JP2001343384A - Method for hybridizing sample chip and method for dehybridizing - Google Patents

Abstract

(57) Abstract: A method and a method for hybridizing a sample chip, in which a DNA probe and an analysis DNA are hybridized and dehybridized in a short time so that the gene expression mode can be analyzed efficiently. Provide a hybridizing method. Provided are a method for hybridizing a sample chip and a method for dehybridizing a sample chip, which can perform an analysis operation with high accuracy using a low-concentration DNA for analysis and can reduce the analysis cost. An analysis sample is hybridized to a number of sample probes arranged and fixed on a sample chip at a minute interval. At this time, a voltage is applied between the first electrode and a second electrode disposed at a predetermined distance from the first electrode in a state where the sample chip set on the first electrode is heated. The sample probe of the sample chip set on one electrode is charged to (+) charge, and the analysis sample between the two is charged to (-) charge, and the analysis sample is dielectrically adsorbed to the sample probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for analyzing a sample probe (hereinafter referred to as a DNA probe) such as a DNA probe or an RNA probe arranged on a glass substrate. Hereinafter, for analysis D
NA), or dissociate the DNA for analysis multiplied by the DNA probe (Dehybridi
ze) The present invention relates to a method for hybridizing and dehybridizing DNA.

[0002]

For example, when analyzing gene expression modes (mutations such as gene expression level and gene deficiency) in cells and tissues, for example, thousands to tens of thousands /
Using a microchip or a DNA chip (hereinafter, referred to as a DNA chip) on which DNA probes are arranged in a dot array at a density of cm ² ,
The DNA for analysis, which is prepared by extracting from the cell or tissue to be analyzed and labeled with a radioisotope or a fluorescent substance, is attached to the A probe. Since the DNA itself has a complementary relationship, the DNA probe and the D
When the NAs are of the same type, they bind to each other, and when the NAs are different, they become non-bonded.

However, since DNA has a helical structure, it takes a long time (about 6 to 12 hours) for the DNA probe and the DNA for analysis to naturally bind to each other. Has a problem that it cannot be performed efficiently. For this reason, for example, it takes a lot of time to specify pathological cells or to specify an effective drug for a disease, which causes a problem that an appropriate treatment or the like cannot be performed.

In addition, a DNA probe for analysis is used for analysis.
In order to efficiently multiply A, it is necessary to use a high-concentration DNA solution for analysis. However, since only a small amount of DNA for analysis can be collected and the cost is high, the analysis work is efficient and low. Couldn't do it to cost.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned conventional drawbacks.
It is an object of the present invention to provide a method for hybridizing a sample chip and a method for dehybridizing a DNA probe, in which the NA probe and the DNA for analysis can be hybridized and dehybridized in a short time to efficiently analyze gene expression modes. is there.

Another object of the present invention is to provide a method for hybridizing a sample chip which can perform an analysis operation with high accuracy using a low-concentration DNA for analysis and can reduce the analysis cost. It is to provide a dehybridizing method.

[0007]

A first aspect of the present invention is to provide a method for hybridizing an analysis sample to a large number of sample probes arranged and fixed at minute intervals on a sample chip. A voltage was applied between the first electrode and a second electrode disposed at a predetermined distance from the first electrode in a state where the sample chip set in was heated, and the sample chip was set on the first electrode. The sample probe of the sample chip is charged to (+) charge, and the analysis sample between the two is charged to (−) charge, and the analysis sample is dielectrically adsorbed to the sample probe.

According to a second aspect of the present invention, when an analysis sample hybridized to a large number of sample probes arranged and fixed at small intervals to a sample chip is dehybridized, the sample is set on the first electrode. A sample of the sample chip set on the first electrode by applying a voltage between the first electrode and the second electrode disposed at a predetermined distance from the first electrode while the sample chip is heated. The probe and the hybridized analysis sample are charged to (-) charge, and the analysis sample charged to (-) charge is dielectrically dissociated to the second electrode side.

[0009]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of the present invention will be described below with reference to the drawings. First, an example of an apparatus that embodies the method of the present invention will be described. In FIG. 1, a table 1 incorporates a heating member 3 such as an electric heater or a Peltier element, and a first electrode plate 5 is mounted on the table 1. . An appropriate number of DNA chips 7 are placed on the first electrode plate 5, and the heating member 3 heats the electrode plate 5 to a predetermined temperature described later.

On the other hand, a second electrode plate 9 is provided above the first electrode plate 5 at a predetermined distance from the first electrode plate 5, for example, 2 to 2.
They are relatively arranged at intervals of about 00 mm. The distance between the first electrode plate 5 and the second electrode plate 9 is adjusted according to a voltage applied between both electrodes, which will be described later. First and second electrodes 5.9
The space between them constitutes a dielectric and may be an air layer or an insulating material.

The first electrode plate 5 and the second electrode plate 9 are composed of two circuits 3
The first electrode plate 5 and the second electrode plate 9 are switched to (+) and (-) by the switching operation of the switch 11, which is connected to the DC power supply 13 via the switch 11 having a contact structure.

As shown in FIG. 2, a DNA chip 7 is placed on a glass substrate 7a such as a slide glass, for example, with a thickness of 200 μm.
A large number of DNA probes 7b are arranged in a dot pattern at a density of several thousand to several tens of thousands / cm ² at intervals of.
The DNA chip 7 is prepared in various types in which DNA probes are fixed for each cell or tissue.

After arranging a single or a plurality of DNA chips 7 corresponding to the type of DNA to be analyzed on the first electrode plate 5, the DNA chips 7 are extracted from the cells or tissues to be analyzed in the first electrode plate 5. Then, the sample is filled with a buffer containing analysis DNA labeled with a radioisotope or a fluorescent substance so that it can be attached to the DNA probe 7b. Note that the liquid surface of the buffer must be in non-contact with the second electrode 9.

Next, in the above state, the heating member 3 is controlled to generate heat, and the DNA probe 7b and the buffer containing the DNA for analysis are switched to a temperature of about 50 to 80 ° C., preferably about 65 ° C. as a preferred example. The first electrode plate 5 is turned (+) by the switch 11.
When a predetermined voltage is applied between the first electrode plate 5 and the second electrode plate 9 by switching the second electrode plate 9 to (-), the DNA probe 7b is converted to (+) charge and analyzed. For D
Dielectric NA to (-) charge.

The voltage applied between the first electrode plate 5 and the second electrode plate 9 is appropriately set and adjusted according to the distance between the first electrode plate 5 and the second electrode plate 9 and the dielectric constant between them. When the distance between the first electrode plate 5 and the second electrode plate 9 is about 2 to 5 mm, the applied voltage is set to about 2 to 5 V, and when the distance is about 200 mm, the applied voltage is set to about 200 to 300 V. Even when the distance between the two is large, if the dielectric constant between the two is high, the applied voltage can be lowered.

The DNA for analysis contained in the buffer solution in the first electrode plate 5 has a helical structure. When heated to about 65 ° C., the DNA is converted from a helical state to a linear state.
b.

In a state where most of the DNA for analysis is denatured into a substantially linear shape by heating, the first electrode plate 5 and the second electrode plate 9
When a predetermined voltage is applied during this time, each DNA probe 7b of the DNA chip 7 set on the first electrode plate 5 and charged to (+) charge as described above is charged to (-) charge. The DNA for analysis is attracted and adsorbed.

The DNA probe 7b and the DNA for analysis
Are the same, the dielectrically adsorbed DNA probe 7b and the DNA for analysis hybridize, and if they are different, the DNA probe 7b and the DNA for analysis are kept unbound.

[0019] DN by heating and applying voltage
The hybridizing time between the A probe 7b and the DNA for analysis is a voltage applied between the first electrode plate 5 and the second electrode plate 9 when the dielectric constant is constant as shown in FIG.
It may be adjusted so that the DNA probe 7b and the DNA for analysis hybridize in seconds or minutes.

After the above operation, a buffer solution is applied to the DNA chip 7 taken out from the first electrode plate 5 to remove analysis DNA not bound to the DNA probe 7b. Is irradiated with light to cause a fluorescent substance labeled on the hybridized DNA for analysis to emit light, thereby specifying the DNA for analysis from the hybridized DNA probe 7b.

Next, the dehybridizing method will be described. After the detection of the analysis DNA hybridized to the DNA probe 7b is completed, the DNA chip 7 is placed in a buffer solution (for dehybridization, the analysis DNL is contained in the buffer solution).
(A is not included) is set on the first electrode plate 5 in which the DNA probe 7b and the DNA for analysis are heated to about 80 to 1 by controlling the heating of the heating member 3.
Heat to 00 ° C, preferably about 95 ° C. Thereby, the glass substrate 7a and the hybridized DN for analysis are used.
Denature A from double stranded to single stranded.

When the changeover switch 11 is operated in the above-mentioned heating state so that the first electrode plate 5 becomes (-) and the second electrode plate 9 becomes (+), and a predetermined voltage is applied between the two, DN
The A probe 7b and the DNA for analysis are charged to (-) charge. At this time, the DNA probe 7b is
Because it is fixed to, it is transformed into a single strand by heating,
(−) The DNA for analysis charged to the electric charge is attracted to the second electrode 9 side, so that the DNA is dehybridized from the DNA probe 7b.

In this embodiment, the DNA probe 7b is
The DNA probe 7b is charged to (+) charge while being heated at 5 to 70 ° C., and the analysis DNA suspended in the buffer is charged to (−) charge, thereby electrically connecting the analysis DNA to the DNA probe 7b. Hybridization can be achieved by attracting and adsorbing the DNA, the hybridization time can be shortened to the unit of seconds or minutes, and the detection time of the DNA for analysis is greatly reduced, and the measurement operation is performed efficiently. be able to.

Conversely, when the DNA for analysis hybridized to the DNA probe 7b is dehybridized, the DNA probe 7b is heated to about 90 to 95 ° C. to convert the hybridized DNA from the single strand to the single strand. DNA probe 7 charged to (-) charge while denatured into a structure
b and only the DNA for analysis from the DNA for analysis
By pulling to the side, the bond between the two can be released and dehybridization can be performed, and the dehybridization time can be greatly reduced.

Further, since the DNA probe 7b is charged to (+) charge and the DNA for analysis is forcibly dielectrically adsorbed, the concentration of the DNA for analysis adjusted to the buffer solution can be reduced, and a very small amount of the DNA can be obtained. Even for DNA for analysis, the analysis operation can be performed with high accuracy and the analysis cost can be reduced.

In the above description, the surface of the second electrode 9 is made of a dielectric material,
For example, by covering with a synthetic resin sheet or the like, or covering the liquid surface of the buffer for suspending the DNA for analysis with the synthetic resin sheet, the DNA for analysis can be obtained even if the distance from the first electrode 5 is very small.
Can be reliably dielectrically charged to a (-) charge.

In the above description, the method of heating and applying an electric field in a state where the DNA chip is immersed in the buffer containing the DNA for analysis has been described.
Requires a large amount of a buffer solution containing In order to avoid this, a buffer solution containing the DNA for analysis is minutely dropped onto the surface of the DNA chip, and then the buffer solution containing the DNA for analysis is spread out by a cover glass set on the surface of the DNA chip. Is also good.

In the above description, the method of providing an electric field by providing a gap between the liquid surface of the buffer stored in the first electrode and the second electrode also in the dehybridizing method,
In this case, the second electrode may be immersed directly in the buffer.

[0029]

The present invention provides a DNA probe and a DN for analysis.
A can be hybridized and dehybridized in a short period of time to efficiently analyze the gene expression mode. In addition, the analysis operation can be performed with high accuracy using a low-concentration analysis DNA, and the analysis cost can be reduced.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing an example of an apparatus for realizing a DNA hybridizing method and a DNA dehybridizing method.

FIG. 2 is a schematic diagram of a DNA chip.

FIG. 3 is a chart showing a relationship between an applied voltage and a hybridizing time.

[Explanation of symbols]

3-heating member, 5-first electrode, 7-DNA chip, 7a
-Glass substrate, 7b-DNA probe, 9-second electrode,
13-DC power supply

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) C12N 15/00 FF term (reference) 4B024 AA11 AA20 CA01 CA09 CA11 HA14 HA19 4B063 QA01 QA18 QQ42 QQ52 QR32 QR35 QR56 QR84 QS03 QS34 QS39 QX02 QX07

Claims (4)

[Claims] When a sample for analysis is hybridized to a large number of sample probes arranged and fixed at a small interval to a sample chip, the sample chip set on the first electrode is heated in a state where the sample chip is heated. A voltage is applied between one electrode and a second electrode disposed at a predetermined distance from the first electrode to charge the sample probe of the sample chip set on the first electrode to (+) charge. A method for hybridizing a sample chip, in which the sample for analysis is charged to a negative (-) charge and the sample for analysis is dielectrically adsorbed to a sample probe. 2. A sample chip set on a first electrode is heated when a sample for analysis hybridized to a number of sample probes arranged and fixed at minute intervals on the sample chip is dehybridized. In the state, the first electrode and the second electrode which is disposed at a predetermined interval with respect to the first electrode.
A voltage is applied between the electrodes, the sample probe of the sample chip set on the first electrode and the hybridized analysis sample are charged to (−) charge, and the analysis sample charged to (−) charge is charged to the (−) charge. A method for dehybridizing a sample chip to be dielectrically dissociated toward two electrodes. 3. The method for hybridizing and dehybridizing a sample chip according to claim 1, wherein the sample probe is a DNA probe or an RNA probe. 4. The method for hybridizing and dehybridizing a sample chip according to claim 1, 2 or 3, wherein the sample for analysis is labeled with a fluorescent substance, a radioactive substance or a magnetic substance.