JP2001108676A - Dna chip manufacturing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To shorten the time required for manufacturing DNA chips and to reduce manufacturing cost. SOLUTION: A PCR reaction liquid 6 is bought into contact with a glass board surface 2 to which oligonucleotides 4a and 4b are fixed (B) to synthesize oligonucleotides 8a and 8b complementary to the oligonucleotides 4a and 4b by an in-situ PCR reaction (c). Then the PCR reaction liquid 6 is removed (D), a nylon film surface 10 is arranged opposite to the glass board surface 2, and a power source device 12 is arranged (E). Then a dissociation liquid is introduced between the glass board surface 2 and nylon film surface 10 to dissociate the oligonucleotides 4a and 4b from the oligonucleotides 8a and 8b, and a voltage is impressed between the glass board surface 2 and nylon film surface 10 by the power source device 12 to move the oligonucleotides 8a and 8b to the side of the nylon film surface 10, to fix them at locations corresponding to the oligonucleotides 4a and 4b (F), and to obtain a replica DNA chip (G).

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, the above DNA
In any of the chip manufacturing methods, there is a problem that it takes a long time to manufacture one DNA chip and the manufacturing cost increases. When a large number of DNA chips having the same oligonucleotide matrix are produced, it is necessary to repeat the same operation for each DNA chip to produce one chip at a time, and there is a problem that the production cost is not reduced. Therefore, an object of the present invention is to reduce the manufacturing time and the manufacturing cost when manufacturing a plurality of DNA chips having the same oligonucleotide matrix.

[0006]

The method for producing a DNA chip of the present invention includes the following steps. (A) performing a PCR reaction using a plurality of oligonucleotides immobilized on a previously prepared DNA chip as a template,
A step of synthesizing an oligonucleotide complementary to the plurality of oligonucleotides, (B) a step of moving the complementary oligonucleotide to another solid phase surface side, and (C) a step of synthesizing the complementary oligonucleotide with the DNA A step of immobilizing the plurality of oligonucleotides on the surface of the other solid phase corresponding to the positions of the plurality of oligonucleotides immobilized on the chip.

A previously prepared DNA chip (hereinafter referred to as a master DNA chip) is prepared. Master DNA
A replication reaction is performed using the oligonucleotide immobilized on the chip as a template. Here, the replication reaction refers to synthesizing an oligonucleotide complementary to the oligonucleotide. After synthesizing complementary oligonucleotides, those complementary oligonucleotides are transferred to another solid phase surface side. A DNA chip having an oligonucleotide matrix immobilized on another solid phase surface corresponding to the position of the oligonucleotide matrix of the master DNA chip and complementary to the oligonucleotide matrix of the master DNA chip (hereinafter referred to as a replica DNA chip)
Get. If a DNA chip having an oligonucleotide matrix complementary to the desired oligonucleotide matrix is used as the master DNA chip, a replica DNA chip having the desired oligonucleotide matrix can be mass-produced in a short time and at low cost. .

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the step (A), PC is used as a replication reaction.
It is preferable to include an operation of removing the PCR reaction solution used in the PCR reaction before transferring the complementary oligonucleotide to another solid phase surface in step (B) using the R reaction. The transfer of the complementary oligonucleotide to the other solid phase surface in the step (B) is preferably an operation of electrically transferring the complementary oligonucleotide. as a result,
Complementary oligonucleotides can be reliably transferred to another solid phase surface side.

[0009]

FIG. 1 is a flowchart showing one embodiment. However, this example does not limit the scope of the claims of this specification. (A) Master DN with oligonucleotide matrix complementary to desired oligonucleotide matrix
Prepare A chip. Oligonucleotides 4a and 4b are immobilized on the glass substrate surface 2 of the master DNA chip. Although a large number of oligonucleotides are fixed on the glass substrate surface 2 in addition to the oligonucleotides 4a and 4b, they are not shown.

(B) An in situ PCR reaction is performed using the master DNA chip as a template. As an apparatus for performing an in situ PCR reaction, for example, TaKaRa PCR Thermal Cycler MP
(Manufactured by Takara Shuzo Co., Ltd.) can be used. PCR buffer, primers, dNTP (deoxynucleotide triphosphate), DNA polymerase, etc.
A solution required for the in situ PCR reaction is mixed to prepare a PCR reaction solution 6, and the glass substrate surface 2 is brought into contact with the PCR reaction solution 6.
Thereafter, in order to prevent evaporation of the PCR reaction solution 6, the glass substrate surface 2 is sealed with a sealing member (not shown).

(C) After heating and cooling,
u Allow the PCR reaction to take place. On the glass substrate surface 2, oligonucleotides 8a and 8b complementary to the oligonucleotides 4a and 4b are synthesized, and the oligonucleotides 4a and 4b are synthesized.
Hybridize to b. (D) For all oligonucleotides immobilized on the glass substrate surface 2, after complementary oligonucleotides are formed, the glass substrate surface 2 is washed to remove the PCR reaction solution 6.

(E) A nylon film surface 10 (another solid surface) is disposed so as to face the glass substrate surface 2. A power supply device 12 for applying a voltage between the glass substrate surface 2 and the nylon film surface 10 is disposed with the glass substrate surface 2 side as a cathode and the nylon film surface 10 side as an anode. A dissociation solution (not shown) containing a denaturant such as urea or formamide is introduced between the glass substrate surface 2 and the nylon membrane surface 10, and the hybridized oligonucleotides 4a and 8a, and the oligonucleotides 4b and 8b are hybridized. At the same time, the power supply device 12 is turned on to apply a voltage between the glass substrate surface 2 and the nylon film surface 10 to move the oligonucleotides 8a and 8b to the nylon film surface 10 side. Here, the means for dissociating the oligonucleotides 4a and 8a, 4b and 8b may be heat denaturation. (F) The oligonucleotides 8a and 8b that have come into contact with the nylon film surface 10 are physically adsorbed to the positions corresponding to the oligonucleotides 4a and 4b by the adsorption action of the nylon film surface 10.

(G) The nylon DNA surface 10 is separated from the glass substrate surface 2, the nylon film surface 10 is washed to remove the dissociation solution, and the replica DNA chip having the desired oligonucleotide matrix formed on the nylon film surface 10. Get. 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.

In this embodiment, a nylon membrane is used as the oligonucleotide matrix forming region of the replica DNA chip. However, the present invention is not limited to this, and the oligonucleotide may be immobilized on a glass or other polymer membrane. Any solid phase can be used. In addition, as a method of immobilizing the oligonucleotide on the surface of the solid phase, there is a method of using the charge of the oligonucleotide to electrostatically bond to a solid phase surface-treated with a polycation, or introducing a functional group into the oligonucleotide. Alternatively, other methods such as a method of covalently bonding to a solid phase surface-treated with a substance having a covalent bond to the functional group may be used. Further, it is preferable that the master DNA chip is manufactured by, for example, the first method or the second method described in the related art. Instead of the master DNA chip on which oligonucleotides are immobilized, a DNA chip on which peptide nucleic acids are immobilized may be used. What is a peptide nucleic acid?
It is an artificial nucleic acid in which a phosphodiester bond of DNA is converted into a peptide bond.

[0015]

According to the method for producing a DNA chip of the present invention,
A PCR reaction is performed using the plurality of oligonucleotides immobilized on the master DNA chip as a template to synthesize a complementary oligonucleotide, and the complementary oligonucleotide is placed at the position where the plurality of oligonucleotides are fixed on the master DNA chip. Correspondingly, the replica DNA chip was immobilized on the solid phase surface, so that the replica DNA chip having an oligonucleotide matrix complementary to the oligonucleotide matrix of the master DNA chip was used.
NA chips can be mass-produced in a short time and at low cost.

[Brief description of the drawings]

FIG. 1 is a flowchart showing one embodiment.

[Explanation of symbols]

 2 Surface of glass substrate 4a, 4b Oligonucleotide 6 PCR reaction solution 8a, 8b Complementary oligonucleotide 10 Nylon membrane 12 Power supply

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[Procedure amendment]

[Submission date] October 20, 2000 (2000.10.
20)

[Procedure amendment 1]

[Document name to be amended] Drawing

[Correction target item name] Fig. 1

[Correction method] Change

[Correction contents]

FIG.

Claims (3)

[Claims] 1. A method for producing a DNA chip comprising the following steps. (A) performing a replication reaction using a plurality of oligonucleotides immobilized on a DNA chip prepared in advance as a template,
A step of synthesizing an oligonucleotide complementary to the plurality of oligonucleotides, (B) a step of moving the complementary oligonucleotide to another solid surface, and (C) a step of synthesizing the complementary oligonucleotide with the DNA A step of immobilizing the plurality of oligonucleotides on the surface of the other solid phase corresponding to the positions of the plurality of oligonucleotides immobilized on the chip. 2. The step (A) is performed as the replication reaction.
The DNA chip according to claim 1, further comprising an operation of removing a PCR reaction solution used in the PCR reaction by using a PCR reaction before transferring the complementary oligonucleotide to another solid phase surface in the step (B). Manufacturing method. 3. The DNA according to claim 1, wherein the transfer of the complementary oligonucleotide to another solid phase surface in the step (B) is an operation of electrically transferring the complementary oligonucleotide. Chip manufacturing method.