WO2010059006A2

WO2010059006A2 - MEMS CHIP FOR CHROMATIN IMMUNOPRECIPITATION (ChIP) AND CHIP METHOD USING SAME

Info

Publication number: WO2010059006A2
Application number: PCT/KR2009/006903
Authority: WO
Inventors: 오현직; 윤태중; 이상훈; 이보연; 황을문
Original assignee: 주식회사 서린바이오사이언스
Priority date: 2008-11-24
Filing date: 2009-11-23
Publication date: 2010-05-27
Also published as: WO2010059006A3; KR20100058057A

Abstract

The present invention relates to an MEMS chip for chromatin immunoprecipitation (ChIP) which replaces conventional EP tubes used for ChIP experiments and which performs ChIP experiments, and to a ChIP method using same. The MEMS chip for ChIP comprises: an injection port through which a sample including a protein that is bound with DNA and antibody is introduced; a chamber communicated with the injection port, and having a plurality of beads positioned on the inner surface thereof, wherein a specific protein that binds with the antibody is fixed to the beads; and a discharge port communicated with the chamber, and formed on the other side of the injection port so as to discharge the introduced sample. Accordingly, the MEMS chip of ChIP can replace the conventional ChIP technology that uses EP tubes, and thus can carry out experiments with a small quantity of sample, with less amount of experimentation time, and have less fluctuation in experimental results, and also can carry out ChIP experiments even at room temperature.

Description

Concentrated microchip for HS IP and how to execute HS IP using the same

The present invention relates to a concentrated microchip for ChIP and a method of performing ChIP using the same. More specifically, the concentrated microchip for ChIP to perform a ChIP experiment by replacing the EP tube used in the existing ChIP experiment and a method of performing ChIP using the same. It is about.

Functional genomics is being conducted as a research stage after the Human Genome Project. Functional genomics is the study of gene function and regulation in the cell or signaling system.

To study this field of functional genomics, it is important to detect the binding of the transcription factor and DNA promoter moiety that play the most important role in gene regulation. DNA chip technology, which is mainly used to study functional genomics, has the advantage of comparing the whole gene expression at the same time, but it has the disadvantage that the binding of transcription factor and promoter cannot be directly analyzed because only DNA and DNA binding are examined.

In order to solve this problem, recently, in the field of cell biology, a method of combining a transcription factor and a promoter using CHIP (CHROMATIN IMMUNOPRECIPITATION) technology and applying it to a DNA chip to determine how gene expression is regulated by a specific transcription factor is used. have.

The purpose of the ChIP technique is to regulate the expression of transcription factors that regulate gene expression by binding to a promoter, which is a specific sequence.

In detail, such ChIP technology, 1) E.P. Inserting the cells into the tube and treating formaldehyde to bind the DNA and the DNA binding protein, 2) inserting the cell lysate into the cells and cutting the DNA into fragments of 200 to 1 kb using a sonicator, 3 ) Put the ChIP binding solution in the lysate containing the DNA fragment and remove the protein and DNA that binds to the beads nonspecifically by adding agarose beads or Sepharose beads, 4) the specific protein ( Example: Add an antibody of acetylated histone H3) and react overnight (DNA and DNA-binding protein, the antibody of the protein binds), 5) Add agarose or Sepharose beads fixed with protein A or G Reaction step (DNA and DNA binding protein, antibody of the protein, agarose or Sepharose beads all bound), 6) high salt, low salt, lithium chloride immune complex washing buffer and TE buffer Washing the beads (washing DNA, protein, etc. not involved in the binding of step 5), 7) separating the washed beads from the DNA-protein-antibody from the beads, 8) Dissociating DNA and protein by treating sodium chloride and protease (K), 9) purely separating DNA using phenol / chloroform, and 10) PCR using specific gene primers. The PCR procedure was performed using the ChIP technique, or, in place of the step 7), 7-1) processing and boiling the SDS sample buffer in the washed beads, followed by Western blot.

In the end, the conventional E.P. ChIP technology using the tube has the advantage of easy identification of transcription factors, etc., but it requires a large amount of sample, the experiment time is very long and complex, the fluctuation of the experimental results is severe, and should be performed at about -4 ℃ temperature Experimental conditions such as this was very difficult.

The present invention has been made to solve the above problems, the object of the present invention is to provide a conventional E.P. It is possible to provide a concentrated microchip for ChIP that can perform ChIP experiments at room temperature while reducing experiment time and reducing the experiment time.

Another object of the present invention is to provide a concentrated microchip for ChIP capable of detecting two or more types of target DNA or proteins at a time in the ChIP technique.

Still another object of the present invention is to provide a method of performing ChIP using ChIP-enriched microchips, which simplify the experimental conditions and the like by using the ChIP-enriched microchips of the present invention.

The present invention is an injection port to which a sample containing a protein combined with DNA and antibodies to achieve the above technical problem; A chamber in communication with the inlet and having a plurality of beads in which a specific protein for binding the antibody is fixed to a surface thereof; And a DNA enrichment MEMs chip for ChIP (Chromatin immunoprecipitation), which is in communication with the chamber and includes an outlet formed at the other side of the injection port to discharge the injected sample.

The specific protein is preferably protein A or protein G, the beads may use agarose beads or Sepharose beads, the average diameter of the beads may be used 45 ~ 165 ㎛. In addition, the beads may be provided with 6μg / 30μl ~ 12μg / 30μl.

The fine chip is preferably installed on the outer periphery of the chamber and the sample is discharged, and may further include a discharge path in communication with the discharge port, more preferably, is installed between the discharge path and the chamber and the bead It is also possible to further include a discharge hole having a smaller passage.

Furthermore, a cover for sealing the chamber may be combined, in which case the cover may use glass or transparent plastic.

In addition, the multi-enriched microchip for ChIP according to another aspect of the present invention, the injection port is a sample containing two kinds of proteins, each of which is coupled to different DNA and antibody; A first chamber in communication with the inlet and having a plurality of beads fixed therein, the first specific protein being bound to the antibody; A second chamber connected to the first chamber by a partition wall and having a plurality of beads coated therein with a second specific protein binding to the antibody; A first discharge passage connected to a portion of an outer circumference of the second chamber so that the sample introduced into the first chamber is supplied to the second chamber; A second discharge passage connected to an outer circumference of the second chamber while being spaced apart from the first discharge passage; And an outlet for discharging the sample introduced into the second chamber.

The partition wall preferably further includes a discharge hole provided between the first discharge path and the first chamber and the second discharge path and the second chamber and having a passage smaller than the bead, wherein the second chamber includes a sample. An auxiliary inlet for supplying the second chamber may be further provided.

On the other hand, the first protein and the second protein is preferably a different kind of protein because it can bind to different antibodies preferably.

Further, the first chamber and the second chamber may be detachable from each other.

Further, preferably, a partition wall having a discharge hole formed between the second chamber and the discharge port may be further provided so that the sample introduced into the second chamber may be discharged to the discharge port.

ChIP performing method using a concentrated microchip for ChIP according to another aspect of the present invention, 1) adding formaldehyde to the cell to combine DNA and DNA binding protein, 2) destroying the cell and chopped the DNA Cutting, 3) preparing a sample by injecting an antibody binding to the DNA binding protein, 4) injecting the sample into the inlet of the concentrated microchip for ChIP of the present invention, and 5) Washing the beads therein, and 6) treating the washed beads with an elution buffer to elute the DNA.

In this case, the DNA preferably refers to all DNA that binds to Acetylated Histone H3 such as GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and in this case, the sample may preferably be added to 600 ~ 1000ul, more preferably 100ul of cell sonication sample (2.5X10 ⁶ cells) and 900ul of IP dilution buffer may be added to step 4).

In this case, the sample may preferably be added @ ~ @ μl.

Steps 4) to 6) may be preferably performed at 15 to 30 ° C., and the sample may be added to the inlet for 5 to 30 minutes in step 4).

Step 5) is preferably a) 0.5 ml of low salt immune complex wash buffer b) 0.5 ml of high salt lmmune complex wash buffer, c) lithium chloride immune complex 0.5 ml of washing buffer (LiCl immune complex wash buffer) and d) 0.5 ml of TE buffer; washed sequentially, each washing time is 30 seconds to 2 minutes and the d) step may be performed twice.

After step 6), preferably, Western blot or PCR may be performed.

In the present invention, when performing the ChIP experiment through the enriched microchip for ChIP of the present invention, DNA and protein can be detected as well as the number of beads used even when a small amount of sample is added as compared to using a conventional EP tube. It can be reduced to less than half, and the average of four days in the existing ChIP experiments can be reduced by up to 50% because the time of use of the chip of the present invention is reduced to about two days. In addition, it is possible to reduce the amount of expensive antibodies, reagents, samples, etc., thereby reducing the cost of experiments, and can easily perform the ChIP experiments using quantified reagents and materials, thereby ensuring consistency of experimental results. Furthermore, it is possible to produce a large amount by soft lithography and the like, and thus the manufacturing cost is low.

1 is a plan view of a concentrated microchip for ChIP according to an embodiment of the present invention.

2 is a plan view of a concentrated microchip for ChIP according to an embodiment of the present invention.

3 is a plan view of a concentrated microchip for ChIP according to an embodiment of the present invention.

Figure 4 is a perspective view of the concentrated microchips for ChIP without the cover according to an embodiment of the present invention.

5 is a perspective view of a concentrated microchip for ChIP in the state that the cover is attached according to an embodiment of the present invention.

6 is a cross-sectional view of a concentrated microchip for ChIP according to an embodiment of the present invention.

* Explanation of symbols for main parts of the drawings

2, 2 ', 12: injection hole 3: chamber 3': first chamber

4, 11: bead 5, 16:

reservoir

6, 13 outlet

7: discharge path 8: bulkhead 10: second chamber

14 second discharge path 15 second partition 30 glass

32: discharge hole

A first embodiment of the DNA enrichment MEMs chip (ChIP) for Chromatin immunoprecipitation (ChIP) is an injection hole into which a sample containing a protein coupled with DNA and an antibody; A chamber in communication with the inlet and having a plurality of beads in which a specific protein for binding the antibody is fixed to a surface thereof; And a discharge port communicating with the chamber and formed at the other side of the injection hole so that the injected sample is discharged.

The second embodiment includes an injection port into which a sample containing two kinds of proteins to which different DNA and antibodies are respectively bound is introduced; A first chamber in communication with the inlet and having a plurality of beads fixed therein, the first specific protein being bound to the antibody; A second chamber connected to the first chamber by a partition wall and having a plurality of beads coated therein with a second specific protein binding to the antibody; A first discharge passage connected to a portion of an outer circumference of the second chamber so that the sample introduced into the first chamber is supplied to the second chamber; A second discharge passage connected to an outer circumference of the second chamber while being spaced apart from the first discharge passage; And an outlet for discharging the sample introduced into the second chamber.

Hereinafter, with reference to the accompanying drawings the present invention will be described in more detail.

Conventional E.P. ChIP technology using tubes has the advantage of easy identification of transcription factors, but requires a large amount of sample, the experiment time is very long and complicated, the fluctuation of the experimental results is severe, and should be performed at about -4 ℃ temperature, etc. The experimental condition of was very difficult.

Therefore, the enrichment microchips (enrichment MEMs chip) for ChIP according to an embodiment of the present invention is an injection hole into which a sample containing a protein coupled with DNA and antibodies; A chamber in communication with the inlet and having a plurality of beads in which a specific protein for binding the antibody is fixed to a surface thereof; And a discharge port communicating with the chamber and formed at the other side of the injection hole so that the injected sample is discharged. The problem of the tube was overcome.

Specifically, FIGS. 1 and 2 are plan views of the enriched microchips for ChIP according to the first embodiment of the present invention. More specifically, the present invention utilizes semiconductor processing techniques such as etching to form a structural shape on a silicon wafer, thereby providing PDMS. It can be manufactured by soft lithography method to form channels, chambers, barrier ribs, etc. of desired structure in the poured PDMS.

The injection port (2) into which the sample containing the protein bound to the DNA and the antibody is processed and communicated with the injection port (2), and a plurality of beads in which a specific protein to bind the antibody is fixed to the surface ( The chamber 3, in which 4) is located, and the chamber 3 are in communication with each other, and an outlet 6 formed on the other side of the injection port 2 is formed to discharge the injected sample.

The injection hole 2 may be shaped to protrude outward as shown in FIG. 1, and may be shaped to be recessed inwardly as shown in FIG. 2. In addition, the sample injected into the inlet comprises a DNA-protein-antibody complex generated by binding a target DNA and a protein, then crushing the same, and adding an antibody capable of binding to the protein. Antibodies used in conventional ChIP experiments can be used.

The chamber 3 in communication with the inlet 2 has a plurality of beads 4 fixed therein (coated) with a protein capable of binding to a specific antibody therein. The material of the beads 4 may be agarose or sepharose, and protein A or protein G capable of binding to a target antibody (an antibody of DNA-protein-antibody complex) in the sample on its surface is a conventional method. Is applied. The diameter of the beads 4 may be used 45 ~ 165 ㎛. In addition, the beads 4 may be provided with 6㎛ / 30μl ~ 12㎍ / 30μL and the number of beads (4) is only about half the number of beads used in the conventional ET tube reaction time This can drastically reduce.

On the other hand, the microchip of the present invention is preferably installed on the outer periphery of the chamber 3 may further include a discharge path (7) for discharging the sample passed through the bead (4) and the discharge path (7) is an outlet In communication with (6), the sample may be discharged. In addition, a discharge hole 32 is formed between the chamber 4 and the discharge path 7 so that the sample can escape, but the beads cannot escape, and the discharge hole 32 is discharged from the chamber 3. It can be formed by installing a partition 8 of appropriate height between the furnaces 7. Furthermore, a cover 30 for sealing the chamber 3 may be combined, in which case the cover may be glass or transparent plastic.

On the other hand, the partition 8 and the discharge hole 32 is also located between the chamber 3 and the reservoir 5, through which the sample passing through the bead 4 is collected in the reservoir 5, the outlet 6 It can be discharged to the outside through. Also, in the present invention, the beads 4 are injected through the inlet 2 or the beads 30 are directly placed in the chamber 3 before the cover 30 is coupled to the chamber 3, and then the cover 30 is coupled. It can be injected by letting it.

In addition, a fine pump or a fine syringe pump (not shown) may be further provided around the outlet 6 to facilitate the passage of the sample.

Next, the operation of the concentrated microchip for ChIP according to the embodiment of the present invention described above will be described. First, a sample containing a small amount of DNA introduced into the inlet 2 and a protein bound to the antibody reaches the chamber 3. Since the chamber 3 is filled with a plurality of beads 4, the sample flows on the surface of the beads 4. At this time, the fluid pressure by the sample is applied to the surface of the bead 4, since the bead 4 is filled in the chamber 3, a phenomenon in which the sample is subjected to a large pressure in the outflow possible direction occurs. At this time, in the present invention, although the bead 4 is pushed forward and both sides in the direction in which the sample flows out, the bead 4 is located between the chamber 3 and the discharge path 7 as shown in FIG. Since the discharge through the discharge hole 32 is a space between the partition wall 8 and the cover 6 and the diameter of the discharge hole 32 is smaller than the diameter of the bead 4, d2 bead (4) Is prevented from leaving the chamber (3). Therefore, the sample is able to go to the discharge passage 7 and the reservoir 5 through the discharge hole 32 formed in the partition 8 through the pores of the beads 4. Accordingly, the sample collected through the reservoir 6 is discharged to the outlet 6. Also, in this process, the beads 4 are distributed to the partition 8 on the outer circumferential side of the chamber 3 and the partition 8 between the chamber and the discharge port 6, so that the beads 4 are pushed to one side and excessive pressure is applied. It is to be prevented from being deformed, damaged or difficult to discharge due to the compactness of the beads 4, so that stable and effective reaction induction is possible. In addition, in this process, the present invention is very small as the bead 4 is 45㎛ ~ 165㎛ or less, the surface area of the beads 4 is very large. In the present invention, a protein A or protein G, which can selectively bind to the target antibody, is conjugated to a sufficiently large surface area of such beads 4.

Therefore, all of the specific antibodies of the sample which come into contact with the surface of the bead 4 and then are discharged by the above-described process will be able to bind to the protein A or G applied to the surface of the bead 4, and thus the sample. Certain proteins can be quickly captured, immobilized and then released while effectively reacting with the antibody. Thereafter, the DNA-protein-antibody complex bound to the beads 4 may be washed with water and dissociated appropriately to detect a desired DNA or protein.

The present invention is a microchip having a very small size of the internal structure, so that a very small amount of the sample passes continuously, it is possible to efficiently obtain a sufficient reaction even if there are very few samples that pass through and react. In addition, in the present invention, the height of the partition wall 8 may be adjusted according to the diameter of the beads 4, and the number of beads 4 placed in the chamber 3 may also be adjusted. In addition, the present invention can repeatedly mass-produce a large number of concentrated microchips for ChIP by etching a plurality of structures on the silicon wafer.

3 is a plan view of a multi-enriched microchip for ChIP according to a second embodiment of the present invention. The same parts as those of the first embodiment in the configuration of the second embodiment will be replaced with the above description and described mainly on the differences.

The multi-enriched microchip for ChIP according to the second embodiment of the present invention is an injection hole into which a sample containing two kinds of proteins in which different DNA and the first second antibody are respectively coupled, is in communication with the injection hole, and Is connected to the first chamber (3 '), the first chamber (3') and the partition wall (8) in which a plurality of beads (4) in which the first specific protein binding to the antibody is fixed is located, A second chamber 10 in which a plurality of beads 11 coated with a second specific protein that binds an antibody are placed, and the sample introduced into the first chamber 3 ′ is supplied to the second chamber 10. The first chamber 7 connected to the outer circumferential portion of the first chamber 3 ′ and a portion of the outer circumferential portion of the second chamber 10, and the second chamber 10 spaced apart from the first discharge passage 7. And a second discharge passage 14 connected to an outer circumference of the) and a discharge port 13 for discharging the sample introduced into the second chamber 10.

In other words, the feature of the second embodiment of the present invention is that two chambers 3 ', 10 are arranged in series, and the sample passing through the first chamber 3' passes through the second chamber 10 again. In one experiment, two types of DNA or proteins are detected.

The injected sample passes through the beads 4 in the first chamber 3 ', and the antibody bound to the target DNA or protein binds to the protein applied to the beads 4. Subsequently, the remaining sample flows into the second chamber 10 through the first discharge path 7 in communication with the outer circumferential portion of the first chamber 3 ′ and a portion of the outer circumferential portion of the second chamber 10, or the first chamber It flows into the 2nd chamber 10 through the discharge hole 32 located in the partition 8 of the lower end of 3 '.

The sample introduced into the second chamber 10 through the first chamber 3 'passes through the bead 11 in the second chamber 10, at which time the bead 11 in the second chamber 10 Its size and material are the same as the beads 4 in the first chamber 3 '. However, the type of protein applied on the bead 11 is different from the beads 4 in the first chamber 3 '(coating a protein capable of binding another antibody) and the applied protein is first By selecting a protein that binds to a different type of antibody than the antibody reacted in the chamber 3 ', as a result, other types of DNA or protein that were not detected in the first chamber 3' can be detected.

Then, the remaining amount of the sample passing through the bead 11 in the second chamber 10 is collected in the reservoir 16 through the second discharge path 14 in communication with the outer circumferential portion of the second chamber 10, and then the discharge port. After being discharged to 13 or through a discharge hole 32 located in the partition 8 of the lower end of the second chamber 10, the reservoir 16 is collected and discharged to the discharge passage 13.

Meanwhile, the second chamber 10 may further include an auxiliary inlet 12 for directly supplying the sample and the beads 11 to the second chamber 10 as needed, and further, each chamber 3 '. , The first chamber 3 'and the second chamber 10 may be formed to be easily attached to and detached from each other (not shown) in order to facilitate washing and dissociation. As the detachable means, all means used for detachable normal chamber may be used. Specifically, a magnetic material, a fastening part (such as a bolt), an interference fit, a bellows tape, and the like may be used, but are not limited thereto.

A third embodiment of the present invention is a method of performing ChIP using a concentrated microchip for ChIP, comprising: 1) adding formaldehyde to a cell to bind DNA and a DNA binding protein, and 2) destroying the cell and slicing the DNA. Cutting, 3) preparing a sample by injecting an antibody binding to the DNA binding protein, 4) injecting the sample into the inlet of the concentrated microchip for ChIP of the present invention, and 5) Washing the beads therein, and 6) treating the washed beads with an elution buffer to elute the DNA.

In detail, steps 1) and 2) are the same as those of the conventional ChIP method. That is, the target DNA or protein, preferably a transcription factor, or the like is combined with the addition of formaldehyde, and then the cells are destroyed and the DNA is crushed. At this time, the DNA portion bound to the protein remains uncut.

Thereafter, in step 3), an antibody capable of specifically binding to the protein is added to bind to the protein, thereby preparing a sample including the same. This makes it possible to detect DNA and protein even when using fewer samples than when using conventional EP tubes, and to reduce the number of beads used to less than half. If the chip of the present invention is used, the test time can be reduced by up to 50% since it is shortened by about 2 days. In addition, it is possible to reduce the amount of expensive antibodies, reagents, samples, etc., thereby reducing the cost of experiments, and can easily perform the ChIP experiments using quantified reagents and materials, thereby ensuring consistency of experimental results. Furthermore, it is possible to produce a large amount by soft lithography and the like, and thus the manufacturing cost is low.

At this time, the sample may preferably be added to the cell sonication sample 100ul (2.5X10 ⁶ cells) and 500 ~ 1,000μl IP dilution buffer.

Thereafter, as the step 4), the sample is injected into the inlet of the concentrated microchip for ChIP of the present invention, in which case the sample may be introduced into the inlet for 5 to 30 minutes, but is not limited thereto. The time can be adjusted accordingly.

After injecting the sample, if the remaining amount of the sample exits through the outlet provided in the concentrated microchips for ChIP, as in step 5) to wash the beads inside the microchips. At this time, the washing method is preferably a) 0.5 ml of low salt immune complex wash buffer b) 0.5 ml of high salt lmmune complex wash buffer, c) washing of lithium chloride immune complex 0.5 ml of buffer (LiCl immune complex wash buffer) and d) 0.5 ml of TE buffer; washed sequentially, each washing time is 30 seconds to 2 minutes and the d) step may be performed twice. Among them, the low salt immune complex washing buffer and the high salt immune complex washing buffer are buffers used in a conventional ChIP experiment. Specifically, 1) the low salt immune complex washing buffer is 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris. -A washing buffer consisting of HCl, pH 8.1, 150 mM NaCl can be used. 2) The high salt immune complex washing buffer may be a washing buffer consisting of 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl, and 3) Lithium chloride immune complex washing buffer Is a washing buffer consisting of 0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1, 4) TE buffer is 10mM Tris-HCl, pH 8.0, A buffer consisting of 1 mM EDTA can be used.

On the other hand, in the general ChIP washing, 500μl of the washing solution is added to each step, followed by centrifugation after stirring for 3 ~ 5 minutes. The ChIP-enriched microchip of the present invention is added to 250 μl of the washing solution step by step in step 5), and the washing is completed within 1 minute.

In addition, in the conventional ChIP method, the ChIP-enriched microchips, which are complicated in the experimental procedures such as stirring and centrifugation, are simply passed through the solution, and the washing step is completed.

Furthermore, in the process of extracting the histone complexes attached to the beads and extracting the pure DNA after the washing step, the conventional ChIP method generates a lot of impurities (other precipitates other than proteins), while the ChIP-enriched microchips show less impurities. This is superior to the usual ChIP method.

Thereafter, in step 6), the washed beads are treated with an elution buffer to elute the DNA to detect desired DNA and protein. At this time, the type of eluent used may be an eluent used in a conventional ChIP experiment. At this time, step 6) is performed in the state that beads are contained in the ChIP enrichment chip. Specifically, after washing the beads in the channel, the histone complex attached to the beads is eluted to obtain a final solution sample.

After step 6), preferably, Western blot or PCR may be performed to determine the sequence and characteristics of the detected DNA or protein in detail.

Hereinafter, the present invention will be described in more detail with reference to Examples. This example is intended to explain the present invention more specifically through the most preferred embodiments, and the scope of the present invention is not limited to these examples.

Example 1 Preparation of Sample

Cells (293 cells, 293F cells, HeLa cells) were treated with formaldehyde at a concentration of 1% to cross-link DNA with histones and DNA binding proteins. Thereafter, the cells were dispensed by 5 × 10 ⁶ cells, and 100 μl (1 mM) of lysis buffer (SDS lysis buffer: 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH8.1) was added and the cells were disrupted.

Thereafter, 2.5 μl of an antibody (Anti-acetyl-Histon H3 (Lys18)) capable of binding to a DNA binding protein was added to the sample, followed by reaction for 3 hours.

1 μg / 30 μl to 12 μg / 60 μl of the size of 45 μm to 165 μm treated with protein A was added to the chamber of the DNA-enriched microchip for ChIP shown in FIG. 1 and incubated for 30 minutes. Thereafter, the sample prepared in Example 1 was flowed into the inlet of the microchip for 15 minutes.

After all of the remaining sample was discharged through the outlet, first, the protein A beads / antibody / DNA (chromatin) complex inside the DEMc chip was washed while flowing for 1 minute in the following steps 1 to 4.

(1) low salt immune complex washing buffer (0.5 ml, one wash, at room temp, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), 2) high salt immune complex Washing buffer (0.5ml, one wash, at room temp, 0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), 3) Lithium chloride immune complex washing buffer (0.5ml , one wash, at room temp, 0.25M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.14), 4) TE buffer (0.5ml, two washes, at room temp , 10 mM Tris-HCl, pH 8.0, 1 mM EDTA)

The target DNA (chromatin) was reacted by flowing 250ul elution buffer (1% SDS, 0.1M NaHCO ₃ ) to the protein A beads / antibody / DNA (chromatin) complex inside the washed DEMc chip for 1 minute. Eluted.

Example 3 Perform Western Blot

(1) First, using 15% SDS polyacrylamide gel, it was operated for 100V, 1 hour. After washing, the 2X SDS sample buffer was added to the sample prepared through Example 2 or the whole cell extract (WCE), heated for 10 minutes, and then spin-down to use only the supernatant. The washed sample is loaded with 20 µl and WCE is loaded with 10 µl. Then, 50 μl of 1X sample buffer was added to 0.5 μl of IgG, and heated for 10 minutes to load 10 μl.

The transfer set was then set, filled with transfer buffer (48mM Tris base, 39mM Glycine, 20% MeOH, 0.0375% SDS), and the protein in the gel was transferred to PVDF membrane at 100V for 1 hour. Then, the membrane was placed in 5% skim milk-TBST and reacted for 1 hour.

Then, incubated for 2 hours with 1 'antibody (Ab as used previously) at 1: 3000 in 5% skim milk-TBST (10 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.05% Tween). Rinse 3 times with 15min each. Then, 1 'antibody b (Anti-rabbit IgG) was added at 1: 3000 to 5% skim milk-TBST, and then incubated for 1 hour, and washed 3 times with TBST. Thereafter, TMB solution was added to Membrane and cultured until color development was detected.

Figure 7 is an electrophoresis picture showing the results of the Western blot. Specifically, from the left side of the photograph, (1) a normal ChIP western blot positive sample, (2) a normal ChIP western blot negative sample, (3) a ChIP concentrated microchip western blot positive sample, and (4) a ChIP concentrated microchip western blot negative Samples were followed by (5) Whole cell extract and (6) Anti-acetyl H3 histone IgG.

As a result, the confirmation of the target protein Acetylated-histone H3 (17.3KDa) can determine whether the desired histone + DNA complex is included. As a result of the above experiment, the histone band did not appear in the sample without the antibody, and the sample with the antibody showed the desired band in both the conventional ChIP method and the ChIP-enriched microchip. This can be confirmed by western blot whether the protein A beads and histone antibodies have correctly captured the desired DNA + histone complex. It was able to capture exactly the same amount as the Tube method.

Example 4 PCR

(1) Example 2 10 μl of 5M NaCl was added to a sample and heated at 65 ° C. for 4 hours to release Histone-DNA crosslink.

(2) 5 μl of 0.5 M EDTA, 10 μl of 1M Tris-HCl pH 6.5, and 1 μl of 10 mg / ml Proteinase K were reacted at 45 ° C. for 1 hour.

(3) DNA was extracted by phenol / chloroform extraction and ethanol precipitation.

(4) A primer of the GAPDH gene of the following sequence ( Product Size: 166 bp) was prepared in advance and PCR was performed.

GAPDH For: 5´-TACTAGCGGTTTTACGGGCG-3´

GAPDH Rev: 5´-TCGAACAGGAGGAGCAGAGAGCGA-3´

(5) 2.5% agarose gel electrophoresis was performed to confirm that GAPDH was correct.

8 is an electrophoresis result carried out through the above-described method. In FIG. 8, lane 1 is a DNA size marker 50Kb ladder, lane 2 is a conventional ChIP PCR negative sample (using an EP tube), lane 3 is a conventional ChIP PCR positive sample using an EP tube, and lane 4 is a ChIP of the present invention. Concentrated microchip negative sample, 5 lanes are 5. ChIP concentrated microchip positive sample.

The target gene described above used the intracellular GAPDH gene and a band was formed at the size of 166 bp. A typical ChIP negative sample is a sample without an antibody, and a typical ChIP positive sample is a sample with an antibody.

The electrophoresis resulted in a GAPDH gene band of 166bp size in the conventional ChIP positive sample and the ChIP enriched microchip positive sample. Thus, the same experimental results as the conventional ChIP chip method can be obtained using the ChIP enriched microchip. .

The present invention is a conventional E.P. It is a very useful invention for the bio industry because it can replace the ChIP technology using a tube and can perform experiments with a small amount of sample, reduce the experiment time, and perform the ChIP experiment at room temperature with little variation of the experimental results.

Claims

An injection port into which a sample including DNA and a protein bound to the antibody is injected; a chamber in communication with the injection port, and having a plurality of beads in which a specific protein to bind the antibody is fixed to a surface thereof; And a discharge port communicating with the chamber and formed at the other side of the injection hole so that the injected sample is discharged. 5. The concentrated microchip for ChIP (Chromatin immunoprecipitation) comprising a.
The concentrated microchip for ChIP according to claim 1, wherein the specific protein is Protein A or Protein G.
The concentrated microchip for ChIP according to claim 1, wherein the beads are agarose beads or sepharose beads.
The concentrated microchip for ChIP according to claim 1, wherein the beads have a diameter of 45 µm to 165 µm.
The concentrated microchip of ChIP according to claim 1, further comprising a discharge path installed at an outer circumference of the chamber to discharge the sample and communicate with the discharge port.
6. The concentrated microchip of ChIP according to claim 5, further comprising a discharge hole installed between the discharge path and the chamber and having a passage smaller than the bead.
The concentrated microchip for ChIP according to claim 1, wherein a cover for sealing the chamber is coupled.
8. The concentrated microchip as claimed in claim 7, wherein the cover is glass or transparent plastic.
The concentrated microchip for ChIP according to claim 1, wherein the beads are 6 µg / 30 µl to 12 µg / 30 µl.
An injection port into which a sample including two proteins in which different DNAs and antibodies are respectively bound is injected; a first port in which a plurality of beads in communication with the injection port are fixed, and a first specific protein to which the antibody binds is fixed is located A second chamber connected to the first chamber by a partition wall and having a plurality of beads coated with a second specific protein to bind the antibody therein; the sample introduced into the first chamber is transferred to the second chamber A first discharge path connected to a portion of an outer circumference of the second chamber to be supplied; a second discharge path connected to an outer circumference of the second chamber while being spaced apart from the first discharge path; And a discharge hole for discharging the sample introduced into the second chamber.
The multi-enrichment for ChIP according to claim 10, further comprising a discharge hole installed between the first discharge path and the first chamber and the second discharge path and the second chamber and having a passage smaller than the bead. Fine chip.
The multi-enriched microchip for ChIP according to claim 10, wherein the partition wall has a discharge hole so that the sample introduced into the first chamber is discharged to the second chamber.
The multi-concentrated microchip for ChIP according to claim 10, wherein the second chamber further comprises an auxiliary inlet for supplying a sample to the second chamber.
The multi-enriched microchip for ChIP according to claim 10, wherein the first specific protein and the second specific protein bind to different kinds of antibodies, respectively.
11. The multi-enriched microchip for ChIP according to claim 10, wherein the first chamber and the second chamber are detachable.
The multi-enriched microchip for ChIP according to claim 10, wherein the antibodies bound to the two or more proteins are different from each other.
The method of claim 10, wherein the multi-enriched microchip for ChIP, characterized in that it further comprises a partition formed with a discharge hole between the second chamber and the discharge port so that the sample introduced into the second chamber can be discharged to the discharge port. Multi-enriched microchip for ChIP.
1) adding formaldehyde to the cell to bind DNA and DNA binding protein; 2) destroying the cell and cutting the DNA finely; 3) preparing a sample by adding an antibody binding to the DNA binding protein. 4) injecting the sample into the inlet of the concentrated microchip for ChIP of any one of claims 1 to 14; 5) washing the beads inside the microchip; And 6) eluting the DNA by treating the washed beads with an elution buffer.
The method of claim 18, wherein the DNA is a DNA capable of binding to a transcription factor or an acetylated histone H3. 19.
19. The method of claim 18, wherein the sample is injecting 600 ~ 1000μL ChIP enriched microchips.
19. The method of claim 18, wherein steps 4) to 6) are performed at 15 to 30 ° C.
19. The method of claim 18, wherein the sample is added to the inlet for 5 to 30 minutes in the step 4).
The method of claim 18, wherein step 5) comprises: a) 0.5 ml of low salt immune complex wash buffer b) 0.5 ml of high salt lmmune complex wash buffer, c) chloride Washing sequentially with a lithium immune complex wash buffer (LiCl immune complex wash buffer) 0.5ml and d) 0.5ml TE buffer; each washing time is 30 seconds to 2 minutes, step d) is characterized in that is performed twice ChIP performing method using concentrated microchips.
19. The method of claim 18, wherein after the step 6), Western blot is performed.
19. The method according to claim 18, wherein after step 6), PCR is performed on the eluted DNA.