JP2002243735A - Method and apparatus for producing probe carrier, and apparatus for analyzing sequence nucleic acid sequence - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To create various kinds of nucleic acid sequences in a short time with a simple device. SOLUTION: A carrier on which a nucleic acid sequence is arranged and a plurality of kinds of synthesis reaction reagents for synthesizing nucleic acid are prepared, and the carrier is brought into contact with the synthesis reaction reagent to synthesize the nucleic acid on the carrier. This synthesis process is sequentially repeated while sequentially selecting the corresponding synthesis reaction reagents according to the nucleic acid sequence to be prepared. As a result, a probe carrier in which nucleic acid sequences corresponding to the order of contact are synthesized on the carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an apparatus for analyzing a nucleic acid sequence, and a method and an apparatus for preparing a probe carrier used for analyzing the nucleic acid sequence.

[0002]

2. Description of the Related Art As a method for analyzing a nucleic acid sequence which is a gene, a so-called DNA microarray or DNA chip in which a large number of nucleic acids (probes) having different sequences are arranged at a high density is prepared and analyzed. A method for hybridizing a nucleic acid (target) is known.

[0003] There are two types of DNA microarrays depending on the method of preparation. One is that many types of oligo DN
A is a type in which A is synthesized at a predetermined position on a glass substrate using lithography technology, and is known as a synthetic DNA chip. The other one is a type in which oligo DNA synthesized in advance is spotted on a glass substrate, and is called an attached DNA microarray.

[0004] Nucleic acids include four types of bases constituting DNA, namely, adenine (A), guanine (G), cytosine (C), and thymine (T), and the property that these bases bind complementarily is used. Thereby, DNA analysis can be performed. For example, adenine (A) and thymine (T),
Guanine (G) and cytosine (C) bind complementarily, respectively. By utilizing this property, it is possible to selectively detect DNA complementary to the oligo DNA arranged on the DNA microarray.

[0005]

However, the conventional method for preparing a carrier on which a nucleic acid (probe) used for analyzing a nucleic acid sequence is arranged has problems in terms of equipment and time, and it is difficult to simply prepare it. For example, in a type called a conventional synthetic DNA chip, a large number of sequences can be synthesized at a high density in a short time because a large number of sequences are synthesized simultaneously using lithography technology according to the sequence information. There is an advantage. However, since this synthetic DNA chip is prepared using a lithography technique, there is a problem that an expensive and large-scale manufacturing apparatus is required as in a semiconductor manufacturing apparatus. In the lithography technique, since a resin used for masking and an etching reagent for removing the resin are used, a device must be devised so that the synthesis reaction of the nucleic acid is not inhibited by the resin or the etching reagent.

In a type called an attached DNA microarray, since oligo DNAs are individually synthesized in advance, a conventionally known synthesis reaction can be used as it is. However, the paste type D
In the preparation of NA microarrays, there is a problem that it takes an enormous amount of time to prepare various types of nucleic acid sequences because oligo DNAs are individually synthesized. Further, there is also a problem that it is difficult to obtain a large number of high-density arrays because pins used for attaching the oligo DNA to the substrate are worn. In addition, since a large-sized apparatus and a long time are required to prepare a probe carrier, analysis of a nucleic acid sequence using these conventional probe carriers requires time for analysis.

Accordingly, the present invention has been made to solve the above-mentioned conventional problems, and has as its object to prepare various types of nucleic acid sequences with a simple apparatus in a short time, and to analyze a nucleic acid sequence in a simple and short time. The purpose is to:

[0008]

SUMMARY OF THE INVENTION The present invention provides a method of arranging nucleic acid sequences on a carrier by a simple method and apparatus without using a lithography technique requiring an expensive and large-scale manufacturing apparatus. And a method for preparing a probe carrier, an apparatus for preparing a probe carrier, a probe carrier, and an apparatus for analyzing a nucleic acid sequence using a probe carrier. .

A first aspect of the present invention is a method for producing a probe carrier. The method for producing a probe carrier of the present invention comprises preparing a carrier on which a nucleic acid sequence is arranged, and a plurality of types of synthesis reaction reagents for synthesizing the nucleic acid, and contacting the carrier with the synthesis reaction reagent to convert the nucleic acid onto the carrier. Let them be combined. This synthesis process is sequentially repeated while sequentially selecting the corresponding synthesis reaction reagents according to the nucleic acid sequence to be prepared. As a result, a probe carrier in which nucleic acid sequences according to the order of contact are synthesized on the carrier can be produced.

Here, the carrier is a support member for holding the nucleic acid, and can be any material that does not affect the nucleic acid. The light-transmitting material such as glass fiber or plastic fiber is used for the probe of the present invention. Suitable for production and nucleic acid sequence analysis. The shape of the carrier is thread-like, rod-like,
It can be plate-shaped, cylindrical, spherical, or the like. The nucleic acid is D
In addition to the four bases that constitute NA, such as adenine (A), guanine (G), cytosine (C), and thymine (T), the base can be an amino acid, and the four bases can be used to analyze the nucleic acid sequence to be analyzed. By making the complementary strand a nucleic acid sequence,
A nucleic acid sequence to be analyzed can be analyzed, and a polypeptide (protein) can be synthesized by using amino acids, and a protein to be analyzed can be analyzed.

According to the method for producing a probe carrier of the present invention, a conventional DNA chip lithography technique
A nucleic acid sequence can be synthesized by a simple and short-time treatment of simply bringing a carrier into contact with a synthesis reaction reagent without using microarray pins.

A second aspect of the present invention is a probe carrier. The probe carrier of the present invention is prepared by preparing a plurality of carriers in which a kind of nucleic acid sequence is arranged on at least one end side of one light-transmitting carrier, and aligning the plurality of carriers so that the ends are in the same direction. It is arranged. The plurality of carriers can be rearranged in an arbitrary shape such as a rectangular shape, a circular shape, and a band shape. According to this probe carrier, a type of nucleic acid sequence is arranged on each carrier, and by identifying the probe carrier that has reacted with the target nucleic acid, the target nucleic acid is analyzed from the nucleic acid sequence created on the probe carrier. And the identification of the nucleic acid sequence is facilitated. Further, by preparing and rearranging each probe carrier corresponding to the type of the nucleic acid sequence to be analyzed, the analysis of the nucleic acid sequence can be made more efficient.

A third aspect of the present invention is an apparatus for producing a probe carrier. The apparatus for preparing a probe carrier according to the present invention includes a plurality of synthesis reaction tanks each including a nucleic acid synthesis reaction reagent for each synthesis reaction reagent, and a configuration including moving means for relatively moving the synthesis reaction tank and the plurality of carriers. I do. The synthesis reaction tank prepares a nucleic acid synthesis reaction reagent for each reagent. For example, when four types of bases of adenine (A), guanine (G), cytosine (C), and thymine (T) are used as nucleic acids, Then, four synthesis reaction vessels containing each base are prepared. In addition, each synthesis reaction tank is provided with a processing portion for synthesizing a carrier, in addition to holding a nucleic acid such as a base to be synthesized. This processing portion can include sub-processing such as cleaning and chemical activation as necessary.

[0014] The moving means can move a synthesis reaction tank or a plurality of supports in a lump, and can also select and move an arbitrary support (including a plurality of supports) from the plurality of supports. Thus, the selected carrier is brought into contact with the selected synthesis reaction reagent. The transfer means synthesizes nucleic acids sequentially on the carriers by repeating an operation of bringing an arbitrary carrier selected from a plurality of carriers into contact with a synthesis reaction reagent in a selected synthesis reaction tank, thereby preparing a probe carrier. The moving procedure of the moving means can be set in a program in advance, for example, and the carrier or the synthesis reaction tank can be moved according to the program.

A fourth aspect of the present invention is an apparatus for analyzing a nucleic acid sequence. The nucleic acid sequence analyzing apparatus of the present invention comprises: a light detecting means for detecting, per carrier, transmitted light transmitted through a probe carrier obtained by synthesizing a nucleic acid sequence on a light-permeable carrier; and an analyzing means for analyzing a nucleic acid sequence of a target nucleic acid. And a configuration including: The target nucleic acid or the nucleic acid sequence on the carrier is provided with a fluorescent label, and the target nucleic acid and the nucleic acid sequence on the carrier are caused to emit fluorescence by a hybridization reaction.
Since the light detection means of the present invention can detect each carrier, the carrier that has reacted with the target nucleic acid can be identified by detecting luminescence. By specifying the carrier, the nucleic acid sequence arranged on the carrier can be specified.

The analyzing means specifies the nucleic acid sequence arranged on the carrier by specifying the carrier detected by the light detecting means, and specifies the nucleic acid sequence of the target nucleic acid in a complementary relationship from the nucleic acid sequence. Thereby, the nucleic acid sequence of the target nucleic acid can be analyzed. According to the nucleic acid sequence analysis device of the present invention, a nucleic acid sequence complementary to a target nucleic acid can be identified by a simple process of simply detecting a luminescent carrier. The sequence can be analyzed.

In another embodiment of the apparatus for preparing a probe carrier of the present invention, a photoreaction can be applied to a synthesis reaction in a synthesis reaction tank. In this embodiment, in the reaction in the synthesis reaction tank, the photoprotecting group is removed by ultraviolet rays. According to this embodiment, a plurality of carriers are kept in contact with the synthesis reaction reagent in the synthesis reaction tank at a time. A combining process can be performed. According to this, the operation of selecting and moving a carrier according to the presence or absence of nucleic acid synthesis can be eliminated, and the mask formation of the lithography technique required for the conventional photoreaction can be eliminated. Can be.

In an embodiment in which a photoreaction is applied to this synthesis reaction, one preferred embodiment of a carrier is a rod-shaped body of a light-transmitting material,
Alternatively, it is a hollow cylindrical body made of a light transmissive material and provided with a light shielding material on the outer peripheral surface. In this carrier, a light-emitting means is provided at the end opposite to the side on which the nucleic acid sequence is synthesized, and light such as ultraviolet light is incident from the end to irradiate the position where the nucleic acid sequence is synthesized with ultraviolet light, and the nucleic acid sequence is synthesized. Perform processing. The nucleic acid sequence is formed on the inner peripheral surface of the cylindrical body, and the light-shielding material provided on the outer peripheral surface can suppress synthesis in the adjacent carrier.

[0019]

Embodiments of the present invention will be described below in detail with reference to the drawings. In the following, FIGS.
The first embodiment of the apparatus for producing a probe carrier will be described with reference to FIGS. 5 to 7, and the second embodiment of the apparatus for producing a probe carrier will be described with reference to FIGS.
And an apparatus for analyzing a nucleic acid sequence will be described with reference to FIG. FIG. 9 shows another embodiment of the apparatus for producing a probe carrier, and FIG. 10 shows an embodiment of the carrier.

First, the first of the apparatus for producing a probe carrier is described.
1 will be described with reference to the schematic configuration diagram of the preparation apparatus in FIG. 1, the flowchart of the procedure for synthesizing the nucleic acid sequence on the carrier in FIG. 2, and the schematic diagram illustrating the procedure for synthesizing the nucleic acid sequence on the carrier in FIGS. I do. In the first embodiment, the nucleic acid can be immobilized on a carrier by applying a synthesis by a chemical bond such as a covalent bond or a synthesis using a photoreaction. Therefore, in the following description, a synthesis example using a chemical bond such as a covalent bond will be described with reference to FIG. 3, and a synthesis example using a photoreaction will be described with reference to FIG.

The probe carrier 2 includes a plurality of carriers 1, and each carrier 1 is provided with one kind of nucleic acid sequence. The probe carrier producing apparatus 10 individually synthesizes a nucleic acid on each carrier 1 constituting the probe carrier 2 and arranges the nucleic acid sequence. The type of the nucleic acid sequence is arbitrary, and may be different for each carrier 1 or may be the same.

The preparation apparatus 10 includes a plurality of synthesis reaction tanks 11.
(11a-11d) and moving means 12 (12a, 12b)
Is provided. The synthesis reaction vessels 11 (11a to 11d) are provided with synthesis reaction reagents used for nucleic acid synthesis for each synthesis reaction reagent.
For example, when four types of bases of adenine (A), guanine (G), cytosine (C), and thymine (T) are used as nucleic acids, each of the synthesis reaction tanks 11a to 11d performs each synthesis reaction. A tank is provided, and each base is fixed to a carrier in the synthesis reaction tank. In addition, each synthesis reaction tank is provided with a processing portion for synthesizing a carrier, in addition to holding a nucleic acid such as a base to be synthesized. In addition, this synthesis reaction tank 11
Can include sub-treatments such as washing and chemical activation as needed.

The moving means 12 relatively moves the probe carrier 2 and the synthesis reaction tank 11 and causes the selected carrier 1 to undergo a synthesis reaction in the selected synthesis reaction tank 11. Here, an example is shown in which the synthesis reaction tank 11 is fixed, and the probe carrier 2 is moved with respect to the synthesis reaction tank 11 by the moving means 12. The moving means 12
A first moving means 12a for moving the probe carrier 2 and a second moving means 1 for moving a specific carrier 1 in the probe carrier 2
2b. The first moving means 12a is a synthesis reaction tank 1 for performing synthesis by moving the entire probe carrier 2.
Select 1. On the other hand, the second moving means 12b selects the carrier 1 from the probe carrier 2, moves to the synthesis reaction tank 11 selected by the first moving means 12a, and causes only the specific carrier to synthesize nucleic acid.

The movement of the moving means 12 is controlled by a drive control means 13 controlled by a control means 14. The control means 14 operates in accordance with an operation procedure set by an input means 16 or the like and stored in a storage means 15. Can be controlled. In the flowchart of FIG. 2, first, a nucleic acid sequence to be arranged on each carrier is set. For example, when analyzing a nucleic acid sequence of DNA, a sequence of four types of bases, adenine (A), guanine (G), cytosine (C), and thymine (T), which are complementary to the nucleic acid sequence, is set. (Step S1).

Based on the set nucleic acid sequence, moving means 1
2 is set. This driving procedure includes selection of the synthesis reaction tank 11 by the first moving means 12a and selection of the carrier by the second moving means 12b. The set driving procedure can be stored in the storage means 15 and read out by the control means 14. it can. The driving procedure can be set from the input means 16 or a setting means (not shown) can be provided to set the driving procedure based on the nucleic acid sequence input from the input means 16 (step S2).

The probe carrier 2 is formed, for example, by holding the carrier 1 in a number corresponding to the type of the nucleic acid sequence to be prepared by a holding means (not shown), and is set on the second moving means 12b (step S3). The moving means 12 is driven. The control means 14 sequentially reads the drive procedure program stored in the storage means 15 and
3, the first moving means 12a and the first moving means 12
b is driven (step S4 to step S8). As a result, the preparation device 10 can automatically prepare a preset nucleic acid sequence.

Next, an example of synthesis using a chemical bond such as a covalent bond will be described with reference to FIG. In this example, a probe carrier 2 composed of four carriers 1A to 1D is used with four bases of adenine (A), guanine (G), cytosine (C), and thymine (T) to form 1A to 1D. T for each carrier
Although an example is shown in which nucleic acid sequences of G, TG, TA, and TG are prepared, the number of carriers and the length of bases to be prepared can be arbitrary.

When the probe carrier 2 is at the position of the synthesis reaction tank 11a (FIG. 3A), the probe carrier 2 is moved from the synthesis reaction tank 11a by the first moving means 12a to thymine (T).
Is moved onto the synthesis reaction tank 11b for synthesizing the probe carrier, and the entire probe carrier 2 is moved to the synthesis reaction tank 11b by the second moving means 12b. In the synthesis reaction tank 11b,
Carriers 1A-1D come into contact with thymine (T) and bind covalently or otherwise. In addition, this synthesis reaction includes processes such as washing and chemical activity as necessary. Thereby, thymine (T) is fixed to the carriers 1A to 1D (FIG. 3B).

After the thymine (T) is fixed to the carriers 1A to 1D, the entire probe carrier 2 is separated from the synthesis reaction tank 11b by the second moving means 12b (FIG. 3 (c)). Next, the first
The synthesis reaction tank 11 for synthesizing guanine (G) from the synthesis reaction tank 11b using the transfer means 12a to synthesize the entire probe carrier 2
c, and the carriers 1A, 1B, 1D selected from the probe carrier 2 are moved to the synthesis reaction tank 11c by the second moving means 12b. In the synthesis reaction tank 11c, the carriers 1A, 1B, and 1D except the carrier 1C come into contact with guanine (G) and bind in the same manner as described above. by this,
Thymine (T) and guanine (G) are immobilized on the carriers 1A, 1B, 1D in the order of TG. The carrier 1C remains in a state where only thymine (T) is immobilized (FIG. 3).
(D)).

After guanine (G) is fixed on the carriers 1A, 1B, 1D, the carriers 1A, 1B, 1D are fixed by the second moving means 12b.
B and 1D are separated from the synthesis reaction tank 11c (FIG. 3 (e)).
Next, the entire probe carrier 2 is moved from the synthesis reaction tank 11c to the synthesis reaction tank 11a for synthesizing adenine (A) by the first movement means 12a.
Only carrier 1C selected from probe carriers 2 by b is moved to synthesis reaction tank 11a. In the synthesis reaction tank 11a, only the carrier 1C comes into contact with adenine (A), and the binding is performed in the same manner as described above. Thus, thymine (T) and adenine (A) are immobilized on the carrier 1C in the order of TA (FIG. 3 (f)). After immobilizing adenine (A) on the carrier 1C, the carrier 1C is separated from the synthesis reaction tank 11a by the second moving means 12b (FIG. 3 (g)).

Thus, a nucleic acid sequence in the order of TG is created on the carriers 1A, 1B and 1D, and a nucleic acid sequence in the order of TA is created on the carrier 1C. In addition, on the surface of each carrier 1,
An amino group is formed by a silane treatment as a pretreatment for performing a nucleic acid synthesis treatment.

Next, an example of synthesis by a photoreaction will be described with reference to FIG. In this example, as in the example of FIG. 3 described above, the probe carrier 2 including the four carriers 1A to 1D is
An example of preparing a nucleic acid sequence of TG, TG, TA, TG on each of the carriers 1A to 1D using four types of bases of adenine (A), guanine (G), cytosine (C), and thymine (T). Is shown. The number of carriers synthesized by the photoreaction and the length of the base to be prepared can be arbitrarily determined.

An amino group is formed on the surface of each carrier 1 by silane treatment, and a photoprotective molecule X is bound thereto (FIG. 4).
(A)). The probe carrier 2 is moved by the first moving means 12a.
Is moved onto the synthesis reaction tank 11b for synthesizing thymine (T), and the entire probe carrier 2 is moved to the synthesis reaction tank 11b by the second moving means 12b. Synthetic reaction tank 1
In 1b, ultraviolet rays (hμ in the figure) are applied to carriers 1A to 1D.
(FIG. 4 (b)) to remove the photoprotecting group to expose the amino group (FIG. 4 (c)). Then, the thymine (T) with the exposed amino group and the photoprotecting group X is added. React the base. As a result, thymine (T) having the photoprotecting group X is formed on the surfaces of the carriers 1A to 1D (FIG. 4D).

After immobilizing thymine (T) on the carriers 1A to 1D, the entire probe carrier 2 is separated from the synthesis reaction tank 11b by the second moving means 12b, and then the first moving means 12a
The whole probe carrier 2 is moved from the synthesis reaction tank 11b to the synthesis reaction tank 11c for synthesizing guanine (G), and the carriers 1A, 1B, 1D selected from the probe carrier 2 are synthesized by the second moving means 12b. Tank 11
Move to c. In the synthesis reaction tank 11c, the carrier 1C
The carriers 1A, 1B and 1D excluding the above are irradiated with ultraviolet rays (hμ in the figure) to remove the photoprotective groups and expose the amino groups, and then the guanine (G) having the exposed amino groups and the photoprotective groups X )) (FIG. 4 (e)). Thus, thymine (T) and guanine (G) with photoprotecting group X
Are formed on the surfaces of the carriers 1A, 1B, 1D. It should be noted that only thymine (T) having the photoprotective group X was formed on the carrier 1C (FIG. 4 (f)).

After guanine (G) is fixed to the carriers 1A, 1B and 1D, the carriers 1A, 1B and 1D are fixed by the second moving means 12b.
B and 1D are separated from the synthesis reaction tank 11c, and then the entire probe carrier 2 is moved by the first moving means 12a.
1c is moved onto the synthesis reaction tank 11a for synthesizing adenine (A), and only the carrier 1C selected from the probe carrier 2 is moved by the second moving means 12b.
Move to a. In the synthesis reaction tank 11a, the carrier 1
Ultraviolet rays (hμ in the figure) on the carrier 1C excluding A, 1B and 1D
To expose the amino group by removing the photoprotective group of the carrier 1C, and then reacting the exposed amino group with the base of adenine (A) having the photoprotective group X (FIG. 4).
(G)). As a result, thymine (T) and adenine (A) having a photoprotecting group X are formed on the surface of the carrier 1C.
Note that thymine (T) and guanine (G) with a photoprotecting group X are formed on the carriers 1A, 1B, and 1D (FIG. 4).
(H)).

Next, a second embodiment of the apparatus for preparing a probe carrier will be described with reference to the schematic configuration diagram of the preparation apparatus shown in FIG. 5 and the schematic view illustrating the procedure for synthesizing a nucleic acid sequence on the carrier shown in FIGS. I do. The second embodiment is a method of immobilizing nucleic acids using a photoreaction, in which a plurality of carriers are collectively brought into contact with a synthesis reaction reagent in a synthesis reaction tank, and light is selectively transmitted to the light-transmissive carriers. The photoreaction is selectively performed by sending, and the configuration for selecting and moving the carrier as in the first embodiment is not required.

The probe carrier 2 used in the second embodiment has a plurality of carriers 1 as in the first embodiment, and each carrier 1 is provided with one kind of nucleic acid sequence. The nucleic acid is synthesized and the nucleic acid sequence is arranged. The carrier 1 used in the second embodiment is formed of a light transmitting material.

The production apparatus 20 includes a plurality of synthesis reaction tanks 21.
(21a to 21d) and moving means 22 (22a to 22d)
And light emitting means 27. Synthesis reaction tank 21 (21a-2)
1d) includes a synthesis reaction reagent used for nucleic acid synthesis for each synthesis reaction reagent. As in the first embodiment, each of the synthesis reaction tanks 11a to 11d is provided with, for example, four types of bases of adenine (A), guanine (G), cytosine (C), and thymine (T) in each synthesis reaction tank. Then, each base is fixed to a carrier in the synthesis reaction tank.

The moving means 22 is for relatively moving the probe carrier 2 and the synthesis reaction tank 11 and causing the selected carrier 1 to perform a synthesis reaction in the selected synthesis reaction tank 11. The moving means 22 includes a first moving means 22a for moving the probe carrier 2 between the synthesis reaction tanks, and a second moving means 22b for moving the probe carrier with respect to each synthesis reaction tank. The first moving means 22a selects the synthesis reaction tank 21 for performing synthesis by moving the entire probe carrier 2. On the other hand, the second moving means 22b moves the entire probe carrier 2 to the synthesis reaction tank 21 selected by the first moving means 22a, and brings the probe carrier 2 into contact with the synthesis reaction reagent at once.

A light emitting means 27 is provided on the opposite side of the probe carrier 2 from the reaction side, and the light emission control means 28 controls light emission for each carrier. The moving means 22 is driven and controlled by a drive control means 23 controlled by a control means 24 in the same manner as in the first embodiment.
Is controlled by the light emission control means 23 controlled by the control means 24. The control means 24 is an operation and light emission procedure set by the input means 26 and stored in the storage means 25, or an operation and light emission procedure set by the setting means (not shown) based on the nucleic acid sequence inputted from the input means 26. Is controlled based on.

Next, an example of synthesis according to the second embodiment will be described with reference to FIGS.
This will be described with reference to FIG. In this example, as in the first embodiment described above, the probe carrier 2 composed of the four carriers 1A to 1D is provided with adenine (A), guanine (G), cytosine (C), and thymine (T). 1A to 1
An example is shown in which nucleic acid sequences of TG, TG, TA, and TG are prepared on each carrier of D. The number of carriers synthesized by the photoreaction and the length of the base to be prepared can be arbitrarily determined.

An amino group is formed on the surface of each carrier 1 by silane treatment, and a photoprotective molecule X is bound thereto (FIG. 6).
(A), FIG. 7 (a)). Synthetic reaction tank 21 for synthesizing thymine (T) from probe carrier 2 by first moving means 22a
Then, the entire probe carrier 2 is moved to the synthesis reaction tank 11b by the second moving means 22b, and is brought into contact with the thymine (T) base. In the synthesis reaction tank 11b, ultraviolet light (hμ in the figure) is irradiated from the light emitting means 27 using the light transmittance of the carriers 1A to 1D (FIG. 6B, FIG. 7).
(B)), the photoprotecting group is removed to expose the amino group (FIG. 7 (c)), and then the exposed amino group is reacted with a base of thymine (T) having a photoprotecting group X. As a result, the thymine (T) with the photoprotecting group X was converted to the carriers 1A to 1D
(FIG. 6 (c), FIG. 7 (d)).

After the thymine (T) is fixed to the carriers 1A to 1D, the entire probe carrier 2 is separated from the synthesis reaction tank 21b by the second moving means 22b, and then the first moving means 22a
The entire probe carrier 2 is moved from the synthesis reaction tank 21b to the synthesis reaction tank 21c for synthesizing guanine (G), and further, the entire probe carrier 2 is moved to the synthesis reaction tank 11c by the second moving means 22b, and guanine ( G)
Contact with base. In the synthesis reaction tank 21c, the carrier 1
The carriers 1A, 1B and 1D excluding C are irradiated with ultraviolet rays (hμ in the figure) from the light emitting means 27 using the light transmittance of the carriers.
The photoprotecting group is removed to expose the amino group, and then the exposed amino group is reacted with a base of guanine (G) having a photoprotecting group X (FIGS. 6 (d) and 7 (e)). As a result, thymine (T) and guanine (G) with a photoprotecting group X are formed on the surfaces of the carriers 1A, 1B, and 1D. Note that only thymine (T) with the photoprotecting group X is formed on the carrier 1C (FIGS. 6 (e) and 7 (f)).

After immobilizing guanine (G) on the carriers 1A, 1B and 1D, the entire probe carrier 2 is separated from the synthesis reaction tank 11c by the second moving means 22b, and then the entire probe carrier 2 is moved by the first moving means 22a. To the synthesis reaction tank 11
The probe carrier 2 is moved onto the synthesis reaction tank 21a for synthesizing adenine (A) from c, and the entire probe carrier 2 is moved to the synthesis reaction tank 21a by the second moving means 22b. In the synthesis reaction tank 21a, the carrier 1C excluding the carriers 1A, 1B, and 1D is irradiated with ultraviolet light (hμ in the figure) from the light emitting means 27 using the light transmittance of the carrier to remove the photoprotecting group of the carrier 1C. The amino group is exposed, and then the exposed amino group is reacted with a base of adenine (A) having a photoprotecting group X (FIGS. 6 (f) and 7 (g)). As a result, thymine (T) and adenine (A) with photoprotecting group X are converted to carrier 1C.
Formed on the surface. Note that thymine (T) and guanine (G) having a photoprotecting group X were provided on the carriers 1A, 1B, and 1D.
Are formed (FIGS. 6 (g) and 7 (h)).

According to the second embodiment, a plurality of carriers can be brought into contact with the synthesis reaction reagent at a time, and a mechanism for selecting and moving the carriers as in the first embodiment is unnecessary. can do.

Next, the nucleic acid sequence analyzer of the present invention will be described with reference to FIG. 8 (a) and 8 (b), the analyzer 30 has a holder 31 on the side opposite to the side on which the nucleic acid sequence is arranged, with the probe carrier 2 of the present invention at the end.
And the light detection means 32 detects the light emission from each carrier 1 for each carrier. The analyzing means 33 includes the light detecting means 32
By specifying the carrier to be detected by the method, the carrier that has reacted with the target nucleic acid can be specified, and the nucleic acid sequence of the target nucleic acid can be complementarily analyzed from the nucleic acid sequence formed on the carrier. Note that each carrier 1 on the holder 31 side and the light detecting means 32
Means that they are directly connected as shown in FIG.
Alternatively, as shown in FIG. 8B, they can be coupled by using an optical fiber or other light guide.

In order to apply the carrier 1 prepared by the preparation method of the present invention to the analyzer of the present invention, one kind of nucleic acid sequence is arranged on at least one end of one light-transmitting carrier.
As shown in FIG. 8 (c), the carrier 1 can be produced by aligning the ends in the same direction and rearranging a plurality of them, thereby holding the carrier 1 with the holder 31. 8 (d)).

In the analysis of a nucleic acid sequence by the nucleic acid sequence analyzing apparatus, a fluorescent label is provided on a target nucleic acid or a nucleic acid sequence on a carrier, and fluorescence is emitted by a reaction by hybridization between the target nucleic acid and the nucleic acid sequence on the carrier. Light emission is detected by light detection means. Since the light detection means of the present invention can detect each carrier, by detecting luminescence, it is possible to identify the carrier that has reacted with the target nucleic acid, and by identifying the carrier, the nucleic acid sequence arranged on the carrier can be identified. Identify. The analysis means specifies the nucleic acid sequence arranged on the carrier by specifying the carrier detected by the light detection means, and specifies the nucleic acid sequence of the target nucleic acid in a complementary relationship from the nucleic acid sequence. Thus, the nucleic acid sequence of the target nucleic acid is analyzed. According to the nucleic acid sequence analyzer of the present invention, the identification of a nucleic acid sequence in a complementary relationship with a target nucleic acid can be performed by a simple process of simply detecting a luminescent carrier,
Nucleic acid sequences can be analyzed easily and in a short time.

FIG. 9 is a schematic view showing another embodiment of the apparatus for producing a probe carrier of the present invention. In this embodiment, the moving means 1
2 is a rotation example, and as shown in FIG. 9A, the first moving unit 12a causes the second moving unit 12b to rotate.
Is rotated, whereby the synthesis reaction tanks 11a-1a-1
The combination of the probe carrier 2 for 1d is changed.
The operation of the preparation apparatus can be the same as that of the first embodiment. For example, in FIG. 9B, a predetermined carrier is brought into contact with a synthesis reaction reagent in the synthesis reaction tank 11a to synthesize nucleic acids ( 9 (c)), the second moving means 12b is moved by the first moving means 12a (FIG. 9 (d)), and FIG. 9 (e).
In, a predetermined carrier is brought into contact with the synthesis reaction reagent in the synthesis reaction tank 11b.

FIG. 10 shows an example of the structure of a carrier applied to the present invention. The carrier 1a shown in FIG. 10 (a) is a rod-shaped body formed of a light-transmitting material, on which a nucleic acid sequence is arranged. When the nucleic acid is immobilized using a photoreaction, it can be performed by irradiating ultraviolet rays or the like from the outside of the carrier 1a, or by irradiating ultraviolet rays from one end and transmitting the inside. The carrier 1b shown in FIG. 10 (b) is a hollow cylindrical body formed of a light transmissive material, and has a nucleic acid sequence arranged on its outer peripheral surface or inner peripheral surface. When one end of the hollow is brought into contact with the synthesis reaction reagent, the synthesis reaction reagent can be automatically attached to the inner peripheral surface by capillary action. Note that irradiation with ultraviolet light or the like can be performed in the same manner as in FIG.

The carrier 1c shown in FIG.
The carrier 1b shown in (b) is provided with a light shielding material and a light protecting group on the outer peripheral surface. According to this configuration, it is possible to prevent ultraviolet light transmitted through the inside of the carrier 1b from leaking to the outside,
It is possible to suppress the reaction of the ultraviolet rays to the adjacent carrier, and make only the specific carrier photoreact. The carrier 1d shown in FIG. 10D is formed by forming a light-transmitting material into a porous material. According to this, the light transmittance can be obtained, and the area in which the nucleic acid sequence is formed can be increased.

[0052]

As described above, according to the method and apparatus for preparing a probe carrier of the present invention, various types of nucleic acid sequences can be prepared in a short time with a simple apparatus. Further, according to the nucleic acid sequence analysis device of the present invention, a nucleic acid sequence can be analyzed in a short time with a simple device.

[Brief description of the drawings]

FIG. 1 is a schematic configuration diagram for explaining a first embodiment of a probe carrier producing apparatus according to the present invention.

FIG. 2 is a flowchart of a procedure for synthesizing a nucleic acid sequence on a carrier in the first embodiment of the apparatus for producing a probe carrier of the present invention.

FIG. 3 is a schematic diagram illustrating a procedure for synthesizing a nucleic acid sequence on a carrier in the first embodiment of the apparatus for producing a probe carrier of the present invention.

FIG. 4 is a schematic diagram illustrating a procedure for synthesizing a nucleic acid sequence on a carrier in the first embodiment of the apparatus for producing a probe carrier of the present invention.

FIG. 5 is a schematic configuration diagram for explaining a second embodiment of the apparatus for producing a probe carrier according to the present invention.

FIG. 6 is a schematic diagram illustrating a procedure for synthesizing a nucleic acid sequence on a carrier in the second embodiment of the apparatus for producing a probe carrier of the present invention.

FIG. 7 is a schematic diagram illustrating a procedure for synthesizing a nucleic acid sequence on a carrier in the second embodiment of the apparatus for producing a probe carrier of the present invention.

FIG. 8 is a schematic diagram for explaining the nucleic acid sequence analyzing apparatus of the present invention.

FIG. 9 is a view showing another embodiment of the apparatus for producing a probe carrier of the present invention.

FIG. 10 is a view showing a form example of a carrier applied to the present invention.

[Explanation of symbols]

1, 1A, 1B, 1C, 1D, 1a, 1b, 1c, 1d
... Carrier, 2 ... Probe carrier, 10, 20 ... Preparation device, 1
1, 11a, 11b, 11c, 11d, 21, 21a,
21b, 21c, 21d: synthesis reaction tank, 12, 22: moving means, 12a, 22a: first moving means, 12b, 22
b: second moving means, 13, 23 ... drive control means, 14,
24 ... control means, 15, 25 ... storage means, 16, 26 ...
Input means 27 light-emitting means 28 light-emitting control means 30
... Analyzer, 31 ... Holder, 32 ... Light detecting means, 33 ...
Analysis means, 34 ... light guide.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 37/00 102 C12N 15/00 FA

Claims (4)

[Claims] 1. A method for producing a probe carrier, comprising synthesizing a nucleic acid sequence on a carrier by successively repeating contact between the carrier and a nucleic acid synthesis reaction reagent to produce a probe carrier. 2. A probe carrier, wherein a plurality of carriers having a kind of nucleic acid sequence arranged on at least one end side on one light-transmitting carrier are rearranged with their ends aligned in the same direction. 3. A method according to claim 1, further comprising: a plurality of synthesis reaction tanks each including a nucleic acid synthesis reaction reagent for each synthesis reaction reagent; and moving means for relatively moving the synthesis reaction tank and the plurality of carriers. An apparatus for producing a probe carrier, wherein an operation of bringing an arbitrary carrier selected from a plurality of carriers into contact with a synthesis reaction reagent in a selected synthesis reaction tank is repeated to create a probe carrier. 4. A method according to claim 1, further comprising: a light detecting means for detecting a transmitted light transmitted through a probe carrier obtained by synthesizing a nucleic acid sequence on a light-permeable carrier for each carrier; and an analyzing means for analyzing a nucleic acid sequence of a target nucleic acid. The light detecting means detects light emission due to the reaction between the nucleic acid sequence on the carrier and the target nucleic acid, and the analyzing means detects the nucleic acid of the target nucleic acid in a complementary relationship from the nucleic acid sequence on the carrier detected by the light detecting means. An apparatus for analyzing a nucleic acid sequence, comprising identifying a sequence and analyzing a nucleic acid sequence of the target nucleic acid.