JP4402321B2 - Nucleotide sequence determination or nucleic acid quantification method using DNA chip - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method capable of performing highly quantitative hybridization at once and an apparatus for carrying out the method.
[0002]
[Prior art]
A DNA chip has hundreds to hundreds of thousands of DNA probes arranged in an array on a solid surface of about 1 to ² cm ² , and can detect and screen DNA quickly and at low cost. By using this, it is possible to instantaneously determine whether or not the target gene is present in the sample.
[0003]
[Problems to be solved by the invention]
However, the base sequence on the DNA chip is relatively short, about 25 bases, and there are many types of DNA on one substrate, each having a different base sequence, and having different optimum hybridization temperatures. Often there are problems with specificity.
[0004]
Therefore, an object of the present invention is to provide a method capable of performing highly quantitative hybridization at once using a DNA chip and an apparatus for carrying out the method.
[0005]
[Means for Solving the Problems]
As a result of intensive studies aimed at achieving the above object, the present inventors have focused on the fact that the specificity of hybridization becomes an important factor. Then, a means for arranging a plurality of base sequences having the same sequence in a line on the DNA chip and providing a temperature gradient in the direction of the arrangement, or a means for changing the temperature with respect to the base sequence on the DNA chip with time is used. For example, the inventors have found that hybridization with high quantitativeness can be performed at one time, and completed the present invention.
[0006]
That is, the first reference example of the present invention is a base sequence determination or nucleic acid quantification method using a DNA chip in which a plurality of base sequences having the same sequence are immobilized in one row, wherein the same base sequence is Provide a temperature gradient in the direction of immobilization, label the base sequence to be detected, hybridize on the DNA chip, then wash with buffer solution, and then label each base sequence immobilized in one row. The present invention provides a method for determining a nucleotide sequence using a DNA chip or a method for quantifying a nucleic acid, characterized in that the amount of nucleic acid of the nucleotide sequence to be detected is quantified by comparing the intensities.
[0007]
Hybridization is preferably performed at a high temperature if importance is placed on specificity (selectivity), but there is a problem that strength (hybridization strength) is lowered. On the other hand, if it is performed at a low temperature, the strength increases, but there is a problem that the specificity decreases. Therefore, it is preferable to perform hybridization under conditions that allow hybridization with only the target base sequence at as low a temperature as possible. However, when a conventional DNA chip is used, it is difficult to find such conditions.
As described above, if measures are taken to provide a temperature gradient in the direction in which the same base sequence is immobilized, the intensity of each base sequence can be easily detected, and the nucleic acid amount of the target base sequence can be specifically determined. Can be quantitatively determined.
[0008]
A second reference example of the present invention is a method for determining a nucleotide sequence or quantifying a nucleic acid using a DNA chip, wherein the nucleotide sequence to be detected is labeled, then hybridized at a low temperature on the DNA chip, and then the buffer solution is changed over time. Using a DNA chip characterized by quantifying the amount of nucleic acid of the base sequence to be detected by washing with the buffer solution while raising the temperature and monitoring the intensity of the label of each base sequence during the temperature raising process A method for determining a base sequence or quantifying a nucleic acid is provided.
In this method, the temperature of each base sequence immobilized on a DNA chip is raised over time. When hybridized at a low temperature, it can hybridize with a sequence other than the target base sequence. Subsequently, if washing is performed with a buffer whose temperature has been increased over time, the hybridization intensity decreases as the mismatch increases. Therefore, if such means is used, a temperature characteristic pattern characteristic to each base sequence can be obtained. Further, by using this method, it is possible to easily detect the temperature at which the intensity variation of the label becomes the largest. Since such temperature is considered to have the highest specificity, the amount of nucleic acid of the base sequence can be easily quantified by comparing their intensities.
[0009]
The third reference example of the present invention has a buffer storage container, a heating means for the buffer, a flow path for flowing the buffer over the DNA chip, and a means for measuring the intensity of the label. An apparatus for use in the above-described nucleotide sequence determination or nucleic acid quantification method is provided.
By using such an apparatus, it is possible to easily raise and lower the temperature of each base sequence immobilized on a DNA chip, and to perform highly quantitative hybridization at a time.
[0010]
The present invention relates to a nucleotide sequence determination or nucleic acid quantification method using a DNA chip, which is a light source that is hybridized on a DNA chip while raising or lowering the hybridization temperature over time, and the DNA chip is used as a laser sheet. The present invention provides a method for determining a base sequence or quantifying a nucleic acid using a DNA chip, wherein the base sequence in the form of a double strand is detected by irradiating the DNA chip from the plane direction of the DNA chip.
The base sequence to be detected becomes a double strand by hybridizing with the base sequence immobilized on the DNA chip, and is immobilized on the DNA chip. In the case of a single strand, it is suspended in the hybridization solution and is not fixed to the DNA chip. Therefore, if the light source made into a laser sheet is irradiated to the DNA chip from the planar direction of the DNA chip, only two strands can be detected, and the nucleic acid amount of the base sequence can be easily quantified.
[0011]
A fourth reference example of the present invention is a method for determining a base sequence or quantifying a nucleic acid using a DNA chip, wherein hybridization is performed on a DNA chip while raising or lowering the hybridization temperature with time, and a double strand. The present invention provides a method for determining a base sequence or quantifying a nucleic acid using a DNA chip, wherein only the base sequence thus obtained is detected using electrochemical means.
By using electrochemical means, only a double-stranded base sequence can be detected with high accuracy.
[0012]
A fifth reference example of the present invention is a method for determining a nucleotide sequence or quantifying a nucleic acid using a DNA chip, wherein hybridization is performed on a DNA chip while the hybridization temperature is lowered from high temperature over time, and only double strands are used. The present invention provides a method for determining a nucleotide sequence or quantifying a nucleic acid using a DNA chip, which comprises adding a reagent that specifically detects a DNA to a hybridization solution and detecting a double-stranded nucleotide sequence.
Such a method uses a reagent that specifically detects only two strands. If a reagent that specifically detects only two strands is used, unlike the case of the fourth invention, a double-stranded base sequence can be detected even when light is irradiated from the top surface of the DNA chip. .
[0013]
DETAILED DESCRIPTION OF THE INVENTION
In the first reference example of the present invention, first, a plurality of base sequences having the same sequence are immobilized in a line on a DNA chip. The base sequences thus immobilized in a row are immobilized on a plurality of types of DNA chips. Next, a temperature gradient is provided in the direction in which the same base sequence is immobilized. The temperature gradient can be appropriately set in consideration of the closeness of the optimum hybridization temperature of different base sequences on the DNA chip. In addition, when the optimum hybridization temperature of different base sequences is approximate, it is preferable to take a small temperature interval between the base sequences.
[0014]
The temperature gradient is preferably set by the following method. First, the direction of the same arrangement | sequence is fixed to a perpendicular direction for a DNA chip. If the direction of the same array is fixed in the horizontal direction, the rate of diffusion of temperature is faster than the rate of diffusion of substances, and thus hybridization cannot be performed sufficiently. Then, either one or both of a heating unit at the top and a cooling unit at the bottom are provided. Thereby, a temperature gradient can be formed in the vertical direction.
[0015]
Next, the base sequence to be detected is labeled and then hybridized on a DNA chip. Examples of the labeling method include a method using a fluorescent reagent and a method using a radioisotope. Among these, a method using a fluorescent reagent is preferable. For hybridization, a hybridization solution may be added onto the DNA chip. The reaction time can be appropriately set according to the length of the sequence to be hybridized. The buffer is then washed to remove the background.
[0016]
On the DNA chip obtained in this way, the portion of the same base sequence arranged in a line that is maintained at the optimum hybridization temperature has a specific and maximum labeling intensity. The amount of nucleic acid can be quantified with high accuracy.
[0017]
In the second reference example of the present invention, first, a base sequence to be detected is labeled and then hybridized at a low temperature on a DNA chip. The labeling method is the same as described above. Here, “at low temperature” means a temperature at which the base sequence to be detected can hybridize with all base sequences on the DNA chip. That is, in the second invention, the base sequence to be detected is hybridized with all base sequences on the DNA chip. This utilizes the fact that even a base sequence with low complementarity will hybridize at low temperatures.
[0018]
Next, the DNA chip is washed with the buffer while raising the temperature of the buffer over time. The method for heating the buffer solution is not particularly limited. For example, the method for directly heating the buffer solution storage container, the method for heating the buffer solution storage container to the DNA chip, or the DNA chip and its surroundings directly. The method of heating etc. are mentioned. In order to raise the temperature with time, the strength of the heating means may be increased with time. The temperature rising rate is preferably constant. The background is removed by washing the DNA chip with buffer.
[0019]
Next, the change in label intensity with time of each base sequence on the DNA chip is measured. Here, even a base sequence with low complementarity is hybridized at a low temperature, but dissociates from a double strand with many mismatches as the temperature rises. Then, at a certain specific temperature, the intensity variation of the label of each base sequence on the DNA chip becomes the largest. Since the specificity is considered to be greatest at this temperature, the amount of nucleic acid is quantified from the intensity of the label at that temperature.
[0020]
FIG. 1 shows an example of an apparatus for carrying out the second reference example. In FIG. 1, the apparatus 1 includes a buffer storage container 2, a line 3 of the buffer solution on the DNA chip, a flow path 5 on the DNA chip, a light source 6, and a detector 7. The buffer is heated in the buffer storage container 2 and / or the line 3 to the DNA chip 4. The temperature of the buffer solution is raised from a low temperature over time. After completion of the hybridization at a low temperature, the buffer solution is fed onto the DNA chip (buffer solution channel 5) while the temperature is raised over time. After the background is removed by washing, the label intensity is measured by the detection unit 7 using the light source 6.
[0021]
FIG. 2 shows another example of an apparatus for carrying out the second reference example. The buffer heating unit 8 is located above the DNA chip. The buffer solution is heated by the heating unit 8 when passing through the buffer channel 5. The heating intensity in the heating unit 8 is adjusted so that the temperature of the buffer solution is raised from a low temperature over time. In FIG. 2, an apparatus in which a light source 6 ′ and a detector 7 ′ are on the same side with respect to an object to be measured is illustrated using an optical fiber as a marker intensity measuring apparatus. However, the measuring device is not limited to this.
[0022]
The present invention is to detect and determine a double-stranded base sequence by irradiating a DNA chip with a light source in the form of a laser sheet from the planar direction of the DNA chip. That is, by irradiating the DNA chip from the planar direction of the DNA chip, only those hybridized on the DNA chip are detected. By performing detection using such a method while performing hybridization while raising or lowering the temperature over time, it is possible to rapidly perform highly quantitative hybridization at once without washing and removing the background. .
[0023]
In the fourth reference example of the present invention, hybridization is first performed on a DNA chip while the temperature is raised or lowered over time, and then only a base sequence that has become a double strand on the DNA chip is subjected to electrochemical means. It is to detect. As a result, it is possible to rapidly perform highly quantitative hybridization at a time without washing and removing the background.
[0024]
An example of the detection method by electrochemical means is shown below. Recently, a novel ligand FND (ferrocenylnaphthalenediimide derivative) having an extremely high ability to discriminate double-stranded DNA and showing a stable electrochemical response has been developed. This has a structure in which a ligand substituent protrudes into both the main groove and the minor groove of a DNA double helix by threading intercalation into double-stranded DNA. These substituents are obscured to stabilize the DNA double helix and at the same time make the ligand difficult to dissociate from the DNA double helix. However, since there is no such effect on single-stranded DNA, it exhibits extremely high binding specificity for double-stranded DNA.
[0025]
The concept of a DNA detection system using the ligand FND is shown in FIG. First, a thiolated DNA probe is bound to a gold electrode, and hybridization with a sample DNA fragment is performed. Then, measurement is performed with a solution containing the ligand, and the redox response of the ligand is measured by an electrochemical method. This quantifies the amount of double strand formation of the target gene. By using differential pulse voltammography (DPV) as an electrochemical method, a current accompanying oxidation of the ligand flows in the vicinity of 460 mV. The current value due to the concentration of the ligand on the electrode increases according to the amount of double strands formed.
[0026]
In the fifth reference example of the present invention, hybridization is first performed on a DNA chip while the hybridization temperature is lowered from a high temperature over time. Next, a reagent that specifically detects only double strands is added to the hybridization solution, and the base sequence that has become double strands is detected. Examples of the reagent that specifically detects only two strands include SYBR Green I (registered trademark: Molecular Probes, Inc). Although detection can be performed by irradiating UV of 254 nm, for example, it is not limited to this method.
[0027]
Next, reference examples of the present invention will be described in more detail with reference examples, but the reference examples of the present invention are not limited to the following examples.
[0028]
Reference Example 1
Seven types of base sequences represented by SEQ ID NOs: 1 to 7 are arranged in a row on the DNA chip, respectively, and the DNA chip is fixed so that the row direction is the vertical direction. 1 L of seawater is filtered through a 0.2 μm Nuclepore filter, and RNA is extracted from bacteria on the filter. This RNA is decomposed with magnesium chloride, dephosphorylated with alkaline phosphatase, and then the ribose of the nucleic acid is oxidized to an aldehyde form. Reaction with lysamine rhodamine ethylenediamine, followed by reduction with a Schiff base, followed by column purification to prepare 30-50mer labeled RNA.
Labeled RNA is hybridized to the chip. Add 3 μL of labeled RNA (1 μg / μL) to the hybridization buffer to 35 μL. After filtration purification, heat at 94 ° C. for 3 minutes. After cooling, 30 μL is placed on the DNA chip on the slide glass, and the cover glass is gently placed from the end. The temperature gradient of the DNA chip is 20 to 80 ° C., and hybridization is performed for 6 hours. After hybridization, the sample is washed with a washing buffer, and the fluorescence signal is detected by a detector and analyzed.
[0029]
Reference Example 2
The labeled RNA prepared above is hybridized on a DNA chip at 20 ° C. After hybridization, the temperature is gradually raised from 20 ° C. to 95 ° C. in a washing buffer (1 × SSC, 1% SDS), and during that time, the fluorescence of DNA hybridized to the chip is scanned. Curves as shown in FIG. 4 (six in FIG. 4) are obtained for each probe. Since each curve is most distant (the vertical line portion in FIG. 4) is considered to have the highest specificity, the temperature is quantified.
[0030]
【The invention's effect】
By using the present invention, the problem of low specificity in the conventional DNA chip is solved, and hybridization with high quantitativeness can be performed at a time. In addition, by using the present invention, the fourth reference example and the fifth reference example , it is possible to rapidly perform highly quantitative hybridization without washing and removing the background.
[Sequence Listing]
SEQUENCE LISTING
<110> MITSUBISHI HEAVY INDUSTRIES, LTD
<120> Method of determining base sequence by DNA chip
<130> 200101079
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[Brief description of the drawings]
FIG. 1 shows an example of an apparatus for carrying out a second reference example.
FIG. 2 shows an example of an apparatus for carrying out a second reference example.
FIG. 3 shows the concept of a DNA detection system using a ligand FND.
4 is a graph showing the relationship of fluorescence intensity with time in Reference Example 2. FIG. The vertical line indicates the point (temperature) at which each curve is farthest away. In FIG. 4, there are six curves, but the number of curves is equal to the number of base sequences on the DNA chip.
[Explanation of symbols]
1: Device used in the base sequence determination or nucleic acid quantification method of the second reference example of the present invention 2: Buffer storage container 3: Line of buffer solution on DNA chip 4: DNA chip 5: Flow path 6 on DNA chip 6 ′: Light source 7, 7 ′: Detection unit 8: Heating unit 9: DNA sensor 10: Counter electrode 11: Temperature sensor 12: DNA probe modified electrode 13: Reference electrode (Ag / AgCl ₂ )
14: Electric field solution 15: Thermostatic bath

Claims (1)

  A method for determining a nucleotide sequence or quantifying a nucleic acid using a DNA chip, wherein hybridization is performed on a DNA chip while raising or lowering a hybridization temperature with time, and a light source formed into a laser sheet is applied to the DNA chip. A method for determining a nucleotide sequence or quantifying a nucleic acid using a DNA chip, comprising: irradiating from a planar direction of the DNA chip to detect a double-stranded base sequence.

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