JP2001188067A - Dna chip manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To shorten the manufacturing time of DNA chips and to reduce manufacturing cost. SOLUTION: Template oligonucleotide 2 is fixed to the inner wall surface of a capillary tube 4 (A), and a large number of capillary tubes 4 in which template oligonucleotide 2 is fixed to the inner wall surfaces are prepared. Then their one-end sides are two-dimensionally arranged and bundled. A PCR reaction liquid 6 is filled in the capillary tubes 4 (B) to synthesize complementary oligonucleotide 8 by the PCR reaction with the template oligonucleotide 2 as a template (C). Than the PCR reaction liquid 6 is removed D, and the template oligonucleotide 2 is dissociated from the oligonucleotide 8 (E). One faces of the capillary tubes 4 are brought into contact with the surface 10 of a nylon film to make a part of an eluate 12 adhere to the surface 10 of the nylon film and to move the oligonucleotide 8 to the side of the surface 10 of the nylon film and fix it to the surface 10 of the nylon film (F).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a DNA chip in which a large number of oligonucleotides are aligned and immobilized on a solid phase surface.

[0002]

2. Description of the Related Art As a method for monitoring the state of gene expression, there is a method using a DNA chip. A DNA chip is a device in which a large number of oligonucleotides (also referred to as DNA probes) corresponding to various genes are aligned and immobilized on a solid phase surface to form an oligonucleotide matrix. In the method using a DNA chip, cDNA (complementary DNA) of mRNA (messenger RNA) as a sample is synthesized using a PCR reaction (Polymerase Chain Reaction), and after fragmentation, each fragment is labeled with a fluorescent label. Label the fragment. These labeled fragments are brought into contact with a DNA chip, and hybridized to oligonucleotides immobilized on the DNA chip. The labeled fragment is retained on the oligonucleotide whose sequence is complementary, and the excess labeled fragment is removed by a washing operation. Thereafter, the amount and position of the retained labeled fragment are detected using a fluorescence microscope, and the type of the corresponding oligonucleotide is examined. This method is suitable for monitoring the expression of a gene whose sequence is known.

[0003] DNA chip manufacturing methods are roughly classified into the following three types. In the first method, a plurality of linkers modified with a protecting group that can be removed photochemically are arranged to be bound to the surface of a solid phase via an amino group. By applying photolithography technology used in semiconductor manufacturing technology, light is irradiated through a mask capable of irradiating only a desired linker fixing position to remove a protective group. Next, a monomer having a protecting group that can be removed photochemically is introduced, and the first coupling reaction is performed. As a result, the oligonucleotide is extended by that portion. By repeating photolithography and monomer introduction, a desired oligonucleotide matrix is formed.

In the second method, an oligonucleotide to be immobilized is prepared in advance, a small amount of the oligonucleotide is dropped on a solid surface such as glass or a polymer film, and immobilized by covalent bonding at the position. For example, if an isothiocyanate group is introduced on the surface of the solid phase and the terminal of the oligonucleotide is an amino group, the oligonucleotide can be easily immobilized at the isothiocyanate group fixing position by covalent bonding. In the third method, an oligonucleotide to be immobilized is prepared in advance, a small amount of the oligonucleotide is dropped on a solid phase surface such as glass or a polymer film, and the oligonucleotide is immobilized at the dropping position by an adsorption action.

[0005]

However, in the above first method, photolithography and introduction of monomers must be repeated each time the oligonucleotide is extended, which is a long time for producing a DNA chip. There is a problem that it takes time and the manufacturing cost is increased. In the above second and third methods, in either method, the PC
Multiple types of oligonucleotides amplified by the R reaction, etc. are taken into a dedicated spotting device, sequentially arrayed and dropped on the solid surface, and it takes a long time to prepare a DNA chip. There was a problem that becomes high. Therefore, an object of the present invention is to reduce the manufacturing cost by reducing the manufacturing time of a DNA chip.

[0006]

The method for producing a DNA chip of the present invention includes the following steps. (A) a step of immobilizing a template oligonucleotide on the inner wall of one or more capillary members; (B) a step of performing a replication reaction in the capillary member to synthesize an oligonucleotide complementary to the template oligonucleotide; (C) disposing another solid phase surface on one end side of the capillary member, and moving the complementary oligonucleotide to the other solid phase surface; and (D) transferring the complementary oligonucleotide to the other solid phase surface. Step of immobilizing on the surface of a solid phase.

[0007] A template oligonucleotide is immobilized on the inner wall of one or more capillary members, and a replication reaction is performed using the template oligonucleotide as a template. Here, the term “replication reaction” refers to the synthesis of an oligonucleotide complementary to a template oligonucleotide, such as a PCR reaction. After synthesizing a complementary oligonucleotide,
The complementary oligonucleotide is transferred to another solid phase surface and immobilized on another solid phase surface. When a plurality of capillary members are used, a DNA chip having a desired oligonucleotide matrix can be produced by aligning and immobilizing complementary oligonucleotides on another solid phase surface.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the step (A), a plurality of capillary members are used, and in the step (C), complementary oligonucleotides are simultaneously transferred to another solid-phase surface by two or more capillary members. Is preferred. As a result, the manufacturing time can be reduced. A DNA chip can be prepared by arranging and immobilizing a plurality of complementary oligonucleotides on the surface of another solid phase at once or by dividing the oligonucleotide into a plurality of times. However, the replication reaction in step (B) may be performed for each of the plurality of capillary members, or may be performed simultaneously.

In the step (A), one end side of a plurality of capillary members is two-dimensionally arranged and used. In the step (B), a replication reaction is simultaneously performed on all the capillary members. Transfer of complementary oligonucleotides to the solid phase surface of all the capillary members at the same time,
In the step (D), it is preferable that all complementary oligonucleotides are simultaneously immobilized on another solid phase surface. As a result, the manufacturing time can be further reduced.

When a large number of DNA chips having the same oligonucleotide matrix are produced by a conventional production method, it is necessary to repeat the same operation for each DNA chip to produce one chip at a time, and the production cost does not decrease. There was a problem. Then, step (C)
Is an operation of transferring only a part of the complementary oligonucleotide synthesized in the step (B) to another solid-phase surface, and after the step (D), 1 or It is preferable to include a step (E) of sequentially moving the remaining complementary oligonucleotides to the surfaces of the plurality of solid phases and immobilizing them. As a result, a large amount of DNA chips having the same oligonucleotide matrix can be easily produced. It is preferable that the transfer of the complementary oligonucleotide to the other solid phase surface in the step (C) is an operation of electrically transferring the complementary oligonucleotide. As a result, the complementary oligonucleotide can be reliably transferred to another solid phase surface.

[0011]

FIG. 1 is a flowchart showing one embodiment. However, this example does not limit the scope of the claims of this specification. (A) A template oligonucleotide 2 having a sequence complementary to an oligonucleotide to be immobilized on a DNA chip is introduced into a hollow capillary tube (capillary member) 4 and immobilized on the inner wall surface of the capillary tube 4. The immobilization of the template oligonucleotide 2 was carried out by introducing an amino group into the end of the template oligonucleotide 2 and introducing an isothiocyanate group to the inner wall surface of the capillary tube 4, and by covalent bonding. However, the immobilization method includes introducing a functional group other than an amino group into the oligonucleotide and subjecting the inner wall of the capillary tube 4 to a surface treatment with a substance having a covalent bond to the functional group so that the oligonucleotide is covalently bonded. Another method may be used, such as a method in which the inner wall of the capillary tube 4 is subjected to a surface treatment with a polycation to make electrostatic coupling by utilizing the charge of the nucleotide.

(B) A number of capillary tubes 4 having different template oligonucleotides 2 immobilized on the inner wall are prepared, and one end side thereof is two-dimensionally arranged and bundled. However, only one capillary tube 4 is shown in FIG. PCR using template oligonucleotide 2 as a template
Perform the reaction. As a device for performing a PCR reaction, for example, T
he RapidCycler (a product of IDAHO TECHNOLOGY INC. (USA)) can be used. A PCR reaction solution 6 is prepared by mixing solutions necessary for the PCR reaction, such as a PCR buffer, primers, dNTP (deoxynucleotide triphosphate), and DNA polymerase, and the capillary reaction tube 6 is filled with the PCR reaction solution 6.

(C) A PCR reaction is carried out by performing a heating and cooling treatment. In the capillary tube 4, an oligonucleotide 8 complementary to the template oligonucleotide 2 is synthesized and hybridized to the template oligonucleotide 2. (D) After the oligonucleotide 8 is formed, the inside of the capillary tube 4 is washed to remove the PCR reaction solution 6. (E) A dissociation solution (not shown) containing a denaturant such as urea or formamide is introduced into the capillary tube 4,
Hybridized template oligonucleotide 2
And oligonucleotide 8 are dissociated. The means for dissociating the template oligonucleotide 2 and the oligonucleotide 8 may be heat denaturation.

(F) A nylon membrane surface 10 (another solid-phase surface) is arranged so as to face one end surface of the capillary tube 4, and one end surface of the capillary tube 4 is brought into contact with the nylon membrane surface 10. When one end surface of the capillary tube 4 is brought into contact with the nylon membrane surface 10, a part of the dissociation solution 12 (the solution obtained by dissociating the template oligonucleotide 2 and the oligonucleotide 8 in the step (E)) in the capillary tube 4 Flows out of the capillary tube 4 due to the adhesive force of the nylon membrane surface 10, and the nylon membrane surface 10
Adheres to The oligonucleotide 8 is moved to the nylon membrane surface 10 together with the dissociation solution 12 flowing out of the capillary tube 4 and is immobilized on the nylon membrane surface 10 by the adhesive force of the nylon membrane surface 10. Although not shown, the complementary oligonucleotides synthesized in a number of two-dimensionally arrayed capillary tubes at one end are simultaneously moved to the nylon membrane surface 10 and immobilized to immobilize the DNA chip. Make it. Thereafter, ultraviolet light may be applied to the nylon film surface 10 to covalently hold the oligonucleotides 8a and 8b on the nylon film surface 10.

After the step (F), the inside of the capillary tube 4 is washed so as not to remove the template oligonucleotide 2, and the PCR reaction solution 6 is filled again to remove the oligonucleotide 8
Is synthesized, and the operation of moving and immobilizing the oligonucleotide 8 to another solid phase surface is repeated, whereby a DNA chip having the same oligonucleotide matrix can be easily produced. In this example, after the oligonucleotide 8 was synthesized, the PCR reaction solution was removed. However, when two or more DNA chips having the same oligonucleotide matrix were to be produced, the complementary oligonucleotide was replaced with a PC.
By synthesizing a large number by R reaction and transferring the PCR reaction solution containing complementary oligonucleotides sequentially to multiple nylon membranes without removing the PCR reaction solution and immobilizing it, it has the same oligonucleotide matrix A DNA chip can be easily manufactured. Also,

Oligonucleotide 8 is applied to nylon membrane surface 1
0, the other end of the capillary tube 4 (the side opposite to the side facing the nylon membrane surface 10) is used as a cathode, the nylon membrane surface 10 is used as an anode, and the other end of the capillary tube 4 is used as a nylon. The film may be electrically moved by applying a voltage between the film surfaces 10. Alternatively, the dissociation solution 12 may be extruded by applying pressure from the other end of the capillary tube 4.

In this embodiment, the nylon membrane is used as the oligonucleotide matrix forming region of the DNA chip. However, the present invention is not limited to this, and the oligonucleotide can be immobilized on glass or another polymer membrane. Any solid phase may be used. Instead of the oligonucleotide, an artificial nucleic acid (also referred to as a peptide nucleic acid) in which a phosphodiester bond of DNA is converted into a peptide bond may be used.

[0018]

According to the method for producing a DNA chip of the present invention,
The template oligonucleotide is immobilized on the inner wall of one or more capillary members, and a replication reaction is performed in the capillary member to synthesize an oligonucleotide complementary to the template oligonucleotide. Since the oligonucleotide is transferred to the solid phase surface and the complementary oligonucleotide is immobilized on another solid phase surface, a DNA chip can be manufactured in a short time and at low cost.

Furthermore, using a plurality of capillary members,
If the transfer of the complementary oligonucleotide to the surface of another solid phase is performed simultaneously by two or more capillary members, the production time can be further reduced. further,
One end side of a plurality of capillary members is arranged two-dimensionally, and a replication reaction is performed simultaneously on all the capillary members, and a complementary oligonucleotide is transferred to another solid phase surface simultaneously on all the capillary members. By immobilizing all complementary oligonucleotides simultaneously on other solid-phase surfaces, the production time can be further reduced,
And the manufacturing cost can be reduced. Further, only a part of the complementary oligonucleotides is moved to another solid surface, and the remaining complementary oligonucleotides are sequentially moved to one or more solid surfaces different from the other solid surfaces. If immobilized, a large amount of DNA chips having the same oligonucleotide matrix can be easily manufactured.

[Brief description of the drawings]

FIG. 1 is a flowchart showing one embodiment.

[Explanation of symbols]

 2,8 Oligonucleotide 4 Capillary tube 6 PCR reaction solution 10 Nylon membrane 12 Dissociation solution

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA11 AA20 CA01 CA09 HA14 HA19 4B029 AA07 AA23 BB20 CC03 CC08 CC11 FA12 FA15 4B063 QA01 QA08 QQ42 QR32 QR84 QS02 QS25 QS34 QS39 QX02

Claims (5)

[Claims] 1. A method for producing a DNA chip comprising the following steps. (A) a step of immobilizing a template oligonucleotide on the inner wall of one or a plurality of capillary members, (B) a step of performing a replication reaction in the capillary member to synthesize an oligonucleotide complementary to the template oligonucleotide,
(C) arranging another solid-phase surface on one end side of the capillary member, and moving the complementary oligonucleotide to the other solid-phase surface; and (D) transferring the complementary oligonucleotide to the other solid-phase surface. Step of immobilizing on the surface of a solid phase. 2. The step (A) uses a plurality of capillary members, and the step (C) uses the two or more capillary members to simultaneously transfer the complementary oligonucleotide to the other solid phase surface. The method for producing a DNA chip according to claim 1, further comprising an operation to be performed. 3. The step (A) uses two-dimensionally arranged one end sides of a plurality of capillary members, and the step (B) performs the replication reaction simultaneously on all the capillary members. (C) performs the transfer of the complementary oligonucleotide to the other solid phase surface simultaneously in all the capillary members, and the step (D) includes transferring all the complementary oligonucleotides to the other solid phase. 3. D according to claim 2, comprising the step of simultaneously immobilizing on the phase surface.
Manufacturing method of NA chip. 4. The step (C) is an operation of transferring only a part of the complementary oligonucleotide synthesized in the step (B) to the other solid phase surface, and the step (D) 4. The method according to claim 1, further comprising a step (E) of sequentially moving the remaining complementary oligonucleotides to one or more solid phase surfaces different from the other solid phase surface and immobilizing the remaining complementary oligonucleotides. 3. The method for producing a DNA chip according to item 1. 5. The method according to claim 1, wherein the transfer of the complementary oligonucleotide to another surface of the solid phase in the step (C) is an operation of electrically transferring the complementary oligonucleotide. The method for producing a DNA chip according to the above.