JP3436741B2 - Method for producing polynucleotide array - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a polynucleotide array in which a large number of various types of polynucleotides are immobilized in spots on a support.

[0002]

In the research of gene expression and gene polymorphism analysis, suitable salts (0.45 mol / l sodium chloride,
Polynucleotide array (DNA) in which a large number of multiple types of polynucleotide solutions in which different types of polynucleotides are dissolved in respective solutions containing 0.045 mol / l sodium citrate etc. Chip) is used so that a large number of types of polynucleotide samples can be simultaneously analyzed in a single measurement operation.

However, in the above-mentioned polynucleotide array, after the dry-fixed polynucleotide is dry-fixed, a blocking treatment is performed to prevent adhesion of the test nucleotide to the surface of the support between the polynucleotides. Alternatively, when the solution of the test nucleotide was dropped on the surface of the support for hybridization, the polynucleotide was easily peeled off from the surface of the support, and the test nucleotide could not be measured effectively and with high sensitivity.

This drawback can be solved by subjecting the surface of the support to a treatment such that it easily binds with amino groups and aminating the ends of the polynucleotide to be immobilized to increase the immobilization strength. It was very expensive and time-consuming to aminate and modify the end of the polynucleotide, and the analysis cost increased.

Further, in order to lengthen the reaction time between the surface of the support and the polynucleotide to enhance the immobilization reaction, a treatment using a highly water-absorbing solvent such as dimethyl sulfoxide or ethylene glycol as a solvent in which the polynucleotide is dissolved. A method has been proposed.

In this method, when the polynucleotide is spotted on the support, the spot shape is likely to be non-uniform, the concentration of the polynucleotide is non-uniform, and the nucleotide to be tested is highly sensitive, It was difficult to analyze efficiently.

Further, as a device for spotting a large amount of a polynucleotide solution in a predetermined amount on the surface of a support, a dispensing needle having a longitudinal groove extending in the axial direction at its tip is used, and the needle is placed in the vertical groove. A sample of the polynucleotide solution is sampled and then brought into contact with the surface of the support to spot a predetermined amount of the polynucleotide in a predetermined spot shape.

However, in the spotting method using the above-mentioned dispensing needle, the excess polynucleotide solution adhering to the tip portion flows out at the initial stage of spotting and adheres to the support to increase the spot diameter. Therefore, the polynucleotide spots may be contaminated with each other, and the spot shapes are not uniform, so that the concentration is not uniform, which causes a problem that the test nucleotide cannot be detected with high sensitivity.

Further, in the spotting method using the above-mentioned dispensing needle, one kind of dispensing needle is used to spot various kinds of polynucleotide solutions, but when the dispensing needle is insufficiently washed. The polynucleotide to be spotted was contaminated with the sample and the nucleotide for test could not be accurately analyzed.

The present invention has been invented in order to solve the above-mentioned conventional drawbacks, and its object is to prevent the cross-contamination of polynucleotides spotted on a support, and to prevent various kinds of problems. It is an object of the present invention to provide a method for producing a polynucleotide array capable of effectively and efficiently performing a work of analyzing a test nucleotide by firmly immobilizing the polynucleotide in a substantially uniform concentration state.

Another object of the present invention is to provide a method for producing a polynucleotide array capable of obtaining a sufficient measurement sensitivity by making the spot concentration of a polynucleotide sample spotted on a support substantially uniform. It is in.

[0012]

The invention of claim 1 relates to a method of producing a polynucleotide array by abutting a dispensing needle on the surface of a support to spot a polynucleotide solution and then fixing the solution. 1. Prepare a polynucleotide solution by dissolving the polynucleotide in a solvent containing at least about 10% by volume formamide in water. The polynucleotide prepared as described above is characterized in that the polynucleotide is immobilized on the surface of a support according to the following steps.

(1). The tip of the dispensing needle is washed with a washing solution. (2). The polynucleotide solution is sampled on a dispensing needle whose tip has been washed. (3). The dispensing needle in which the polynucleotide solution is sampled is abandoned to the striking plate to remove the excess polynucleotide solution attached to the tip. (Four). A large number of polynucleotide solutions are spotted by bringing a dispensing needle into contact with the surface of the support. (Five). A large number of polynucleotide solutions spotted on the surface of the support are dried to immobilize the polynucleotides. (6). The support on which the polynucleotide is immobilized is immersed in a block solution to block the support surface between the polynucleotide spots.

Further, the invention of claim 2 relates to a method for producing a polynucleotide array in which a dispensing needle is brought into contact with the surface of a support to spot a polynucleotide solution and then fixed to prepare a polynucleotide array. The polynucleotide is dissolved in a solvent containing at least about 10% by volume glycerol in water to prepare a polynucleotide solution. The polynucleotide prepared as described above is immobilized on the surface of a support according to the following steps. Is characterized by.

(1). The tip of the dispensing needle is washed with a washing solution. (2). The polynucleotide solution is sampled on a dispensing needle whose tip has been washed. (3). The dispensing needle in which the polynucleotide solution is sampled is abandoned to the striking plate to remove the excess polynucleotide solution attached to the tip. (Four). A large number of polynucleotide solutions are spotted by bringing a dispensing needle into contact with the surface of the support. (Five). A large number of polynucleotide solutions spotted on the surface of the support are dried to immobilize the polynucleotides. (6). The support on which the polynucleotide is immobilized is immersed in a block solution to block the support surface between the polynucleotide spots.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the drawings. 1-2, the support 3 of the polynucleotide array 1 may be made of glass, film, plastic (polyethylene, polypropylene, polystyrene, etc.), and suitable examples include those made by Matsunami Glass Industry Co., Ltd. A slide glass (model S9115) can be used.

Surface Treatment of Support The surface of the support 3 is subjected to positive charge treatment or amination treatment. As the positive charge treatment, the surface of the support 3 is surface-treated with poly-L-lysine, aminopropyltriethoxysilane or the like to be positively charged. As the amination treatment, the surface of the support 3 positively charged by the above method is treated with an aldehyde or a succinide so that the amino groups are easily bonded. This amination treatment is effective in promoting the binding of amino groups at the ends of the immobilized polynucleotide.

Preparation of Polynucleotide Solution The polynucleotide solution fixed on the surface of the support 3 is prepared as follows.

The type of polynucleotide (nucleic acid) used in the present invention is not particularly limited, and examples thereof include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). And DN
A and RNA are also not particularly limited, and chromosomal DNA
Derived DNA fragment, mRNA, cDNA, chemically synthesized DN
Either A or chemically synthesized RNA, specifically P
It may be any of CR amplification product, restriction enzyme digested DNA and the like.

The polynucleotide used in the present invention may be derived from any organism. Further, the polynucleotide used in the present invention includes a DNA fragment, a cDNA fragment and a chemically synthesized DNA derived from a chromosomal position known to be associated with disease. Furthermore, the polynucleotide of the present invention may be a DNA fragment containing a part of a specific gene, an mRNA having an abnormal transcription level, or a cDNA derived from the mRNA.

The above-mentioned polynucleotide of the present invention is purified water or 3 × SSC solution (0.45M sodium chloride,
The pH was adjusted to 7.0 with 0.045 M sodium citrate), and 10% by volume of formamide or glycerol was added thereto to adjust the polynucleotide concentration to 0.5 to 50 pmol / μl.
Use the one adjusted in.

To spot the polynucleotide solution on the surface of the support 3, a large number of dispensing needles 5 are used.
However, for example, the dispensing heads 7 arranged in a matrix at intervals of 100 to 200 μm are used. A large number of polynucleotide solutions are spotted by controlling the movement in the XYZ axis directions with respect to the surface of the support 3. Each dispensing needle 5 used for this spotting has a tip diameter of, for example, 100 to 20.
A vertical groove 5a is formed in the axial direction from the tip at 0 μm, and the polynucleotide solution spotted in the vertical groove 5a is absorbed by the capillary phenomenon and stored.

As the dispensing needle 5, a through hole is formed at the tip, a heating member is attached to the outer periphery of the tip, and a polynucleotide solution is stored in a tank provided at the rear end and stays in the through hole. May be spotted by instantaneously heating the heating member to form bubbles and jumping them to the surface of the support 3.

In addition to the above-mentioned bubble pop-out type dispensing needle, the polynucleotide solution may be directly ejected by utilizing deformation of the piezo element or ultrasonic vibration and spotted on the surface of the support 3. Good.

When the polynucleotide solution is spotted on the surface of the support 3 by controlling the movement of the washing / dispensing head 7, first, the tip of each dispensing needle 5 in the dispensing head 7 is washed and spotted. Prevents various types of polynucleotides from being contaminated (carryover).

As a cleaning method, first, the tip of each dispensing needle 5 in the dispensing head 7 is pressed against a cleaning and water absorbing plate such as a porous ceramic plate or a water absorbing paper plate, and the residual poly is retained in each vertical groove 5a. After the nucleotide solution is discharged, the 0.1% sodium dodecylsulfate aqueous solution tank, the ultrapure water tank, and the ethanol tank are immersed in this order for about 1 to 5 seconds. Phase suction is performed and the attached ethanol is dried.

Polynucleotide Sampling Various types of polynucleotide solutions to be fixed on the surface of the support 3 at the tip of each dispensing needle 5 in the dispensing head 7 washed as described above are stored, for example, The solution is immersed in each solution tank of a 384-well multi-plate for about 7 sec., And the polynucleotide solution is absorbed into each longitudinal groove 5a by a capillary phenomenon to sample.

Removal of excess polynucleotide A discarding operation is performed prior to the spotting operation by the dispensing head 7. That is, when the polynucleotide solution is sampled by the dispensing needles 5, the excess polynucleotide solution remains attached to the outer surface of the tip of each dispensing needle 5. For this reason, the tip end surface of each dispensing needle 5 is pressed against a discarding plate such as a porous ceramic plate, a water-absorbent paper plate, or a glass plate several times within about 0.5 to 1 sec. Excess solution adhering to the outer surface of the tip of the dispensing needle 5 is absorbed.

This prevents the excess polynucleotide solution adhering to the tip of the dispensing needle 5 from being spotted at the initial stage of spotting on the surface of the support body 3, and the size of each spot by the spotted polynucleotide solution can be controlled. The concentration of the polynucleotide solution is made substantially uniform to prevent the contamination between spots.

Spotting Next, the dispensing head 7 is attached to the surface of the support 3 by XYZ.
Each dispensing needle 5 in the dispensing head 7 by controlling movement in the axial direction
The front end surface of each is contacted for about 0.5 to 1 sec., And various kinds of polynucleotide solutions are spotted in a matrix. In this spotting work, the dispensing head 7 is X
-In the Y-axis direction, the mutual spacing of each spot is about 100 ~
The movement is controlled to be 200 μm. The spotting environment at that time may be a room temperature environment, but preferably the external environment temperature is about 2 to 10 ° C., preferably about 2 to 6 ° C., and the humidity is kept at 50 to 70%. By doing so, it is possible to prevent the spotted polynucleotide solution from drying in a short time, and to extend the immobilization time.

Specifically, the entire region including the support 3 and the dispensing head 7 is partitioned from the outside air, and a temperature sensor and a humidity sensor are provided in the partitioned internal region to heat and set the temperature and humidity to the above-mentioned set temperature and humidity. A humidifying device is provided for this purpose.

Next, about 1 to about 1 of water vapor is applied to the surface of the support 3 on which a large number of various kinds of polynucleotide solutions are spotted.
After spraying for 3 seconds, the support 3 is placed on a hot plate heated to about 80 to 95 ° C. and dried and fixed. When the tip surface of the dispensing needle 5 is brought into contact with the surface of the support 3 and the polynucleotide solution is spotted, the adhesion state of the polynucleotide solution in each spot becomes non-uniform and the concentration in the spot is not constant. In order to avoid this, after spotting the polynucleotide solution onto the support 3, water vapor is sprayed to reduce the concentration of the spotted polynucleotide solution to make the adhesion state at each spot uniform.

At this time, the formamide-added polynucleotide solution can maintain a certain form because the formamide inhibits nucleotide denaturation. Therefore, the support 3 which has been subjected to the positive charge treatment
When spotted on the surface of, the probability that the phosphoric acid negatively charged in the polynucleotide is electrostatically bonded to the surface of the support 3 is increased.

When spotted on the surface of the support 3 which has been aminated, the probability of binding to the amino group at the end of the polynucleotide is increased. That is, when the polynucleotide is constantly denatured, electrostatic binding or amino group binding is difficult to occur, but the binding probability can be increased by inhibiting the denaturation of the polynucleotide to a certain form.

Furthermore, in the polynucleotide solution to which glycerose is added, the denaturation of nucleotides is not hindered as in the addition of formamide and the nucleotides are constantly denatured. The time can be lengthened to increase the binding probability.

As a result, the fixing strength of the polynucleotide on the surface of the support 3 is increased.

Next, a UV crosslinker (UVC5 manufactured by Hoefer Co., Ltd.) is applied to the surface of the support 3 on which the polynucleotide is fixed.
00) is used to irradiate about 50 to 120 mJ of ultraviolet light to immobilize the polynucleotide.

Blocking Treatment Next, the non-spotting region of the polynucleotide on the surface of the support 3 is subjected to blocking treatment. As the blocking method, 1 to 1.5 wt% succinic anhydride, 9
0% by volume N-methylpyrocinone, 10% by volume 0.2M sodium borate solution (pH: 8.0), the support 3 on which the polynucleotide was immobilized in a block solution adjusted to pH 5 to 6 for about 15 min Do soaking for a while.

Then, the support 3 taken out from the block solution was immersed in hot water at 95 to 99 ° C. for about 60 seconds, and the support 3 was immersed in ethanol.
Air dry the surface.

As another blocking treatment method, a block solution having a pH of 8 to 9 adjusted with an ethanolamine solution is used, and the blocking solution is similarly immersed. That is, the support 3 on which the polynucleotide is spotted is placed at 45 to 65 ° C.
After incubating under saturated steam for 4 to 14 hours, each final concentration is 50 mmol / l ethanolamine,
0.1 M Tris buffer pH: 9.0, 0.1% Sodium dodecylsulfate 10: 30 min. In a block solution adjusted to pH: 8-9, then washed with water and then 50
Immerse in SSC solution containing 0.1% sodium dodecyl sulfate at 15 ° C. for 15 to 60 min. Further, after washing with water, the liquid components adhering to the support 3 are removed and then dried.

Comparative Example 1 In this comparative example, a synthetic oligonucleotide (80 bases, final concentration 25
pmol / μl), 3 × SSC solution, phosphate buffer (pH:
7.5), using a synthetic oligonucleotide solution dissolved in 4 kinds of solvents, ultrapure water, 10 volume% formaldehyde aqueous solution, the immobilization rate for each solvent was verified.

The nucleotide used for hybridization has a base sequence complementary to the synthetic oligonucleotide fixed on the surface of the support 3, and is labeled with a fluorescent dye (Cy3) (80 bases, final concentration 1).
pmol / μl, containing 0.1 g / ml salmon sperm DNA) was used.

As the support 3, a slide glass for DNA chip (trade name: M, manufactured by Matsunami Glass Industry Co., Ltd.) is used.
AS coated slide) was used.

As shown in FIGS. 3 and 4, when the synthetic oligonucleotide is dissolved in formaldehyde as a solvent, the fluorescence intensity is highest as compared with other solvents. This fact indicates that when formaldehyde is used as the solvent for the synthetic oligonucleotide, the amount of the synthetic oligonucleotide immobilized on the surface of the support 3 is the largest.

FIG. 5 shows the support in the case where the blocking process and the hybridization process are applied to the polynucleotide array 1 on which the synthetic oligonucleotide solution in which the synthetic oligonucleotide is dissolved is fixed by using formamide as a solvent. It is the result of 16 trials with respect to the immobilization rate of the synthetic oligonucleotide spots on 3 surfaces, and the average immobilization rate was 97.6% when formamide was used as the solvent.
It was possible to leave a sufficient fixed amount.

COMPARATIVE EXAMPLE 2 FIGS. 6 and 7 show a comparison between the case of performing the discarding prior to spotting and the case of not performing the discarding. In this experiment, a porous ceramic plate was used as the water absorbing plate 9 and the support 3 was used. Λ phage DNA labeled with a fluorescent dye (Cy3) was used as a polynucleotide to be spotted on the surface of.

Then, 116 spots were continuously spotted on the surface of the support 3 by one spotting operation, and then the fluorescence intensity was read by a scanner to form a graph.

As a result, when spotting is performed several times prior to spotting on the surface of the support 3, the fluorescence intensity per spot is more uniform than when no tapping is performed. On the contrary, when the tapping is not performed, the fluorescence intensity at the early stage of spotting is high, and therefore the amount of polynucleotide spots is large.

Comparative Example 3 Using one dispensing needle 5, a fluorescent solution in which 400 fmol / μl of a fluorescent dye (Cy3, Cy5) was dissolved in pure water and pure water were alternately sampled to sample the surface of the support 3 And spot the fluorescent label with a scanner. The spotting cycle is fluorescence sampling → spotting →
The cleaning effect of the dispensing needle 5 is verified by performing cleaning, pure water sampling, spotting, ...

As shown in FIG. 8, spots where fluorescence is detected and spots where fluorescence is not detected appear alternately. Therefore, the inside of the vertical groove 5a is almost completely washed by washing the dispensing needle 5 as described above. This indicates that no fluorescent dye was detected in pure water.

[0051]

INDUSTRIAL APPLICABILITY According to the present invention, a variety of polynucleotides are firmly immobilized in a substantially uniform concentration while preventing cross-contamination of polynucleotides spotted on a support, and the nucleotides for analysis are analyzed. Work can be performed effectively and efficiently. Further, the spot concentration of the polynucleotide sample spotted on the support can be made substantially uniform to obtain sufficient measurement sensitivity.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing a production process of a polynucleotide array.

FIG. 2 is a partial perspective view showing a spotting state of a polynucleotide array.

FIG. 3 is a photograph of a fluorescent state of each solvent observed by a scanner when hybridizing using a polynucleotide dissolved in each solvent.

4 is a graph quantifying the fluorescence intensity of each spot in FIG.

FIG. 5 is a table showing the immobilization rate of polynucleotides according to the number of blocking processes and hybridization processes.

FIG. 6 is a graph showing fluorescence intensity when a dispensing needle is tapped prior to spotting a polynucleotide on a support.

FIG. 7 is a graph showing fluorescence intensity when spotting is performed without tapping.

FIG. 8 is a photograph showing the cleaning effect of a dispensing needle.

[Explanation of symbols]

1-polynucleotide array, 3-support, 5-dispensing needle, 5a-longitudinal groove

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI G01N 37/00 102 C12N 15/00 F

Claims (16)

(57) [Claims] 1. A method for producing a polynucleotide array, which comprises the following items in a method for producing a polynucleotide array by contacting a dispensing needle on the surface of a support to spot a polynucleotide solution and then fixing the solution. 1. A polynucleotide solution is prepared by dissolving the polynucleotide in a solvent containing at least about 10% by volume formamide in water. 2. The polynucleotide is immobilized on the surface of the support according to the following steps. (1). The tip of the dispensing needle is washed with a washing solution. (2). The polynucleotide solution is sampled on a dispensing needle whose tip has been washed. (3). The dispensing needle in which the polynucleotide solution is sampled is abandoned to the striking plate to remove the excess polynucleotide solution attached to the tip. (Four). A large number of polynucleotide solutions are spotted by bringing a dispensing needle into contact with the surface of the support. (Five). A large number of polynucleotide solutions spotted on the surface of the support are dried to immobilize the polynucleotides. (6). The support on which the polynucleotide is immobilized is immersed in a block solution to block the support surface between the polynucleotide spots. 2. A method for producing a polynucleotide array, which comprises the following items in a method for producing a polynucleotide array by contacting a dispensing needle on the surface of a support and spotting a polynucleotide solution. 1. A polynucleotide solution is prepared by dissolving the polynucleotide in a solvent containing at least about 10% by volume glycerol in water. 2. The polynucleotide is immobilized on the surface of the support according to the following steps. (1). The tip of the dispensing needle is washed with a washing solution. (2). The polynucleotide solution is sampled on a dispensing needle whose tip has been washed. (3). The dispensing needle in which the polynucleotide solution is sampled is abandoned to the striking plate to remove the excess polynucleotide solution attached to the tip. (Four). A large number of polynucleotide solutions are spotted by bringing a dispensing needle into contact with the surface of the support. (Five). A large number of polynucleotide solutions spotted on the surface of the support are dried to immobilize the polynucleotides. (6). The support on which the polynucleotide is immobilized is immersed in a block solution to block the support surface between the polynucleotide spots. 3. The method for producing a polynucleotide array according to claim 1, wherein the concentration of the polynucleotide dissolved in the solvent is 0.5 to 50 pmol / μl. 4. The method for producing a polynucleotide array according to claim 1, wherein the polynucleotide is a PCR amplification product derived from a biological gene. 5. The method for producing a polynucleotide array according to claim 1, wherein the polynucleotide is an artificially synthesized oligonucleotide. 6. The method for producing a polynucleotide array according to claim 1, wherein the discarding plate is any one of a porous ceramic plate, a water absorbing paper plate and a glass plate. 7. The washing of the dispensing needle according to claim 1 or 2, wherein the dispensing needle is immersed in a 0.1% sodium dodecylsulfate solution which is brought into contact with a water absorbing material to discharge the polynucleotide solution remaining in the longitudinal groove. A method for producing a polynucleotide array, which comprises the steps of: dipping in pure water, dipping in ethanol, and vapor phase suction drying. 8. The method for producing a polynucleotide array according to claim 1, wherein the environment for spotting the polynucleotide on the surface of the support is a room temperature environment. 9. The method for producing a polynucleotide array according to claim 1, wherein the spotting environment of the polynucleotide on the surface of the support is a temperature of 2 to 10 ° C. and a humidity of 50 to 70%. 10. The method for producing a polynucleotide array according to claim 1 or 2, wherein the surface of the support is positively charged by surface treatment with either poly-L-lysine or aminopropyltriethoxysilane. 11. The method for producing a polynucleotide array according to claim 1, wherein the support surface is aminated by either aldehyde treatment or succinide treatment. 12. The method for producing a polynucleotide array according to claim 1, wherein the block solution is a succinic acid solution adjusted to at least pH 5-6. 13. The method for producing a polynucleotide array according to claim 1 or 2, wherein the blocking solution is an ethanolamine solution adjusted to at least pH 8-9. 14. A method for producing a polynucleotide array according to claim 1 or 2, wherein steam is sprayed on the surface of the support on which the polynucleotide solution is spotted to make the adhered state of the polynucleotide solution uniform at each spot. 15. The method for producing a polynucleotide array according to claim 1, wherein the dispensing needle has a longitudinal groove extending in the axial direction at the tip thereof, and a polynucleotide solution can be sampled in the longitudinal groove. . 16. The support according to claim 1, wherein the dispensing needle is provided with a heating member at a tip portion having a through hole, and the polynucleotide solution in the through hole is instantaneously heated by the heating member to form bubbles. A method for producing a polynucleotide array in which a polynucleotide solution is spotted on the surface.