nucleic acid trapping portion was not arranged just below the cells but directly below, and a peripheral ring-shaped region was provided and the magnetic beads 12 were packed in this portion. By applying an electric field in the direction perpendicular to the substrate plane, nucleic acids such as mRNA are guided by the electrophoretic guide to the bead portion packed in a ring shape and captured by the DNA probe on the bead surface. The following process is the same as that in Example 1. The diameter of the cell trapping part prepared by PDMS was 16 μm, the diameter of the microscope window 1101 immediately below the cell trapping part was 25 μm, and the height was 15 μm. The pore array sheet 35 is also protected by a resist mask at the time of anodization so that pores are not formed in the portion corresponding to the portion immediately below the cell trapping portion. It is also possible to reduce the influence of scattering by the pore array sheet by making the thickness of the microscope window 1101 thicker than the depth of focus perpendicular to the device surface of the microscope optical system without performing anodization patterning. It is.

  In addition, as shown in FIG. 12, the nucleic acid trapping portion can be arranged laterally instead of around the cell. 12B is a cross-sectional view taken along the line A-A ′ in FIG. 12A, and FIG. 12C is a cross-sectional view corresponding to the cross-sectional view taken along the line B-B ′ in FIG. In this example, 1201 is a nucleic acid trapping unit. In this case as well, a DNA probe is immobilized on the beads and mRNA is captured. Other device configurations and sample adjustment methods are the same as in the first embodiment.

  In addition, in FIGS. 11 and 12, the region packed with beads is used as a nucleic acid trapping portion, but in the same manner as in Example 2 in which the pore array sheet is used as a nucleic acid trapping portion, the portion where pores are formed is limited. Needless to say, it is possible to produce a configuration similar to that shown in 11 and 12 without using beads.

  Using such a nucleic acid extraction device, before performing detailed gene expression analysis by crushing cells for gene expression analysis, the shape and fluorescence staining of genes and proteins in high-resolution cells are alive. Quantitative or Raman imaging can be performed, and these imaging data can be associated with gene expression analysis data. A system configuration for realizing this will be described below.

  First, in order to construct a nucleic acid extraction device in FIG. 13, cell samples placed on a planar device (cell array 1320, pore array sheet 1321, etc.) are measured with an optical microscope and gene expression analysis using the above device. The minimum system configuration for measuring cell dynamics in detail by matching the results with individual cell data is shown.

  1200 represents a nucleic acid extraction device and a cell sample placed on the device. 1201 is a flow system for performing mRNA extraction and nucleic acid amplification from cells represented by FIG. In this flow system, by processing cell-derived mRNA, it has a certain length in order to determine the sequence with the next-generation (large-scale) DNA sequencer 1205, and the end is processed before nucleic acid processing. An amplification product containing the tag sequence with information is obtained. Again, arrow 1211 indicates the movement of the amplification product. On the other hand, the cells on the device are observed by the optical microscope 1203 in a form in which the positions of the cells on the device are specified in advance. Here, a thin arrow indicates movement of information. The optical microscope 1203 includes a phase contrast microscope, a differential interference microscope, a fluorescence microscope, a laser-scanning confocal fluorescence microscope, a Raman microscope, a nonlinear Raman microscope (CARS microscope, SRS microscope, RIKE microscope), an IR microscope, and the like. As far as genetic information is concerned, there is little information obtained with these optical microscopes. However, basically, the measurement can be performed in a state where the cells are alive, the time-dependent change of the cells can be measured, and the response of the cells to the stimulus can be measured in real time. By using a device that stores position information on the device, detailed information related to gene expression can be associated with information including temporal changes by a microscope. In order to realize this, an information system 1206 that integrates sequence information 1212 from the next generation (large-scale) DNA sequencer, information 1213 of the optical microscope image, and position information 1214 corresponding to the tag sequence is provided in the system. There is a need. In the present invention, the minimum configuration of the system that integrates the cell measurement information described above is a system 1207 of a portion other than the next generation (large-scale) DNA sequencer 1205. Information input to the DNA sequencer and a sample (nucleic acid) (Amplification product) output.

  FIG. 13 shows an example of a system configuration when a fluorescence microscope is combined as an optical microscope. 1203 is a fluorescence microscope. In cell 1, GFP is expressed in a protein chamber (for example, p53) to be measured, or a fluorescent substance is introduced into a specific protein by immunostaining. The amount of protein expressed in individual cells is correlated with the gene expression data obtained by processing the sample in a nucleic acid extraction device after cell disruption and quantifying it by DNA sequencing. Can take. At this time, in order to recognize the cell, the nucleic acid is stained with DAPI and the cell nucleus is recognized, and the cell position is identified by a fluorescence microscope. Since the protein mass is measured by the expression of GFP, the change with time can be followed, but there are several types of proteins that can be measured simultaneously. On the other hand, gene expression analysis by sequencing can be about 100 at a time, and 1000 types of analysis are possible if a probe is prepared. Therefore, information on detailed gene expression control in the cell can be obtained for each individual cell. However, it is impossible to capture the change with time. However, if the data on what kind of protein expression is obtained in advance by combining the two, information about gene regulation should be estimated from only the protein expression data. Is possible. Information system 1206 executes such an association of fluorescence microscope data and gene expression data and estimation of information related to gene control.

  Next, details of the configuration of the fluorescence microscope 1203 will be described. 1300 is a light source, here a mercury lamp. 1301 is an excitation filter that determines the excitation wavelength, 1302 is a dichroic mirror, and 1303 is an emission filter that selects a light reception wavelength. When a plurality of types of phosphors are introduced into a cell and simultaneously measured, 1301, 1302, and 1303 are selected by the control 1304, and only light from a specific phosphor is measured. The fluorescence image of the cell is obtained by an objective lens 1305, an imaging lens 1306, and a CCD camera 1307. 1308 is a control computer for controlling these and acquiring image data.

Next, the control system of the flow system 1201 will be described. The control computer of the flow system is 1309, which controls the XY stage 1310 to move the microscope image. At this time, the position coordinates on the microscopic image calculated using the position coordinates on the pore array sheet, the array data of the cell recognition tag and the XY stage position coordinates can be made to correspond on the control computer. This control computer 1309 is a cell introduction control device 1311 that controls the introduction of cells into the flow cell system, a differentiation inducing agent that changes the state of the cell, a drug that wants to investigate the response of the cell, a lysate or sample for crushing the cell Reagent control device 1312 that controls the introduction of reagents for processing, cell culture conditions, temperature that controls the temperature cycle during PCR, CO ₂ concentration control device 1313, unnecessary reagents and upper reagents used for cells, medium replacement, etc. The discharge device 1314 and the lower reagent discharge device 1315 for discharging the prepared nucleic acid amplification product are appropriately controlled. The finally obtained nucleic acid amplification product is passed to the next generation (large-scale) DNA sequencing system 1205 for sequence analysis. At this time, emPCR and bridge amplifier for sequencing are assumed to be executed in this system. The positional information of the image and the cell recognition tag sequence information collated on the control computer are sent to the integrated information system 1206 to associate the protein amount obtained from the fluorescence image with the gene expression amount. Furthermore, the temporal change estimation of gene expression analysis data is executed with the same system. This makes it possible to measure the dynamics of the gene expression network.

  This fluorescent microscope is used not only for intracellular measurement, but also for immunofluorescent staining of substances secreted from cells, such as captured cytokines, which are guided into the pore array sheet and captured by antibodies, and measure the amount thereof. Also good. Of course, the gene expression level after disruption may be used in the same manner.

  FIG. 14 shows an example in which a differential interference microscope is combined instead of the fluorescence microscope. The differential interference microscope image only measures the shape without using a fluorescent reagent, but it is one of the measurement methods that has the least influence on cells when cells must be returned to the body, such as in regenerative medicine. When a correspondence can be made between the change in the cell shape obtained from this image and the change in the gene expression, the measurement system is capable of performing detailed cell classification with little cell damage.

  1401 is a light source, here a halogen lamp. 1402 is a polarizer, 1403 and 1404 are a Wollaston filter and a Wollaston prism, respectively. 1405 is a condenser lens, and 1406 is an objective lens.

  FIG. 15 shows an example in which a CARS microscope is used as the optical microscope. The CARS microscope, like the Raman microscope and IR microscope, can obtain a spectrum corresponding to the chemical species of the laser excitation part, and therefore can increase the amount of information on the cell state than the differential interference microscope. However, CARS is a non-linear process, and has a merit that the signal intensity is higher than that of the Raman signal, and a sufficient signal can be obtained with a relatively weak laser excitation intensity, so that damage to cells is small. By correlating such CARS image and gene expression analysis data, it becomes possible to determine the cell state in more detail.

  Reference numeral 1501 denotes a light source, which is a pulse laser (microchip laser) here. This is split into two by a beam splitter 1502, and one is introduced into a nonlinear fiber (photonic crystal fiber) 1503 to generate Stokes light. The other light is used as it is as pump light and probe light, and is condensed on the sample (in the cell) by the water immersion objective lens 1504 to generate anti-Stokes light. Only the anti-Stokes light is transmitted through the high-pass filter 1505, and the coherent anti-Stokes Raman spectrum is acquired through the spectroscope 1506 by the CCD camera 1507 for the spectroscope.

(Example 4)
In this embodiment, the cell trapping portion is not composed of an opening having the same size as the cell, but a substance that chemically captures the cell surface, that is, a substance that chemically binds to the cell surface substance. An example composed of fixed areas is shown. FIG. 16 shows an example in which the cell trapping unit is changed corresponding to Example 1 (FIG. 1). Beads to which proteins such as antibodies that bind to the cell surface are immobilized are arranged in a partial region on the beads used for nucleic acid trapping (capture). The antibody on the bead can add a function of capturing a specific type of cell. For example, a group of antibodies called CD (cluster of differentiation) antibodies includes an antibody group corresponding to the type of cell membrane protein centering on leukocytes. This antibody can be biotinylated, immobilized with streptavidin on the beads, and captured by using the inkjet printer technology on the cell trapping part 2 in Fig. 16 to capture cells with a specific CD classification antigen. It becomes. Of course, it is needless to say that the CD antibody is not directly immobilized on the beads, but may be immobilized on the beads via a biotin-modified secondary antibody. Further, antibodies other than CD antibodies may be immobilized on beads, or molecules that bind to receptors on cells may be immobilized. An example of such a molecule is fibronectin. Fibronectin is known to bind to integrins on cells. By fixing fibronectin on the beads, it becomes possible to capture adherent cells.

  Other examples of substances that chemically bind to cell surface substances include extracellular matrices such as collagen, laminin, elastin and the like.

  Next, an example is shown in which cell capture is chemically performed in a device configuration corresponding to Example 3 (FIG. 2). This is an example in which a substance for capturing cells is fixed to a part of a transparent region of a nucleic acid extraction device. As the capture substance, an antibody may be used as described above, or another substance may be used. FIG. 17 shows a configuration example of the nucleic acid extraction device. Cell capturing antibody 1702 is fixed to cell trapping section 2. The immobilization method is by immobilizing biotinylated antibody and streptavidin in this region. Only cells having the antigen 1703 corresponding to the antibody can be captured.

  Furthermore, an example corresponding to Example 2 (FIG. 7) is shown. As shown in Fig. 18, by immobilizing the antibody in the region 1801 for capturing cells on the pore array sheet 71, a cell trapping part capable of capturing cells having the antigen 1802 corresponding to the antibody is formed. doing. In order to fix the antibody at a specific position, about a few tens of pL of biotinylated antibody was injected using an inkjet printer head. Since the entire device surface is coated with streptavidin by the method shown in Example 3, the antibody is immobilized only in a specific region.

(Example 5)
In Examples 1 to 4, the second strand formation step (steps shown in FIGS. 2 (d)-(e) and 8 (c)-(d)) and the PCR amplification step (FIG. 2 (f) and FIG. 8). (e)) also shows an example performed in the apparatus as shown in FIG. 5, FIG. 10, FIG. 11 or FIG. However, the entire nucleic acid extraction device 1901 or the nucleic acid extraction device divided into multiple devices can be placed in a resin tube 1902 (commonly used 0.2 mL or 1.5 mL capacity tubes) or 96 or 384-well plates. Insertion as shown in FIG. 19 may be performed by mixing the reagent 1903 necessary for 2nd strand synthesis and PCR amplification into this tube. With this configuration, not only can the user freely change the conditions for these processes, but the cell recognition tag and molecular recognition tag can be changed from the 2nd strand synthesis in the tube. Can be inserted at the end opposite to the position shown in Example 1-4.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

  According to the present invention, quantification, sequencing, and molecular identification of biomolecules can be performed on a large number of cultured cells, a large number of immune cells, (in blood) cancer cells, and the like. It is possible to measure whether or not a certain number exists in the living body. This makes it possible to measure early diagnosis of cancer and the heterogeneity of iPS cells.

1: Cell
2: Cell trapping section
3: Flow path
4: Nucleic acid trapping section
5: Flow path
6: Flat substrate
7: Upper reaction area
8: Lower reaction area

Claims (13)

A cell trapping part for fixing one cell each;
A flow path through which the nucleic acid extract for extracting nucleic acid from the cells flows from the top to the bottom through the cell trapping section;
A nucleic acid trapping section that is connected to the cell trapping section via the flow path and is arranged below the cell trapping section and fixes the extracted nucleic acid;
A flow path for discharging the solution after nucleic acid extraction from the nucleic acid trapping part to the opposite side of the cell trapping part,
The cell trapping section, the two flow paths, and the nucleic acid trapping section form a pair in the vertical direction, and a plurality of the pairs are arranged in the plane direction,
The nucleic acid extraction device, wherein the nucleic acid trapping section includes a DNA probe for nucleic acid trap, and the DNA probe includes a cell recognition tag sequence.   The nucleic acid extraction device according to claim 1, wherein the nucleic acid trapping unit includes beads to which a DNA probe for the nucleic acid trap is fixed.   The nucleic acid extraction device according to claim 1, wherein the nucleic acid trapping unit includes a porous membrane in which a DNA probe for the nucleic acid trap is fixed in a pore.   The nucleic acid extraction device according to any one of claims 1 to 3, wherein a substance that chemically binds to a substance on a cell surface is fixed to the cell trapping portion.   4. The nucleic acid extraction device according to claim 2, wherein a part of the DNA probe for the nucleic acid trap includes a sequence for specifying a position on the chip.   4. The nucleic acid extraction device according to claim 2, wherein a part of the DNA probe for trapping the nucleic acid contains a different sequence for each trapped nucleic acid molecule.   The nucleic acid extraction device according to claim 6, further comprising means for introducing an enzyme for reverse transcription of RNA trapped in the nucleic acid trapping section.   The nucleic acid extraction device according to any one of claims 1 to 7, wherein a portion immediately below the cell trapping portion is made of an optically transparent material.   The nucleic acid extraction device according to claim 1, wherein a nucleic acid trapping portion is provided immediately below the cell trapping portion.   The nucleic acid extraction device according to any one of claims 1 to 8, wherein a nucleic acid trapping portion is provided in a region other than immediately below the cell trapping portion.   11. A nucleic acid processing apparatus comprising: the nucleic acid extraction device according to claim 1; and means for introducing a reagent for constructing a cDNA library.   11. A nucleic acid processing apparatus comprising: the nucleic acid extraction device according to claim 1; a reagent for constructing a cDNA library; and a means for introducing a reagent for nucleic acid amplification.   A nucleic acid extraction device according to any one of claims 1 to 10, and a microscope unit for observing cells trapped in the cell trapping unit with a differential interference microscope, a phase contrast microscope, a Raman microscope, or a coherent Raman microscope. A nucleic acid processing apparatus characterized by the above.

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