JP2003339375A - Hybridization method and apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hybridization method and apparatus to improve the hybridization efficiency of a target DNA relative to the probe DNA immobilized on a substrate surface in a DNA chip assay. <P>SOLUTION: A hybridization solution containing fine particles is sealed on a DNA chip with a cover glass and a sealing agent. The hybridization chamber holding the DNA chip is rotated on a rotary shaft. Since the fine particles are moved in the direction of the gravitation force, these particles have an effect to stir the hybridization solution. The hybridization efficiency is increased by the increase of the diffusion distance of the DNA target in the hybridization solution. The method is effective for improving the hybridization efficiency in the DNA chip assay and attains a signal having high quantitative accuracy and reproducibility. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

2. Description of the Related Art A DNA chip is a device in which thousands to tens of thousands of gene DNAs are arranged and immobilized at high density on a glass substrate. Multiple different DNA probes are immobilized at high density on a solid-phase substrate such as a slide glass whose surface is specially processed. DNA
By dropping a fluorescently labeled sample from mRNA extracted from cells or tissues by a reverse transcription reaction onto the chip and performing hybridization, it is possible to analyze the expression information of many genes in parallel on a large scale. With the progress of the human genome project, the focus has been on comprehensively elucidating the functions and networks of all genes existing in the genome, rather than capturing each gene individually.

A way to analyze the action of pharmaceuticals with a DNA chip has also been opened. The mechanism of action of the immunosuppressant FK506 has already been analyzed in detail, and FK506 binds to the FK-binding protein (FKBP), and the FKBP cal
Inhibits cineurin (CN) activation. As a result, transcription factors
It is known that NFAT activation is inhibited and lymphocyte activation is suppressed. Marton et al. Analyzed this point of action using a DNA chip on which more than 6,000 cDNAs close to all yeast genes were immobilized. As a result, 36 genes (about 0.6%) showed more than 2-fold fluctuation due to the action of FK506. The variation profile closely resembled the difference in gene expression profiles between the wild-type and CN-deficient strains. Regarding this achievement, Merton et al., Nature Medicine No. 4 (20
2000) pp. 1293 to 1301 (Marton MJ et
al., Nature Medicine, 4, (1999), 1293-1301).

Further, it has been shown that the prognosis of patients can be estimated by a new classification from the gene expression profile of cancer cells using a DNA chip. Alizad et al. Produced a DNA chip on which 18,000 clones of cDNA were immobilized, and diffuse large B cell lymphoma (DLBCL)
Gene expression was analyzed in cancer cells of patients. As a result, the patients were divided into two groups, one showing an expression profile very similar to the gene expression of germinal center B cells, and the other showing an expression profile very similar to peripheral blood activated B cells. Moreover, the former had a significantly better survival rate than the latter. Such a classification could not be done by the existing pathological diagnosis. Regarding these results, Alizad et al. Nature 403 (2000) pp. 503 to 511 (Alizadeh AA et al., Nature, 403, (2000),
503-511). Under the above circumstances,
Expectations are increasing for DNA chip technology as a comprehensive gene expression monitoring method.

A typical DNA chip is a DNA chip manufactured by Affymetrix. Affymetrix DNA chips synthesize DNA probes in the solid phase,
The probe length can only be extended up to 25 mer. Therefore, the fluorescence detection sensitivity is lower than that of the spot type DNA chip. To overcome this, the Affymetrix experimental protocol rotates the DNA chip at a rate of 60 times per minute during hybridization.

[0005]

The merit of the measurement using the DNA chip is that the expression states of thousands to tens of thousands of genes can be simultaneously analyzed. On the other hand, the disadvantage is that the reproducibility and quantitativeness of the data are low compared with other gene expression analysis techniques such as Northern hybridization, RT-PCR, and body map method. One of the causes of low reproducibility and quantitativeness of the DNA chip is that the efficiency of hybridization of the administered target DNA is low. Since the efficiency of hybridization is low, the fluorescence intensity detected becomes weak, especially for genes with a low expression level, and the quantification / reproducibility of data is significantly reduced.

The efficiency of hybridization on the DNA chip will be calculated below. Generally, the diffusion constant of mRNA in aqueous solution is about D = 10 μm ² / s,
Politz et al. Bulletin No. 95 (1998)
Year) 6043 to 6048 (Politz et al., P
roc. Ntal. Acad. Sci. USA 95 (1998) pp6043-6048)
Report to. In addition, since the time required for hybridization in the analysis using a DNA chip is generally about 6 hours, the distance that the target DNA in the solution can move on the glass slide surface during hybridization is

[Equation 1] <r ² > ^1/2 = <x ² + y ² > ^1/2 = 4Dt ^1/2 = (4
× 10 × 60 × 60 × 6) ^1/2 = 929 [μm]. This is an average of some target DNA molecules from one position during hybridization time r = 929 μm
It means that you can only move. On the other hand, hybridization is performed by spreading the target DNA solution using a cover glass of about 24 x 50 mm on the slide glass. Therefore, even if it is assumed that the target DNA hybridizes to all complementary probe DNAs on glass, the ratio of the target DNAs that contribute to hybridization in one spot on the DNA chip to the total amount of the administered target DNAs is ,

[Equation 2] πr ² / (22 × 50) = π (0.929) ² / (22 × 50) = 2.2
It becomes 6 × 10 ^-3 . This indicates that no more than 0.2% in the target DNA solution contributed to the hybridization.

The DNA chip can be roughly classified into two types: one for spotting cDNA or synthesized oligonucleotides and one for solid-phase synthesis using lithography on a substrate. Although the latter DNA chip is manufactured by Affymetrix, the length of oligonucleotide that can be prepared for solid phase synthesis on a substrate is limited to 25 mer. However, the 25-mer oligonucleotide is a cDNA DNA with a length of 300 mer or longer.
The efficiency of hybridization is extremely reduced when compared to the probe. This is reported by Masuho in "DNA Chip and Latest PCR Method" (Shujunsha), pages 7 to 12. In order to improve this, in the experimental protocol of Affymetrix, the sample cDNA is amplified as cRNA by T7 RNA polymerase, and the DNA chip is rotated at a rate of 60 times per minute in the hybridization. However, even when using the above-mentioned protocol, there is a 3-fold or more variation in the gene with low expression level, and it is difficult to measure the gene expression level for 20% of the budding yeast gene, Murakami et al. Hyundai Kagaku ”(Tokyo Kagaku Dojin) (July 2000 issue), pages 60 to 6
Reported on page 8. Therefore, the low efficiency of hybridization in measurement with a DNA chip is
It has become one of the major challenges.

The object of the present invention is to enhance the hybridization efficiency of 0.2% at the most, which is the limit of the prior art, and
It is an object of the present invention to provide an inexpensive, simple and versatile hybridization method and device capable of obtaining a highly reproducible signal.

[0009]

In order to achieve the above-mentioned object, the hybridization method and apparatus of the present invention are:
Fine particles, a sample, a container for holding a fluid containing the fine particles and the sample, means for fixing a plurality of the containers, means for controlling the humidity of the container, means for keeping the temperature of the container constant, It has means for rotating the container.

That is, the present invention provides the following (1) to (16):
I will provide a. (1) A method for hybridizing a first nucleotide and a second nucleotide complementary to the first nucleotide, wherein the second nucleotide is added to a substrate on which the first nucleotide is immobilized. The above method, characterized in that the solution containing the same is mixed, and the fine particles are used to perform the hybridization while stirring the solution. (2) The fine particles are beads,
The hybridization method according to (1) above. (3) The hybridization method according to (1) above, wherein the fine particles have a diameter of 0.01 μm or more and 10 μm or less.

(4) The hybridization method according to (1) above, wherein the surface of the fine particles is coated with a hydrophilic group. (5) The hybridization method according to (1) above, wherein the specific gravity of the fine particles is higher than that of the solution. (6) The hybridization method according to (1) above, wherein the fine particles are magnetic fine particles.

(7) The hybridization method according to (1) above, wherein the stirring is performed by rotating the substrate. (8) The agitation is performed by moving the fine particles in a solution in a direction of a rotation center of the rotation and in a direction opposite to the rotation center, thereby performing the stirring.
The hybridization method described. (9) The hybridization method according to (1) above, wherein the stirring is performed by vibrating the substrate.

(10) The hybridization method according to (6) above, wherein the stirring is performed by rotating the magnetic fine particles using a stirrer. (11) After the mixing, the solution may be stirred by placing a cover material on the substrate to shield it in order to confine the solution, and rotating and / or vibrating the shielded substrate. , Above (1)
The hybridization method described. (12) A base on which the first nucleotide is immobilized,
A container for holding a solution containing the base, a second nucleotide complementary to the first nucleotide, and fine particles; and a rotation mechanism for rotating the container. Hybridization device.

(13) The hybridization apparatus according to (12) above, further comprising a control mechanism for controlling the rotation direction and rotation speed of the rotation by the rotation mechanism. (14) The hybridization device according to (12), wherein the rotation speed is controlled to 1 rpm or more and 60 rpm or less. (15) The hybridization apparatus according to (12) above, wherein the apparatus further comprises means for controlling the humidity inside the container. (16) The hybridization apparatus according to (12) above, wherein the apparatus further comprises means for controlling the temperature inside the container.

The structure of the present invention will be described in more detail below. 1) Construction of DNA chip Microparticles are mixed in a sample containing fluorescently labeled target DNA, and the microparticles are dispersed in a sample tube by pipetting. The target DNA is dropped on the DNA chip, covered with a cover glass, and the target DNA is spread over the region on the DNA chip where hybridization is to be performed. Target D for DNA chip using sealant
Fix NA. The target DNA and microparticles are sandwiched between a cover glass and a DNA chip, and this is fixed with a sealant.

Specific properties and numerical values of the method using fine particles will be described below. The surface of the fine particles is preferably covered with a hydrophilic group such as a carboxyl group or a hydroxyl group. As a result, non-specific adsorption of the fine particles and the target DNA is prevented, and the hybridization can be efficiently performed without reducing the amount of the target DNA contributing to the hybridization. Moreover, the composition of the fine particles is not particularly limited, and commercially available beads and the like can be preferably used. Magnetic beads are particularly preferably used. It has been confirmed by experiments that polystyrene beads are also effective, and metal powders such as gold particles can obtain almost the same effects.

The diameter of the fine particles is about 0.01-10 μm.
A range of 4 to 10 μm can be used, with a range of 4 to 10 μm being preferred
Yes. The most effective of these was the diameter of 10 μm
It is a fine particle. Here, with a DNA chip and a cover glass
Since the gap formed is about 20 μm, the diameter is large.
If it is too much, movement will be restricted, so it is not so preferable
No The specific gravity of fine particles is higher than that of water.
Is desirable. The specific gravity of the fine particles that can be used is 1 to 20,
1.05-19.3, most preferably about 1.5 g / cm³ And
It Also, the concentration of fine particles is about tens of thousands per μl.
It is preferable to use it once. The slide installation position is
The horizontal direction is more effective than the vertical direction in the plane of the rotation direction.
Target.

2) Hybridization device Hybridization incubator (stirring device)
Is composed of one or a plurality of holding shafts capable of holding a hybridization chamber holding a DNA chip and a rotating shaft for rotating the holding shafts. The holding shaft and the rotating shaft can rotate independently of each other. The rotation speed of the rotary shaft can be set arbitrarily. Further, it is possible to set the rotation speed constant or variable with respect to time. It is also possible to control the rotation speed momentarily by a program. The direction of rotation can also be set arbitrarily.

The temperature at the time of hybridization can be arbitrarily set within the temperature range usually used in hybridization. Further, it is possible to provide a temperature control means capable of keeping constant with an accuracy of ± 0.1 ° C. with respect to the temperature set as the optimum temperature for hybridization.

The rotation speed of the container can be 1 to 60 rpm. The highest hybridization efficiency can be achieved at 60 rpm. It is considered that more effective stirring effect can be obtained by changing the rotation speed by a program or reversing the rotation direction at regular intervals.

Specifically, a fluorescently labeled target DNA is prepared by a reverse transcription reaction, and prehybridization is carried out by heat denaturation at 95 ° C. for 2 minutes. After that, the fine particles are mixed with the hybridization solution containing the target DNA and added dropwise to the DNA chip. Cover with a cover glass and seal the hybridization solution with DN.
Fix to A chip. DN in the hybridization chamber in the hybridization incubator
Fix and seal the A chip. Then, the hybridization chamber is fixed to the holding shaft in the hybridization incubator, and rotation of the holding shaft is started. The hybridization chamber contains a moisturizer containing 3 × SSC to prevent evaporation of the hybridization solution on the DNA chip. The hybridization incubator performs hybridization at an optimum temperature. The hybridization incubator can fix a plurality of hybridization chambers, and has a rotation axis that rotates each chamber. During rotation, the fine particles in the hybridization solution constantly fall in the direction of gravity, which has the effect of stirring the target DNA. As a result, the target in the hybridization solution
The efficiency of hybridization between the DNA and the DNA probe fixed on the glass slide can be increased.
Note that FIG. 8 is a flow chart showing the whole steps.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION A first embodiment of the hybridization method and apparatus of the present invention will be described with reference to FIGS.
4 will be described. The hybridization solution 107 in FIG. 1 contains the target DNA 103 that has been fluorescently labeled, purified, and then heat-denatured. The fine particles 104 are mixed in the hybridization solution 107, and pipetting is performed in the sample tube 102 to uniformly disperse the fine particles 104 in the hybridization solution 107. Since the fine particles 104 have a carboxyl group on the surface, they have the characteristic of not nonspecifically adsorbing to the target DNA 103. Therefore, the amount of hybridization of the target DNA 103 to the probe DNA does not decrease.

Next, the hybridization solution 107
Is dropped on the DNA chip 101, the cover glass 105 is covered, and the hybridization solution 107 is spread over the region on the DNA chip where hybridization is to be performed. The hybridization solution 107 under the cover glass 105 is fixed to the DNA chip 101 using the sealant 106.

A method of fixing the DNA chip 203 to the hybridization chamber 201 will be described with reference to FIG. DNA in the hybridization chamber 201
A humectant 202 containing 3 × SSC (Standard Sodium Citrate) is added to prevent the hybridization solution on the chip 203 from drying. By adding the lid 205 to the hybridization chamber 201, the humidity in the hybridization chamber 201 can be maintained at a saturated vapor pressure and the drying of the hybridization solution can be prevented. In addition, the hybridization chamber 201 is also used in the DNA chip 203 even in the rotating state described later.
Can be firmly held. Although 206 is a stopper, the method of fixing the DNA chip in the chamber is not particularly limited.

A method and apparatus for improving the efficiency of hybridization will be described with reference to FIG. This device is provided with a hybridization incubator 304 and a hybridization chamber 3 for keeping the temperature constant.
A holding shaft 302 for fixing 03 and a rotating shaft 301 for rotating the holding shaft 302. The rotating shaft 301 and the holding shaft 302 can rotate independently in the directions of arrows 311 and 312.

Further, the hybridization incubator 304 can independently and arbitrarily set the rotation speed and the rotation direction of the rotation shaft 301 and the holding shaft 302 at any time by a program. Also, the holding shaft 3
02 can hold a plurality of hybridization chambers 303 at the same time. Further, since the hybridization chamber 303 has a circular shape, the DNA chip can be held in any direction such as vertical or parallel to the rotation direction of the rotation shaft 301.

In FIG. 3, an example in which the DNA chip 306 is arranged horizontally with respect to the rotating direction of the rotating shaft 301 will be described. On the DNA chip 306, the hybridization is progressing with the hybridization solution sealed by the cover glass 305 and the sealant 308. The hybridization chamber 303 holding the DNA chip 306 rotates about the rotation shaft 301 in the direction of the arrow at a speed of 60 rotations per second. The hybridization solution held on the DNA chips 306 and 310 contains fine particles 307 and 309. The particles 307 and 309 fall in the direction of gravity in any rotating state. Therefore, the fine particles 307 and 309 volumetrically eliminate the hybridization solution in the moving region,
It has the effect of stirring the hybridization solution. DN
Compared with the case where the A chip 306 is left stationary, the diffusion distance of the DNA target in the hybridization solution is increased, so that the efficiency of hybridization is increased.
Therefore, the number of obtained signals is increased, which brings about an effect of improving reproducibility and quantitativeness in DNA chip measurement.

In the above example, only the rotation of the rotating shaft has been described, but the efficiency of hybridization can be further increased by adding the rotation of the holding shaft at the same time.

A rotation method different from that of FIG. 3 will be described with reference to FIG. Similarly, the hybridization incubator 405 accurately maintains the hybridization temperature at the optimum temperature. The rotors 401 are arranged in parallel in the hybridization incubator 405 and can rotate in one direction (402). Rotor 401
When the hybridization chamber 404 is grounded above, the hybridization chamber 404 rotates in one direction. In the DNA chip 403 held in the hybridization chamber 404, the fine particles are held in the target DNA as described above. Since the particles fall in the direction of gravity and agitate the target DNA, the hybridization efficiency is increased.

Further, moving the rotor 401 in a seesaw shape as shown by an arrow 406 further increases the efficiency of hybridization. Also, the rotor 401
It is also possible to apply vibration to the, which further increases the efficiency of hybridization. The rotation speed, rotation direction, seesaw-shaped amplitude distance, amplitude speed, and vibration of the rotor 401 can be set to any state at any time.

FIG. 5 illustrates a method of rotating the magnetic fine particles by a stirrer to increase the hybridization efficiency. By the method described above, the magnetic fine particles 503 are already dispersed in the target DNA on the DNA chip. When the magnetic fine particles 503 rotate on the surface of the DNA chip 502, the magnetic fine particles 503 come into contact with the DNA probe, causing problems such as peeling of the DNA probe and inhibition of efficient hybridization. In order to avoid these problems, in this embodiment, the DNA chip 502 is grounded to the stirrer 501 with the cover glass surface facing down.

The magnetic fine particles 503 fall on the surface of the cover glass and rotate as a minute stirrer rotor. Since the target DNA solution is efficiently stirred, the efficiency of hybridization is increased.

A method for increasing the efficiency of hybridization by convection will be described with reference to FIG. High heat source 60
DNA chip 6 with target DNA sealed on 1
03 is grounded. Next, a low heat source 602 such as a Peltier element is grounded on the upper surface of the DNA chip 603. A temperature difference occurs between the upper surface and the lower surface of the DNA chip, causing convection of the target DNA. Since the target DNA solution is efficiently stirred, the efficiency of hybridization is increased.

FIG. 7 shows an example of experimental results of hybridization by the conventional method and the above-mentioned novel method. In the experiment, IFI 56 (gene of interferon-inducible protein 56) was used as a sample, and hybridization was carried out at 62 ° C. for 6 hours in a buffer having the following composition.

[0035]

The vertical axis of FIG. 7 shows the fluorescence intensity. Compared with the conventional method, this method can obtain 5 times the fluorescence intensity. The error bar is the standard deviation of the signal with 20 spots.

[0037]

INDUSTRIAL APPLICABILITY As described in detail above, by using the present invention, the target DNA on the DNA chip can be agitated, the efficiency of hybridization can be improved, and the detection sensitivity of the target DNA can be increased. Further, it has an effect that the hybridization time can be shortened.

[Brief description of drawings]

FIG. 1 shows an example of fixing a hybridization solution to a DNA chip.

FIG. 2 shows an example of a method for immobilizing a DNA chip in a hybridization chamber.

FIG. 3 shows an example of a hybridization device of the present invention.

FIG. 4 shows an example of a hybridization device of the present invention using different rotation methods.

FIG. 5 shows a hybridization method of the present invention using rotation of magnetic fine particles by a stirrer.

FIG. 6 shows a method for increasing the efficiency of hybridization by convection.

FIG. 7 shows an example of an experimental result by the conventional method and the hybridization method of the present invention.

FIG. 8 is a flow chart showing the whole process.

[Explanation of symbols]

101 ... DNA chip, 102 ... Sample tube, 10
3 ... Target DNA, 104 ... Fine particles, 105 ... Cover glass, 106 ... Sealing agent, 107 ... Hybridization solution, 201 ... Hybridization chamber, 202, 204 ... Moisturizing agent, 203 ... DNA chip, 2
05 ... Lid, 206 ... Stopper, 301 ... Rotating shaft, 30
2 ... Holding shaft, 303 ... Hybridization chamber, 304 ... Hybridization incubator,
305 ... Cover glass, 306, 310 ... DNA chip,
307, 309 ... Fine particles, 308 ... Sealing agent, 311 ...
Rotational direction of rotary shaft 312 ... Rotational direction of holding shaft 401 ... Rotor, 402 ... Rotational direction of rotor, 403 ...
DNA chip, 404 ... Hybridization chamber, 405 ... Hybridization incubator,
406 ... Vibration direction of seesaw 501 ... Stirrer, 502 ... DNA chip, 503 ... Magnetic fine particles, 601 ... High heat source, 602 ... Low heat source, 603 ... DN
A tip

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Toshiro Saito             4-6 Kanda Surugadai, Chiyoda-ku, Tokyo             Hitachi, Ltd. Life Science Promotion             Within the business unit (72) Inventor Hiroyuki Tomita             4-6 Kanda Surugadai, Chiyoda-ku, Tokyo             Hitachi, Ltd. Life Science Promotion             Within the business unit (72) Inventor Masatoshi Narahara             4-6 Kanda Surugadai, Chiyoda-ku, Tokyo             Hitachi, Ltd. Life Science Promotion             Within the business unit F-term (reference) 4B024 AA11 CA01 HA14                 4B029 AA07 AA23 BB20 CC03 FA15                 4B063 QA01 QQ79 QR32 QR55 QR66                       QR82 QS34 QS36 QX02

Claims (16)

[Claims] 1. A method for hybridizing a first nucleotide and a second nucleotide complementary to the first nucleotide, wherein the second nucleotide is attached to a substrate on which the first nucleotide is immobilized. The above method, characterized in that a solution containing a nucleotide is mixed, and hybridization is carried out while stirring the solution using fine particles. 2. The hybridization method according to claim 1, wherein the fine particles are beads. 3. The diameter of the fine particles is 0.01 μm or more 1
The hybridization method according to claim 1, wherein the hybridization method is 0 μm or less. 4. The hybridization method according to claim 1, wherein the surface of the fine particles is covered with a hydrophilic group. 5. The hybridization method according to claim 1, wherein the specific gravity of the fine particles is higher than that of the solution. 6. The hybridization method according to claim 1, wherein the fine particles are magnetic fine particles. 7. The hybridization method according to claim 1, wherein the stirring is performed by rotating the substrate. 8. The stirring according to claim 7, wherein the fine particles move in the direction of the rotation center of the rotation in the solution and in the direction opposite to the rotation center to perform the stirring. Hybridization method. 9. The hybridization method according to claim 1, wherein the stirring is performed by vibrating the substrate. 10. The hybridization method according to claim 6, wherein the stirring is performed by rotating the magnetic fine particles using a stirrer. 11. After the mixing, a cover material is placed on the substrate to confine the solution to shield the solution, and the solution is agitated by rotating and / or vibrating the shielded substrate. The hybridization method according to claim 1, wherein 12. A container for holding a base on which a first nucleotide is immobilized, the base, a second nucleotide complementary to the first nucleotide, and a solution containing fine particles, And a rotating mechanism for rotating the container. 13. The hybridization device according to claim 12, further comprising a control mechanism for controlling a rotation direction and a rotation speed of rotation by the rotation mechanism. 14. The hybridization apparatus according to claim 12, wherein the rotation speed is controlled to 1 rpm or more and 60 rpm or less. 15. The apparatus according to claim 12, further comprising means for controlling the humidity in the container.
A hybridization device as described. 16. The apparatus according to claim 12, further comprising means for controlling the temperature in the container.
A hybridization device as described.