Lock-like molecular analysis methods
The method analyzes chain molecules by recording electrophoretic morphology to estimate size without amplification, addressing the time issue in gene analysis and enhancing precision using specific electrophoresis media.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- PICO TECH BIO CO LTD
- Filing Date
- 2024-11-29
- Publication Date
- 2026-06-10
AI Technical Summary
Gene analysis by electrophoresis requires time-consuming amplification processes like PCR, prolonging the overall analysis time.
A method that analyzes chain molecules like DNA and RNA by recording and estimating their electrophoretic morphology during electrophoresis, eliminating the need for amplification by utilizing the worm-like motion and shape changes of molecules in a polymer-filled electrophoresis medium, and correlating these morphological parameters with molecular size.
Enables size estimation of molecules without amplification, reducing analysis time and improving precision through the use of suitable electrophoresis media, such as HEC, PEG, and PEO, allowing for faster and more accurate nucleotide sequencing.
Smart Images
Figure 2026094588000001_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for analyzing chain molecules such as nucleic acids and proteins such as DNA and RNA.
Background Art
[0002] Nucleic acids such as DNA and RNA are formed by the binding of bases such as adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). In order to know the base sequence of a nucleic acid, the base sequence can be analyzed by using a DNA sequencer typified by a capillary DNA sequencer after undergoing a pretreatment called the Sanger method.
[0003] Generally, in a capillary DNA sequencer, a polymer is filled inside a capillary, and nucleic acids are fragmented into a plurality of molecules of different lengths one base at a time by the Sanger method. After labeling the ends of these molecules with a fluorescent substance that emits different fluorescent colors depending on the type of terminal base, each fragmented molecule is analyzed by electrophoresis. As the molecules migrating through the flow path entangle with the polymer filling the flow path, a difference in the mobility of each molecule occurs due to the size (chain length) of each molecule. Therefore, the base sequence of the extension can be inferred based on the mobility of each molecule and the color of the fluorescent substance labeled on the terminal base.
Summary of the Invention
Problems to be Solved by the Invention
[0004] In the analysis of genes by electrophoresis, it is necessary to amplify the target gene by PCR or the like before electrophoresis, but gene amplification takes time, so as a result, gene analysis takes a long time.
[0005] Therefore, an object of the present invention is to provide a method for analyzing chain molecules that does not require amplification processing.
Means for Solving the Problems
[0006] It is known that when chain-like molecules such as DNA fragments are subjected to electrophoresis in a channel filled with a polymer-containing electrophoresis medium, each chain-like molecule moves while changing shape like an earthworm, a phenomenon called "worm motion." This worm motion occurs because the chain-like molecules become entangled with the polymer contained in the electrophoresis medium as they move through it. The inventors of this invention discovered through experiments that the worm motion of chain-like molecules during electrophoresis is dependent on the size of the chain-like molecules, leading to the present invention.
[0007] In other words, the chain-like molecular analysis method according to the present invention is An electrophoresis step in which a chain-like molecule to be analyzed is moved in a predetermined electrophoresis medium, A recording step for recording the electrophoretic morphology of the analyte molecule in the electrophoretic medium during the electrophoresis step, The system includes an estimation step of estimating the size of the analyte based on the electrophoretic morphology of the analyte recorded in the recording step.
[0008] The chain molecule analysis method according to the present invention may include a correlation data preparation step of preparing correlation data between the electrophoretic morphology in a predetermined electrophoretic medium and the size of the chain molecule, and in the estimation step, the size of the target molecule to be analyzed may be estimated using the correlation data.
[0009] Furthermore, the electrophoretic motion may include changes in shape due to worm-like movement.
[0010] Furthermore, the parameters indicating the electrophoretic morphology may include the length a in the electrophoretic direction when the shape of the chain molecule is approximated as an ellipse, and / or the length b in the direction perpendicular to the electrophoretic direction.
[0011] In the above case, the parameter indicating the electrophoretic pattern may be the ratio of length a to length b.
[0012] Furthermore, the parameters indicating the electrophoretic pattern may include the electrophoretic speed. [Effects of the Invention]
[0013] According to the chain-like molecule analysis method of the present invention, the electrophoretic morphology of the analyte molecule is recorded, and the size of the analyte molecule is estimated based on the recorded electrophoretic morphology of the analyte molecule. Therefore, the size of the analyte molecule can be estimated without amplification of the analyte molecule. [Brief explanation of the drawing]
[0014] [Figure 1] This is an image of the electrophoretic channel showing the morphology of chain-like molecules over time during electrophoresis. [Figure 2] This figure shows the time-series changes in an ellipse that approximates the shape of chain molecules during electrophoresis. [Figure 3] This graph shows the time evolution of an ellipse a / b, which approximates the shape of chain molecules during electrophoresis. [Figure 4] This graph shows the results of an investigation into the effect of different electrophoretic media on the electrophoretic morphology of chain molecules. [Figure 5] This is a flowchart illustrating one example of a chain-like molecular analysis method. [Figure 6] This is statistical data on the electrophoretic velocity of chain molecules in a minute section. [Modes for carrying out the invention]
[0015] An embodiment of the chain-like molecular analysis method according to the present invention will be described below with reference to the drawings.
[0016] Figure 1 shows images (a) to (g) in chronological order of the morphology of chain molecules (DNA fragments) undergoing electrophoresis inside a polymer-filled capillary. Focusing on, for example, one chain molecule indicated by an arrow in (a) to (g) of this figure, it can be seen that the chain molecule changes shape while moving in one direction (from left to right) with a worm-like motion.
[0017] FIG. 2 is an image showing the time change of an ellipse approximating the shape of a chain molecule undergoing electrophoresis in the order of (a) to (e) in a time series. By comparing (a) to (e), it can be seen that both the dimension a in the direction parallel to the migration direction of the ellipse approximating the shape of the chain molecule during electrophoresis and the dimension b in the direction perpendicular to the migration direction change with time. In this example, in order to numerically represent the migration pattern, the shape of the chain molecule during electrophoresis is approximated by an ellipse, but the present invention is not limited to this, and it may be approximated by a sphere assuming the radius of gyration of the chain molecule or by a cylindrical rigid body.
[0018] FIG. 3 records the time change of the ratio (a / b) of the dimensions a and b of an ellipse approximating the shape of a chain molecule (DNA fragment) during electrophoresis. Referring to the data in FIG. 3, it can be seen that a / b of the ellipse approximating the shape of the chain molecule during electrophoresis changes periodically with a certain amplitude. As a result of the experiments by the present inventors, it was found that parameters such as the maximum value, minimum value, amplitude, and period of a / b change depending on the size of the chain molecule.
[0019] From the above, by analyzing parameters (for example, the maximum value, minimum value, amplitude, period of a / b) indicating the migration pattern of the analysis target molecule, the size of the analysis target molecule can be estimated. For example, by performing electrophoresis of a standard sample with a known size and obtaining in advance the parameters of the migration pattern during electrophoresis of the standard sample, and creating correlation data between the size of the chain molecule and the parameters of the migration pattern, it can be used for estimating the size of the analysis target molecule. Also, by comparing the parameters of the migration patterns of each chain molecule obtained by electrophoresing a plurality of fragmented chain molecules, the size relationship of each chain molecule can be obtained and used for inferring the nucleotide sequence of the nucleic acid.
[0020] FIG. 4 shows data indicating the time change of a / b of a chain molecule when the same chain molecule is electrophoresed under the same conditions except for the electrophoresis medium, using capillaries filled with HEC (hydroxyethyl cellulose), PEG (polyethylene glycol), and PEO (polyethylene oxide) as the electrophoresis media, respectively. Referring to this data, it can be seen that the electrophoresis form of the chain molecule also changes depending on the type of electrophoresis medium. That is, the correlation between the size of the chain molecule and the parameters of the electrophoresis form also changes depending on the type of electrophoresis medium.
[0021] Therefore, by using an electrophoresis medium suitable for the type of molecule to be analyzed, that is, an electrophoresis medium for which the correlation between the size of the molecule to be analyzed and the parameters of the electrophoresis form is clear, various chain molecules can be analyzed with high precision. The molecule to be analyzed can include biopolymers modified by methylation, phosphorylation, histone modification, etc., and proteins with altered structures. As the electrophoresis medium, solutions in which polymers such as HEC, PEG, and PEO are dissolved, liquid crystals, etc. can be used. Polymers include functional polymers, crosslinked polymers, and non-crosslinked polymers.
[0022] FIG. 5 shows a flowchart of an embodiment of a method for analyzing chain molecules using the above principle.
[0023] First, correlation data between the size of a chain molecule and the parameters (such as the maximum value, minimum value, amplitude, period, etc. of a / b) indicating the electrophoresis form of the chain molecule when a known chain molecule is electrophoresed in a specific electrophoresis medium is prepared (step 101).
[0024] The chain molecule to be analyzed is purified (step 102). Using the Sanger method or the like, the purified chain molecule is fragmented to generate a plurality of molecules with different lengths for each base, and labeling is performed by binding fluorescent dyes different depending on the type of terminal base to each fragmented molecule (step 103).
[0025] Electrophoresis of the labeled molecules is performed, and the electrophoretic morphology of each molecule during electrophoresis is recorded by photographing or imaging (Step 104). Subsequently, the electrophoretic morphology of each recorded molecule is analyzed, and parameters indicating the electrophoretic morphology of each molecule are obtained (Step 105). The size of each molecule is estimated by applying pre-prepared correlation data to the parameters of each molecule obtained from the analysis (Step 106). Since each fragmented molecule is labeled with a fluorescent dye according to the type of terminal base, the type and size of the terminal base of each molecule can be determined, and thereby the base sequence of the chain molecule being analyzed can be estimated.
[0026] Although the movement speed of chain molecules during electrophoresis is not constant because they perform a worm-like motion due to entanglement with the electrophoretic medium, as mentioned above, the worm-like motion of chain molecules has periodicity. Therefore, if the movement speeds over multiple periods are statistically analyzed, differences in movement speed due to differences in the size of the chain molecules will be observed.
[0027] Figure 6 shows statistical data of electrophoresis velocity at a micro-section thickness of approximately 50 μm when using DNA of sizes 100 bp, 200 bp, and 300 bp. Calculating the electrophoresis velocity of each DNA size from this statistical data, we found that the electrophoresis velocity of 100 bp DNA was 376.4 μm / sec, 200 bp DNA was 213.1 μm / sec, and 300 bp DNA was 129.5 μm / sec. Thus, even in micro-sections, the electrophoresis velocity of chain molecules differs depending on their size, demonstrating that separation based on size is possible.
[0028] As described above, by analyzing the electrophoretic morphology of a single chain molecule, it is possible to estimate the size of each fragment with electrophoresis over a much shorter distance and time compared to general electrophoresis that forms bands for each fragment. Thus, using the chain molecule analysis method according to the present invention not only eliminates the need for amplification processes such as PCR, but also significantly reduces the time required for electrophoresis.
Claims
1. An electrophoresis step in which a chain-like molecule to be analyzed is moved in a predetermined electrophoresis medium, A recording step for recording the electrophoretic morphology of the analyte molecule in the electrophoretic medium during the electrophoresis step, A chain-like molecule analysis method comprising: an estimation step of estimating the size of the analyte based on the electrophoretic morphology of the analyte recorded in the recording step.
2. The system includes a correlation data preparation step for preparing correlation data between the electrophoretic morphology of a chain molecule in a predetermined electrophoretic medium and the size of the chain molecule. The chain-like molecule analysis method according to claim 1, wherein the estimation step involves estimating the size of the molecule to be analyzed using the correlation data.
3. The method for analyzing chain molecules according to claim 1, wherein the electrophoretic morphology includes a change in shape due to worm movement.
4. The method for analyzing a chain molecule according to claim 3, wherein the parameters indicating the electrophoretic morphology include the length a in the electrophoretic direction when the shape of the chain molecule is approximated as an ellipse, and / or the length b in a direction perpendicular to the electrophoretic direction.
5. The method for analyzing chain-like molecules according to claim 4, wherein the parameter indicating the electrophoretic morphology is the ratio of length a to length b.
6. The method for analyzing chain molecules according to claim 1, wherein the parameters indicating the electrophoretic morphology include the electrophoretic velocity.