JP3201867B2 - Nucleic acid analysis method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting the type and amount of a nucleic acid, particularly mRNA (messenger RNA).

[0002]

BACKGROUND OF THE INVENTION Blueprints of biological activity are written in DNA. The living body performs various biological activities in response to an external stimulus or the like. At that time, mRNA involved in the activity first increases, and a protein corresponding to the activity is generated to execute the biological activity. That is, active cells may have various m depending on the situation.
It produces RNA, and the type and amount of mRNA in the cell is like a fingerprint of cellular activity.
By knowing the type and amount of NA, it is possible to grasp the state of cells or tissues. In addition, knowing the mRNA that governs various conditions and the proteins that it produces can provide insights into disease treatment and biological control, and detect the type and amount of mRNA from an application point of view. It is important to establish a method. But,
Analyzing active mRNAs is tedious and labor intensive.

[0003] In the conventional method, a method has been employed in which a protein associated with an event of interest is captured and mRNA encoding the protein is examined. According to this method, a DNA (or RNA) probe that hybridizes to the mRNA of interest is prepared. The probe is radioactively labeled. Then, how the mRNA is increased or decreased during various activities of the living body is examined to estimate its role. Northern blot is used for detecting the mRNA. That is, all mRNAs extracted from a living body were separated by agarose gel electrophoresis, and the mRNAs separated on a nylon filter or the like were used.
Is transferred. Next, a radioactively labeled DNA probe that hybridizes to a specific mRNA is sprinkled to hybridize to a target mRNA, and the mRNA is labeled. Thereafter, the position of the labeled mRNA was covered with a photosensitive film and transferred to the photosensitive film, and the presence or absence or increase / decrease of the target mRNA was examined by examining the position.

[0004]

The above-mentioned conventional method uses mRN
Since the presence or absence of A was determined by whether or not the DNA probe hybridized, only one type of mRNA could be examined at a time. However, in vivo, various mRNs
A works while interacting with each other through the protein produced based on it. Therefore, in order to understand biological functions and to develop therapeutic guidelines and diagnostic methods for diseases, it is necessary to know the abundance of various mRNAs in each life cycle. That is, it is necessary to simultaneously detect changes in the abundance of dozens or hundreds of mRNAs as well as a single mRNA, and the development of a method that enables this is desired.

The present invention has been developed in response to such a demand.
MRNA that can simultaneously determine the type and amount of A
The purpose is to provide an analytical method. In this specification, the term “nucleic acid” is used as a term including both mRNA and its c-DNA (complementary DNA).

[0006]

In order to achieve the above object, the present invention provides a method for preparing a plurality of mRNAs or c-Ds of interest.
A plurality of nucleic acids are identified and analyzed simultaneously by changing the mobility of NA (complementary DNA), that is, the DNA probe to be hybridized to each of the nucleic acids so that the nucleic acids can be identified by electrophoresis. As a means for changing the mobility of the DNA probe, the length of the probe may be changed, or a chemical substance that reacts with the amino group may be bonded to a part of the phosphate group or the base part or the sugar part of the probe. By changing the mobility of the probe as a whole, or by changing the length of the linker portion.

[0007] Only the DNA probe hybridized to the nucleic acid is taken out, and the DNA probe hybridized to each of the plurality of nucleic acids is desorbed (released) from the nucleic acid by increasing the temperature from each of the plurality of nucleic acids, and then electrophoresed. DNA probes are separated and detected based on the difference in degree, and the type and abundance of the nucleic acid are examined by performing quantitative and qualitative analysis of these DNA probes.

Although the nucleic acid may be used as a mixture, the nucleic acid may be separated by length separation by agarose gel electrophoresis, fractionated, hybridized with a DNA probe, and analyzed by the same procedure as described above. Further, a plurality of nucleic acids obtained by hybridizing a plurality of DNA probes having different mobilities are separated by agarose gel electrophoresis, and then the DNA probes hybridized to the plurality of nucleic acids are released from each of the plurality of nucleic acids by heating. After that, the DNA probe is electrophoresed, and the DNA probe is separated and detected based on the difference in mobility, whereby a two-dimensional analysis is performed based on the size of the nucleic acid and the size of the DNA probe, and the type and abundance of the nucleic acid are examined.

Each DNA probe is labeled with a radiolabel, a fluorescent substance, a dye, a chemiluminescent substance, or the like. The DNA probes separated by electrophoresis are detected and identified by detecting radiation, fluorescence, light absorption, chemiluminescence, and the like by the labeled substance of each DNA probe.

[0010]

Since the difference in the migration speed of the hybridized DNA probe is used to identify the type of nucleic acid, several hundred probes, and thus several hundred nucleic acids, can be separated and detected at a time. In addition, by labeling the DNA probe with a fluorescent substance, changing the type of the fluorescent substance and changing the emission wavelength, a number close to 1000 can be obtained by a combination of the two factors of the length of the DNA probe and the type of the fluorescent substance (fluorescent wavelength). DN
A probe can be prepared, these different DNA probes are hybridized to each of a plurality of nucleic acids of interest, only the DNA probe hybridized to the nucleic acid is taken out, the DNA probe is released from the nucleic acid by heating, and electrophoresis is performed. Since the DNA probe is separated and detected at a time, the type and abundance of the nucleic acid can be simultaneously known.

[0011]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the present invention will be described with reference to FIGS. In the following description, the analysis target is mRNA, but the same applies even when the analysis target is c-DNA. [Example 1] To remove mRNA from cells, magnetic beads having polythymine oligomer (dT) _n are used. Details of the procedure can be found in the Technical Handbook, Molecular Biology (Dynaviz BioMagnetic Separation System) (Technical ha
ndbook, Molecular Biology (Dynabeads biomagnetic s
eparation system)), or Nucleic Acid Research, Vol. 18, p. 3669, 1990 [Nu
cleic Acid Research 18, 3669 (1990)]. In addition, polythymine oligomer (d
T) A microtiter plate holding _n on its surface may be used [Nature, 357, 519, 19
1992 (Nature 357, 519 (1992))]
Shows a flow of preparing an mRNA sample. As shown in FIG. 1, mRNAs 1 to 3 have polyadenine oligomer (dA) _n and 4 at the 3 ′ end. When magnetic beads 5 having a polythymine oligomer (dT) _n- chain, 6 are added to a biological sample and mixed, the adenine chain hybridizes with the thymine chain, so that the mRNA binds to the magnetic beads 5 like 7-9. I do. After this reaction is performed in a solution, the magnetic beads 5 are fixed to a part of the container 10 by the magnet 12 and the inside of the container is washed to remove anything other than mRNA. The solution thus obtained contains almost all mR
NA is used as a sample.

In this example, the mRNA in the biological sample was hybridized and separated using magnetic beads with 0.1 mg of polythymine oligomer (dT) ₂₅ , but the amount of mRNA attached to the magnetic beads was at most about 0.2 μg. If the average length of mRNA is 2K bases, this is about 2
× 10 ¹¹ molecules. Each mRNA contained in this
Has a copy number of 10 ⁷ -10 ⁸ molecules or less.

Next, as shown in FIG. 2, a DNA probe that hybridizes to the mRNA to be analyzed is selected from a c-DNA (complementary DNA) database. DNA
The probe adjusts the length of the base and the length of the linker and adjusts the gel electrophoresis speed (mobility) corresponding to each mRNA.
Are different from each other, and by performing electrophoretic separation, it is possible to identify to which mRNA the measured DNA probe has hybridized.

The DNA probe may be labeled with a radioactive isotope such as ³² P. Here, a description will be given of a fluorescent label for labeling the DNA probe with a fluorescent substance. For example, a fluorescent substance FITC (fluorescein isothiocyanate: emission wavelength: 515 nm) is bound to the 5 ′ end of each DNA probe via an amino group. About 300 DNA probes were used. Six groups of 50 DNA probes having different electrophoresis speeds by about 2 bases were prepared and used. The length of the DNA probe is from 20 bases to 1
There are up to 20 bases, and inosine and the like are added as the length increases, so that the stability of the hybridoma is not extremely different. Hereinafter, these are referred to as probe set 1 to probe set 6. FIG. 2 shows only the case where the probe set 1 is used for simplification of the description.

The amount of each probe in the DNA probe set is 1 fmol. (Femtomole). The sample is divided into six equal parts as shown in FIG. 2, and probe set 1 to probe set 6 are added to each fraction to hybridize the probe. Next, the magnetic beads and the mR attached thereto were
NA, and fluorescent label D hybridized to mRNA
The NA probe is fixed to a part of the container 10, and excess DNA probe is washed and removed. By this operation, the fluorescently labeled probe binds to the mRNA captured by the magnetic beads as in 101 to 103.

Next, as shown in FIG. 3, probes having different lengths bound to mRNA are separated and analyzed by electrophoresis. m
The DNA probe hybridized to the RNA is placed on the upper end of the capillary gel 201 for separation together with the magnetic beads. When a sample solution is injected into the funnel upper part 202 (sample addition part and negative electrode tank) 202, the DNA probe sinks at the upper end of the capillary together with the magnetic beads 210. After the magnetic beads 210 are localized at the upper end using the magnet 205, this portion is
Heat to 00 ° C. to desorb the DNA probe. Electrophoresis voltage is applied to both ends of the capillary gel 201, and the detached DNA probes 51 to 53 migrate downward. However, since the DNA probes have different migration speeds depending on the type of the corresponding mRNA, Probes can be separated.

The gel used for separation is 6% by weight of polyacrylamide (6% T, 3% C) containing 3% by weight of a crosslinking agent.
It is the same as that used for DNA sequencing [Analytical Chemistry, Vol. 62, p.
Year 0 (Analytical Chemistry 62, 900 (199
0))]. The gel length of the separation part is 25 cm, and one base can be separated up to 400 bases. The fluorescence emitted by irradiating the laser at the lower end of the gel capillary or the place where the DNA probe is extracted during the sheath flow is measured. As the fluorescence photometric system, for example, the one described in Japanese Patent Application No. 3-234427 can be used. In the embodiment shown in FIG. 3, the fluorescently labeled DNA probe eluted from the capillary gel 201 is carried to the lower positive electrode tank 209 by the sheath liquid. Laser light applied to the position (for the sake of simplicity, laser 207 is shown in the drawing)
This excites the labeled phosphor of the probe. Fluorescence emitted from the labeled phosphor is detected by the detector 208 via a lens system and a filter system (not shown). The detected fluorescence intensity corresponds to the amount of mRNA that hybridizes to the DNA probe.

Although FITC is used here as the phosphor 50, TRITC (tetramethylrhodamine isothiocyanate (emission wavelength: 575 nm)) or sulfolordamine 101 (emission wavelength: 615 nm) may be used. Hundreds of probes can be detected at a time by combining the label with the fluorescent substance and the DNA probe length.

During electrophoresis, mRNA stays near the upper end of the gel because mRNA is much longer (average 2K bases) than DNA probe. Therefore, after all the probes have been eluted from the lower end, the mRNA can be migrated in the reverse direction, trapped again on the magnetic beads, and the detection can be attempted again with another probe set. In this case, many mRNAs can be analyzed with a small number of samples. Although the mRNA was directly examined here, the same analysis can be performed by converting the mRNA into c-DNA having better stability than the mRNA. That is, it is also possible to perform complementary chain synthesis using the polythymine oligomer attached to the magnetic beads as a primer and obtain c-DNA attached to the magnetic beads, followed by measurement.

Example 2 In the above example, mRNA was used as a mixture, but after separating mRNA by length, D
The NA probe can be hybridized and analyzed. That is, after the mRNA captured by the magnetic beads is thermally desorbed, agarose gel electrophoresis is performed. At this time, mRN eluted using a short tube-shaped gel
A is fractionated or kept separated in a long gel,
Thereafter, the gel is cut out and fractionated for each mRNA having a different length.

Each of the fractions thus produced is individually
As a sample, using the probe set,
Perform analysis by method. Polythymine oligomer (d
T) _nMagnetic beads and DNA probe set
To allow excess DNA probes to wash.
Clean and remove. Next, as described in FIG.
The DNA probe contained therein was desorbed by heating, and the
The DNA probe is subjected to length separation using a migration path.

According to this example, the size of mRNA and D
Since analysis can be performed two-dimensionally using the size of the NA probe, analysis of thousands of mRNAs can be performed at once.
At this time, if one probe is allowed to hybridize with a plurality of mRNAs, a large number of mRNAs can be analyzed with a small number of probes, and the information can be obtained from two information, the length of the probe and the length of the messenger. The mRNA to which the probe has hybridized can be determined.

In this embodiment, in order to increase the throughput of analysis, a plurality of fractions are simultaneously electrophoresed using a gel electrophoresis apparatus including a plurality of gel capillaries described in Japanese Patent Application No. 3-234427. Line sensor, diode array with image amplifier, or CC
Fluorescence photometry may be performed simultaneously using a D detector or the like.

[Embodiment 3] In the above Embodiment 2, mRNA was fractionated, but it is also possible to separate and measure by two-dimensional electrophoresis without fractionation. The procedure will be described first.
Capture the mRNA using magnetic beads as shown in
Purify. Subsequently, the temperature is raised to dissociate the hybridized adenine chain-thymine chain (polythymine oligomer-polyadenine oligomer hybridization), and only the magnetic beads are removed to obtain mRNA. A DNA probe is added thereto and hybridized to mRNA. The obtained mixture is injected into a capillary gel (agarose 1%) having an inner diameter of about 0.5 to 1 mm.

A length of 10 above the capillary gel inlet
After sealing with a polyacrylamide gel (8% T, 3% C) or a semi-permeable membrane, electrophoresis is first performed in the direction opposite to the electrophoretic separation (on the polyacrylamide gel or the semi-permeable membrane side).
Then, the hybridized DNA probe and the free DNA probe migrate about one order of magnitude faster, so that the free DNA probe passes through the polyacrylamide gel or semipermeable membrane in 1 to 5 minutes and is removed. However, the hybridized DNA probe and mRNA remain on the gel surface or in the gel. Therefore, the hybridomers are separated by setting the electric field to the normal separation direction.

After electrophoresis for a certain period of time, the tube gel is extracted and placed on a polyacrylamide slab gel to perform second-dimensional separation. This separation in the second dimension can be performed with a device similar to the device described in US Pat. No. 4,305,799. That is, after heating the tube-shaped gel part to detach the DNA probe from the mRNA, the DNA probe is electrophoresed in a direction perpendicular to the tube gel, and the length is separated. The length of the DNA probe and the position of the mRNA in the horizontal direction indicate the type of mRNA to which the DNA probe has hybridized, and the amount of the detected DNA probe indicates the amount of mRNA.

[0027] In the above respective embodiments have been described when using a fluorescent label, labeling with dyes silver staining, etc., ³²
Labeling with a radioactive element such as P or labeling with a chemiluminescent material such as peroxidase and luminol or Digoxigen can also be used. Light absorption is used as a detection means when a dye is used as a label.When a radioactive element or a chemiluminescent material is used, the separated DNA probe is held in a gel, and when a chemiluminescent material is used, a luminescent reagent is added. Then, a technique such as transferring a pattern to a photosensitive film is used.

[0028]

As described above, according to the present invention, mR
By varying the migration speed of DNA probes that hybridize to NA, a large number of DNA probes can be analyzed simultaneously, whereby the types and amounts of multiple mRNAs can be measured simultaneously.

[Brief description of the drawings]

FIG. 1 is a flow chart showing the preparation of an mRNA sample.

FIG. 2 is a flowchart for obtaining a labeled probe that binds to mRNA captured by magnetic beads.

FIG. 3 is a conceptual diagram illustrating separation analysis of each probe by electrophoresis.

[Explanation of symbols]

1, 2, 3, ... mRNA, 4 ... polyadenine, 5 ... magnetic beads, 6 ... polythymine oligomer, 7, 8, 9 ... mRNA captured by magnetic beads, 10 ... container, 11 ... reaction solution, 12 ... magnet, 13: magnetic beads, 50: phosphor, 5
1, 52, 53 ... desorbed fluorescent labeled probe, 101,
102, 103: fluorescently labeled probe bound to mRNA captured by magnetic beads, 201: capillary gel, 2
02: funnel-shaped sample addition section / negative electrode tank, 205: magnet, 206: heater, 207: laser oscillator, 208
Detector 209 Positive electrode tank 210 Magnetic beads 2
11—Reaction liquid.

────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroko Furuyama 1-280 Higashi Koigakubo, Kokubunji-shi, Tokyo Inside the Central Research Laboratory, Hitachi, Ltd. (56) References JP-A-6-317586 (JP, A) JP-A-4 JP-A-267898 (JP, A) JP-A-63-229365 (JP, A) JP-A-3-128000 (JP, A) JP-A-2-75958 (JP, A) JP-A-63-122956 (JP, A) JP-A-61-258168 (JP, A) JP-A-62-162752 (JP, A) JP-A-60-256059 (JP, A) JP-A-2-2144752 (JP, A) JP-A-6-260 189796 (JP, A) JP-A-6-153996 (JP, A) JP-A-6-153998 (JP, A) JP-A-6-43159 (JP, A) (58) Fields investigated (Int. ^7, DB name) G01N 27/447 G01N 33/50 G01N 33/58 C12Q 1/68

Claims (16)

(57) [Claims] (1) a step of hybridizing a DNA probe having a different mobility for each of a plurality of kinds of nucleic acids; (2) a step of removing the free DNA probe; and (3) a step of (3) the nucleic acid. (4) a step of separating the DNA probe released in step (3) and separating and detecting the DNA probe in step (3) based on the difference in mobility. Method. 2. The nucleic acid analysis method according to claim 1, wherein
A nucleic acid analysis method, wherein the nucleic acid is obtained by fractionating into different lengths by electrophoresis. 3. The nucleic acid analysis method according to claim 1, wherein the DNA probe is separated by electrophoresis in the step (4). 4. The nucleic acid analysis method according to claim 1, wherein the length of the DNA probe is changed or a substance that chemically modifies the DNA probe is changed. The nucleic acid analysis method, wherein the mobility is varied by the following methods. 5. The nucleic acid analysis method according to claim 1, wherein the DNA probe is released by raising the temperature in the step (3). 6. The nucleic acid analysis method according to claim 1, wherein the DNA probe comprises a radioactive element,
It is labeled with any of a phosphor, a dye, and a chemiluminescent substance, and detects any one of radiation from the radioactive element, fluorescence from the phosphor, light absorption by the dye, and chemiluminescence from the chemiluminescent substance. A nucleic acid analysis method, wherein the DNA probe is detected by the following method. 7. A step of: (1) a step of hybridizing DNA probes having different mobilities for each of a plurality of types of nucleic acids; (2) a step of removing the free DNA probes; and (3) a step (3). (2) separating the nucleic acid hybridized with the DNA probe obtained in 2) by first electrophoresis using an electrophoretic medium; and (4) hybridizing the DNA probe separated by the first electrophoresis. (5) separating the DNA probe released from the nucleic acid obtained in the step (4) and separating and detecting the DNA probe by the second electrophoresis based on the difference in the mobility. A nucleic acid analysis method comprising: 8. The nucleic acid analysis method according to claim 7, wherein
A method for analyzing nucleic acids, wherein the electrophoresis medium is agarose. 9. The nucleic acid analysis method according to claim 7, wherein
A nucleic acid analysis method, wherein the electrophoresis medium used for the second electrophoresis is polyacrylamide. 10. The method for analyzing nucleic acid according to claim 7, wherein the length of the DNA probe is changed or a substance that chemically modifies the DNA probe is changed. The nucleic acid analysis method, wherein the mobility is varied by the following methods. 11. The nucleic acid analysis method according to claim 7, wherein in step (4), the DNA probe is released by raising the temperature. 12. The nucleic acid analysis method according to claim 7, wherein the DNA probe is labeled with any of a radioactive element, a phosphor, a dye, and a chemiluminescent substance. A nucleic acid analysis method, wherein the DNA probe is detected by detecting any of radiation from an element, fluorescence from the phosphor, light absorption by the dye, and chemiluminescence from the chemiluminescent substance. 13. A step of (1) hybridizing DNA probes having different mobilities for each of a plurality of types of nucleic acids; and (2) combining the nucleic acid with which the DNA probes are hybridized and the free DNA probes. (3) electrophoretically separating the DNA probe from the electrophoresis medium, and (3) following the step (2), the DN in the electrophoresis medium.
A step of electrophoretically separating the nucleic acid to which the A probe has hybridized; and (4) prior to the second electrophoresis,
Releasing the DNA probe from the nucleic acid to which the DNA probe has hybridized; and (5) separating the DNA probe released in the step (4) by the second electrophoresis based on the difference in mobility. And analyzing the nucleic acid. 14. The nucleic acid analysis method according to claim 13, wherein the length of the DNA probe is changed, or
A nucleic acid analysis method, wherein the mobility is varied by changing a substance that chemically modifies the DNA probe. 15. The nucleic acid analysis method according to claim 13, wherein the DNA probe is released by raising the temperature in step (4). 16. The method according to claim 13, wherein the DNA probe is labeled with any of a radioactive element, a fluorescent substance, a dye, and a chemiluminescent substance. A diffusion analysis method, wherein the DNA probe is detected by detecting any of radiation from an element, fluorescence from the phosphor, light absorption by the dye, and chemiluminescence from the chemiluminescent substance.